Small Molecule APOL1 Inhibitors as a Precision Medicine Approach for APOL1-mediated Kidney Disease

Small Molecule APOL1 Inhibitors as a Precision Medicine Approach for APOL1-mediated Kidney Disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Small Molecule APOL1 Inhibitors as a Precision Medicine Approach for APOL1-mediated Kidney Disease Brandon Zimmerman, Leslie Dakin, Anne Fortier, Evanthia Nanou, and 29 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3922092/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jan, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Chronic kidney disease affects ~10% of people worldwide and there are no disease modifying therapeutics that address the underlying cause of any form of kidney disease. Genome wide association studies have identified the G1 and G2 variants in the apolipoprotein L1 ( APOL1 ) gene as major contributors to a subtype of proteinuric kidney disease now referred to as APOL1‑mediated kidney disease (AMKD). We hypothesized that inhibition of APOL1 could have therapeutic potential for this genetically-defined form of kidney disease. Here we describe the development of preclinical assays and the discovery of highly potent and specific APOL1 inhibitors with drug-like properties. We provide evidence that APOL1 channel activity drives podocyte injury and that inhibition of this activity stops APOL1-mediated cell death and kidney damage in a transgenic mouse model. These preclinical data, combined with recent clinical data, support the potential of APOL1 channel inhibition for the treatment of AMKD. Health sciences/Nephrology/Kidney diseases/Glomerular diseases/Focal segmental glomerulosclerosis Health sciences/Nephrology/Kidney diseases/Chronic kidney disease/End-stage renal disease Biological sciences/Drug discovery/Target validation Biological sciences/Cell biology/Mechanisms of disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Main Chronic kidney disease (CKD) represents a significant global health burden, and the discovery of disease modifying treatments that address the underlying cause of kidney disease has proven difficult. Genome-wide association studies identified that individuals with recent ancestry from sub-Saharan Africa and chronic kidney disease are more likely to be homozygous or compound heterozygous for the G1 (S342G and I384M) or G2 (N388del:Y389del) variants in the a polipoprotein L1 ( APOL1) gene. Proteinuric kidney disease in the setting of two APOL1 alleles has been termed APOL1‑mediated kidney disease (AMKD) 1,2 . Patients with AMKD progress more rapidly than people with similar clinical features who lack APOL1 genotype 3-5 , and have higher rates of end stage renal disease, transplant, dialysis and death 1,6 . Moreover, inheriting two APOL1 risk variants carries increased risk of proteinuric kidney disease both in the absence of any other condition, as well as in the setting of inflammatory conditions such as interferon treatment, systemic lupus erythematosus, sickle cell disease, HIV, and COVID-19 7,8 . APOL1 is expressed in multiple tissues, most predominantly the liver 9 , where APOL1 is secreted into the bloodstream as part of high-density lipoprotein. Circulating APOL1 protects against Human African Trypanosomiasis, caused by Trypanosoma brucei brucei ( T.b.b. ). The mechanism whereby APOL1 confers resistance is through the formation of ion channels in the parasite membrane resulting in swelling and lysis of T.b.b. 1,10 . The APOL1 G1 and APOL1 G2 variants evolved from the ancestral allele, APOL1 G0, and rose to high frequency in sub-Saharan Africa due to positive selection: they protect against evolved strains of Trypanosoma brucei that evade APOL1 G0 11 . APOL1 is also expressed in the podocyte 12 , a specialized kidney cell involved in glomerular function 13 . Cell-specific podocyte APOL1 variant expression in mice leads to proteinuric nephropathy, providing evidence that renal APOL1 expression is intrinsic to the causal biology of disease progression 14 . In response to inflammation, expression levels of APOL1 in the podocyte increase 15 . The relationship between inflammation and APOL1 expression in the kidney offers a potential explanation as to both why not all people carrying two APOL1 G1/G2 variants develop kidney disease, and why numerous inflammatory conditions are associated with AMKD. The cellular and molecular mechanisms whereby APOL1 G1/G2 expression leads to podocyte cytotoxicity remains unclear. Proposed mechanisms include effects on autophagy, lysosomal permeability, mitochondrial dysfunction, endoplasmic reticulum stress, cation channel formation, 10,16-18 pyroptosis and activation of stress kinases 15,19-21 . While these various cellular processes may be consequential for downstream pathophysiology, the key to addressing the underlying cause of AMKD is to define the root cause whereby the G1 and G2 variants trigger podocyte cell damage. We hypothesized that APOL1 ion channel activity in the podocyte membrane was the source of APOL1-mediated podocyte toxicity and thus AMKD. We based this hypothesis on the high frequency of G1 and G2 variants due to protection from Trypanosoma infection, and that the biological mechanism of APOL1 protection is known to involve formation of ion channels in the parasite membrane. We further hypothesized that a highly specific APOL1 ion channel inhibitor would be effective in AMKD. We reasoned an APOL1 ion channel inhibitor would be likely to have limited on-target safety risk, because there is a published report of an individual with no detectable APOL1 protein expression due to homozygous truncation mutations. This individual was healthy except for a sensitivity to trypanosome infection 22 . Further support for this hypothesis comes from the observation that the APOL1 gene is found only in certain higher primates and is absent in other species, suggesting that APOL1 is not an essential gene in mammals 23,24 . To test the hypothesis that APOL1 ion channel activity drives kidney disease in AMKD, we set out to discover and develop a specific inhibitor of channel function, and then to evaluate the impact of APOL1 inhibition in a clinical trial of patients with AMKD. Here we present the development of assays and discovery of a novel series of oral, small molecule APOL1 inhibitors, exemplified by the clinical candidate inaxaplin (VX-147) 25 . These molecules are potent and specific inhibitors of APOL1 channel function in multiple cellular models. They both prevent proteinuria in an acute mouse model of AMKD and resolve established proteinuria and glomerular histopathology in a chronic mouse model of AMKD. As recently published, a 13-week clinical trial of VX-147 in patients with focal segmental glomerulosclerosis demonstrated a 47.6% reduction in proteinuria 25 . Development of assays to identify small molecule inhibitors of APOL1 channel activity To identify small molecule APOL1 channel inhibitors and characterize their properties and biological consequence, we generated a suite of in vitro assays and in vivo transgenic mouse models. First, using tetracycline (tet)-inducible HEK293 cell lines expressing APOL1 G0 , G1 or G2, we assessed APOL1 channel function by monitoring APOL1-mediated ion flux in a fluorescence-based thallium flux assay. Second, to confirm that inhibition of ion flux seen in the thallium flux assay was directly related to APOL1 ion channel inhibition, we also established a patch clamp electrophysiology assay to directly monitor APOL1-mediated ionic current. Third, to assess the impact of APOL1 ion channel function on cell viability, we generated an APOL1-mediated cell death assay in the same tet-inducible cell lines. Fourth, we rationalized that an APOL1 inhibitor should be able to prevent the only known biological function for APOL1, namely trypanolysis, so we established a trypanosome cell death assay. Finally, to investigate the effect of APOL1 channel inhibition in vivo , we generated transgenic mice homozygous for the APOL1 G2 variant to assess the impact of APOL1 inhibition on APOL1‑dependent proteinuria and renal pathology. Our expectation was that an APOL1 ion channel inhibitor would show efficacy against all these preclinical endpoints. Discovery of small molecule inhibitors of APOL1 channel activity Using the tet-inducible APOL1 G1 cell line, we initiated a high throughput screening campaign using a fluorescence-based thallium flux assay, which monitored APOL1-mediated passage of thallium (a surrogate for potassium ions [K + ]) across the plasma membrane. Compound 1 was our initial hit from the screen, with modest potency but full efficacy at inhibiting APOL1-mediated flux. We embarked on a medicinal chemistry campaign, which brought us to the lead optimization candidate, Compound 2. Further optimization of this candidate gave rise to a series of compounds that included Compound 3 and the clinical candidate, VX-147 (Fig. 1). Characterization of Compounds 1-4 are described in the methods, in Table S1 and Fig. S1-4. APOL1 risk variants lead to increased channel activity We hypothesized that the enhanced toxicity related to APOL1 G1 and G2 was the result of increased ion flux. Indeed, in the thallium flux assay, we saw increased flux activity in cells expressing risk variants relative to APOL1 G0. Using these APOL1-inducible cell lines, we assessed the ability of APOL1 to function as an ion channel by performing automated whole-cell patch clamp recordings. We found that all forms of APOL1 are permeable to monovalent cations (Na + , K + , Cs + ) and had a negative reversal potential when isolating outward currents (Fig. 2A). In addition, we discovered that the outward current amplitude generated by APOL1 G1 and APOL1 G2 was at least two-fold greater, compared to APOL1 G0-mediated currents (Fig. 2B). To ensure the increased current was not due to differential cell surface APOL1 expression, we monitored expression by flow cytometry. Cells expressing APOL1 G0 showed the highest cell surface expression, while cells expressing APOL1 G1 and G2 had significantly lower levels (~50% or less) on the surface (Fig. 2C). Next, we were interested to understand whether K + efflux or Na + influx is the initiating event of APOL1‑mediated toxicity. Therefore, we performed a cell death rescue assay in three different media conditions. We saw robust cell death in standard assay media allowing for Na + and K + flux as well as media only allowing K + efflux, however, when in media where K + efflux was inhibited, cell death was almost completely abrogated (Fig. 2D). Taken together, these experiments support the hypothesis that the cellular toxicity of APOL1 risk variants is due to increased channel activity of the APOL1 protein and associated increases in K + efflux. In vitro characterization of VX-147 To confirm direct inhibition of APOL1 ion channel activity by APOL1 inhibitors, we performed automated whole-cell patch recordings as above with increasing concentrations of VX-147. Using the average peak outward current before and after application, we determined VX-147 potently inhibited the APOL1 ion channel with IC 50 values of 2.3 nM for G0, 1.3 nM for APOL1 G1, and 1.1 nM for APOL1 G2 (Fig. 3A). Next, we evaluated the functional consequence of APOL1 inhibition by the ability of APOL1 to lyse T.b.b.. We found that VX‑147 prevented APOL1-mediated T.b.b. death with essentially equivalent potencies to ion channel inhibition, with EC 50 values of 2.0 nM for APOL1 G0, 2.1 nM for APOL1 G1, and 1.2 nM for APOL1 G2 (Fig. 3B). We further explored whether APOL1 inhibition could rescue cell death in the same HEK293 cells. We saw complete inhibition of APOL1-dependent cell death with EC 50 values of 4.3 nM for APOL1 G0, 2.0 nM for APOL1 G1, and 2.2 nM for APOL1 G2 (Fig. 3C). In conclusion, the fact that VX-147 is equipotent across all of these in vitro functional assays underscores the importance of channel activity as the mechanism of APOL1-mediated cell death. In vivo characterization of APOL1 channel inhibitors Given the robust inhibition of APOL1 in vitro , we sought to explore the functional effect of APOL1 inhibition in vivo. Since model organisms lack endogenous APOL1 expression, we established transgenic mice expressing human APOL1 G2 . We determined the APOL1 G2 transgene inserted multiple times in tandem, and therefore classify these mice as APOL1 G2 multicopy mice (APOL1 G2 mc ). We found that homozygous APOL1 G2 mc were viable, healthy, lived a normal lifespan, and had no baseline phenotype. However, a single injection of interferon-γ (IFNγ), which induces APOL1 kidneyexpression (Fig. S5A), led to significant APOL1‑dependent proteinuria in APOL1 G2 mc compared to control Friend leukemia B virus (FVB) mice (Fig. S5B and 5C). We recently reported prophylactic treatment with VX-147 reduced APOL1-dependent proteinuria in APOL1 G2 mc 25 . Here we show that a close structural analog of VX‑147, Compound 3, with comparable potency (Table 1), was able to prevent APOL1‑mediated proteinuria in this model at 30 mg/kg bid (Fig. S5D, S5E and S5F). Our prophylactic model demonstrated that APOL1 inhibitors could prevent proteinuria, but their impact on pre-established proteinuria and kidney histology was unknown. Therefore, we utilized a more aggressive published proteinuria model to assess the effect of APOL1 inhibition on established proteinuria and kidney pathology. This model entails the hydrodynamic injection of an IFNγ‑expressing plasmid into single copy, homozygous APOL1 G2 mice (APOL1 G2 sc ) 26 . These mice develop proteinuria approximately 5 days after plasmid injection, peaking around day 8 and remaining elevated through the study time course (Fig. 4A). Mice were randomized into two groups based on day 5 urinary albumin-to-creatinine ratio (UACR), and treatment with vehicle or 30 mg/kg bid Compound 3 began on day 6. Compound 3 had a rapid effect at lowering the UACR (Fig. 4A). Analysis of the UACR AUC following compound intervention revealed a 97% reduction over the 8 days of compound administration (Fig. 4B). Microscopic evaluations of kidney tissues showed that mice not injected with the IFNγ plasmid (Fig. 5A and 5D) had no evidence of glomerular or tubular damage. In vehicle-treatedAPOL1 G2 sc , approximately 60% of glomeruli exhibited histomorphologic changes (Fig. 5B, black arrow) while remaining glomeruli were morphologically normal (Fig 5B, white arrow). Glomerulopathy was characterized by segmental to global collapse of capillary lumina (Figure 5B), mesangial hyalinization (Fig. 5E), synechiae between capillary basement membranes and parietal epithelium, and vacuolation and reduplication of parietal epithelial cells (PEC). Tubules were often dilated, lined by attenuated epithelium, and contained eosinophilic proteinaceous fluid (Fig 5B and 5E). In contrast, no findings were noted in the animals treated with Compound 3 (Fig. 5C and 5F). Serial sections of kidneys were immunolabeled for nephrin 27,28 to investigate the effect of systemic inflammation (IFNγ) on glomerulal filtration barrier function. Whole slide imaging and an artificial intelligence (AI) based classifier 29,30 were used to quantify glomerular expression of nephrin. The quantitative scoring was expressed as an H-score, a cumulative assessment of area and intensity. The nephrin H-scores were comparable between the no IFNγ control and Compound 3-treated groups, while the vehicle-treated group had significantly lower nephrin H-scores (Fig. 5G). Based on the microscopic data, we employed a novel quantitative analysis of kidney health using the podocyte exact morphology measurement procedure (PEMP) 31 . This technique uses fluorescence immunolabeling for podocin and integrin α3 and super-resolution microscopy to assess podocyte health based on the filtration slit density (FSD). The FSD represents the ratio of length of the filtration slit for a given glomeruli (podocin staining) relative to its surface area (integrin staining) with high FSD values indicating healthy, intact glomeruli. Representative maximum intensity projections of the kidney filtration slit are shown for naïve, vehicle-treated and Compound 3-treated mice (Fig. 5H, 5I, 5J). Vehicle-treated mice showed significantly reduced FSD when compared to Compound 3-treated mice or naïve mice as summarized in Fig. 5K. Discussion Individuals with proteinuric CKD and two APOL1 risk variants (G1 or G2) have a genetically defined form of kidney disease referred to as AMKD. AMKD occurs in the presence of inflammatory conditions such as interferon treatment, lupus, SCD, HIV and COVID, but may also be observed in the absence of an overt inflammatory comorbidity. People with CKD and two APOL1 risk variants progress more rapidly to end-stage kidney disease relative to those without two APOL1 risk variants. While the genetic evidence demonstrates a key role of APOL1 G1 and G2 genetic variants in the human causal biology of proteinuric non-diabetic kidney disease, the molecular and cellular mechanism whereby APOL1 variants lead to kidney damage has been less clear. APOL1 is expressed in multiple tissues, and various cellular and molecular mechanisms have been proposed as relevant to AMKD. Based on the known mechanism of human biology whereby APOL1 variants protect from Trypanosoma Brucei infection, we hypothesized that APOL1 ion channel function leads to kidney damage. To test this hypothesis we developed multiple assays, conducted a high throughput screen to identify small molecule inhibitors of APOL1-mediated ion flux, and optimized those molecules to identify compounds with the potency, specificity, and drug-like properties suitable to evaluate the impact of APOL1 inhibition in cells, animal models, and, ultimately, AMKD patients. Here we present data that show the highly specific channel blocker, VX-147, directly inhibits APOL1mediated ion flux and prevents both APOL1-dependent trypanosome and mammalian cell death. VX-147 was shown to bind directly to APOL1 with high affinity and broad profiling across a large panel of potential off-targets did not identify any significant activity, indicating that the mechanism of action is due to direct inhibition of the APOL1 ion channel 25 . We report that the mechanism by which APOL1 G1 and G2 variants cause cell death is likely a result of their enhanced channel activity in human cells. We demonstrate that efflux of K + , and not influx of Na + , is responsible for APOL1-mediated cell death. There seems to be many downstream mechanisms by which APOL1-mediated ion flux leads to cell death 10,15–21 , but the root cause, and thus ideal therapeutic target, appears to be the ion channel function of APOL1. In fact, a recent publication highlighted the many proximal and distal events caused by APOL1-mediated ion flux and demonstrated that VX-147 treatment prevented or reversed these downstream events 32 . In transgenic APOL1 mouse models, we used Compound 3, a close analog of VX-147 with comparable potency, to show that blockage of the APOL1 channel can both prevent and reverse APOL1-dependent proteinuria as well as preserve glomerular integrity. This is the first reported evidence demonstrating that functional inhibition of APOL1 can significantly reduce established proteinuria in an in vivo preclinical model. Further evidence in support of the ion channel hypothesis comes from a recent human genetics study demonstrating that the N264K substitution in the protein sequence of APOL1 both decreased APOL1 ion flux and reduced kidney disease incidence in APOL1 G2 carriers 33 . The most important evaluation of the ion channel hypothesis is to perform clinical trials of potent and specific APOL1 inhibitors in AMKD patients. We recently reported Phase 2a proof-of-concept data with VX-147 in patients with two APOL1 risk alleles and proteinuric nondiabetic kidney disease 25 . This 13-week study in 16 individuals demonstrated a 47.6% reduction in proteinuria with stable GFR. An ongoing Phase 2b/3 clinical trial of inaxaplin in patients with two APOL1 risk variants and proteinuric non-diabetic kidney disease will further define the clinical potential of APOL1 inhibition for patients with AMKD. Declarations Acknowledgments: Thank you to all the APOL1 project team members, past and present, who helped further our understanding around APOL1 biology and contributed to the Vertex APOL1 program. We would also like to thank Dr. David Altshuler for advice on the project and comments on the manuscript. Author contributions: Conceptualization: B.Z, L.A.D., A.F., TS, BF, MEB. Methodology: BZ, LAD, AF, EN, AB, NH, SK, JP, TS, BF. Investigation: BZ, LAD, EN, AB, JM, HM, MF, TW, SN, GM, CM, PM, GW, PS, KD, HS, KS, FB, AP, FL, SP, SKP, LH, MH, FD, ON, NH, SK, JP, TS Writing – original draft: BZ, EN, BF, MEB. Writing – review & editing: BZ, LAD, EN, JP, BF, MEB. Competing interests: The authors declare the following competing financial interest(s): All authors listed are current or past employees of Vertex Pharmaceuticals, Incorporated and may hold stock in the company. Materials & Correspondence: Correspondence or material requests should be directed to Brandon Zimmerman or Mark E. Bunnage. Data and materials availability: All data are available in the manuscript or the supplementary material. Materials and Methods Cell line generation and growth Human embryonic kidney (HEK) cell lines containing a tetracycline (tet)-inducible expression system (T‑REx™; Invitrogen, Carlsbad, CA) and pAAVS1‑Puro‑APOL1 G0, pAAVS1‑Puro‑APOL1 G1 or pAAVS1‑Puro‑APOL1 G2 were generated. Individual clones of each cell line were generated and clones G0 DC2.13, G1 DC3.25, and G2 DC4.44 were used in this study. Cell growth medium was Dulbecco's modified Eagle’s medium (DMEM; Gibco, Gaithersburg, MD; Catalog # 11960‑077) supplemented with 10% (v/v) tet‑free fetal bovine serum (FBS; Takara Bio USA, Mountain View, CA; Catalog # 631368), 2 mM L‑glutamine (Gibco; Catalog # 25030‑081), 5 µg/mL Blasticidin S HCl (Gibco; Catalog # A11139‑03), 1 µg/mL puromycin dihydrochloride (Gibco; Catalog # A11138‑03), and 100 U/mL penicillin-streptomycin (Gibco; Catalog # 15140‑122). Electrophysiology HEK293 APOL1-inducible cells were induced with 15 ng/mL of tet for 16‑20 hours. Automated electrophysiology was performed by Qpatch48 (Sophion Biosciences). Cells were perfused with extracellular solution containing: 137 mM NMDG, 5.4 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM Glucose, 10 mM HEPES. The pH was adjusted to 7.2 with NaOH and adjusted to 300-305 mOsm with sucrose. Ouabain (100 μM) was added in the extracellular solution for blockade of Na + /K + ATP-ase. NMDG replaced Na + in the extracellular solution to isolate K + efflux. The intracellular solution contained: 124 mM CsF, 2 mM MgCl 2 , 1 mM CaCl 2 , 11 mM EGTA, 10 mM HEPES, 1 mM ATP pH 7.2, 285-290 mOsm. We used Cs + in the internal solution because APOL1 channels are permeable to Cs + but Cs + blocks endogenous potassium voltage-gated channels. Resistances of the QPatch plate chambers (holes) was 1.5-2.5 MΩ. Steady state inhibition of APOL1 current was measured using a ramp protocol in response to six consecutive compound concentrations (100 pM, 1 nM, 3 nM, 10 nM, 100 nM, 1 μM) with an exposure duration of 5 min to each concentration, after an initial 5 min current recording (control). Each solution exchange of 5 mL volume was performed twice. The voltage protocol applied to the cells was as follows: Resting step: ‑80 mV for 100 ms; test pulse: -100 mV to 80 mV for 200 ms; resting step: -80 mV for 100 ms repeated at 10 s intervals, from a holding potential of -90 mV. Peak outward current was measured at the end of the 200 s test pulse at +80 mV. Data acquisition and analysis was performed using the Sophion QPatch Assay Software (Sophion Biosciences). We continuously monitored the series resistance (MΩ) and the capacitance (pF). Cells were removed from analysis if the series resistance exceeded 15 MΩ, if the peak amplitude current was smaller than 100 pA, and if the reversal potential was smaller than ‑20 mV. The average peak outward currents before and after test compound application were used to calculate the percentage of current inhibited at each concentration. Outward currents in response to each concentration of VX-147 were normalized to its initial control current without VX-147. Concentration-response data were fit by GraphPad Prism using non-linear regression analysis with variable slope (four parameters). Flow cytometry HEK293 APOL1-inducible cells were grown in DMEM with 2% (v/v) doxycycline (dox)-free FBS in 96 well plates (Corning, 3904) at a seeding density of 50,000 cells/well in 100 μL of media and treated +/- 15 ng/mL doxycycline to induce APOL1 expression for 16-24 h. Cells were washed with 100 μL DPBS and detached with 100 μl of 1x Versene (Gibco, 15040066) at 37°C for 15 min. Cells were then resuspended in complete media and transferred to a 96-well plate round bottom plate (Corning, 3799) and pelleted in a tabletop centrifuge at 300 x g for 5 mins. All staining reactions were performed in FACS buffer (DPBS supplemented with 2% FBS and 2mM EDTA). Cells were stained for surface APOL1 expression in 50 μL of a 1:100 dilution of anti‑APOL1 rabbit monoclonal antibody (Abcam, ab252218) in FACS buffer on ice for 30 mins. Cells were washed in 200 μL of FACS buffer twice. Secondary staining was performed in 50 μL of a 1:2000 dilution of secondary goat anti-rabbit Alexa Fluor 647 antibody (Thermofisher, A32733) on ice for 20 mins. Cells were washed in 200 μL of FACS buffer twice. Cells were fixed in 50 μl of 4% paraformaldehyde for 15 min at room temperature. 4% PFA was washed off and replaced with 200 μL of FACS buffer for FACS analysis. Cells were immediately analyzed on a Beckman Coulter CytoFLEX instrument. The Alexa Fluor 647 signal was excited using the 638 nm laser and 660/10 nm BP filter. Cells were analyzed using FACS Diva and FlowJo Softwares. Thallium assay methods HEK293 APOL1-inducible cells were plated in DMEM with 2% (v/v) tet-free FBS with 15 ng/mL tet to induce APOL1 expression for 17-19 h. Cells were seeded into 384 well BioCoat™ Poly-D-Lysine coated, black/clear, flat-bottom, tissue culture-treated microplates at 2.6 x 10 4 cell/well. Loading buffer (HBSS, 20 mM HEPES, 5 mM Probenecid, 0.2 mM Ouabain, and components A and C from the FLIPR potassium assay bulk kit (Molecular Devices; Catalog # R8223) was added to the wells of the cell-containing assay plate. The cells were incubated for 30 minutes at room temperature (23°C) and then compounds were added to the assay plate wells. Compounds were tested with a top concentration of 10 μM followed by a quarter-log dilution series with 20 total points down to 0.312 nM. An active control was used in each assay and the DMSO concentration was kept fixed at 0.2%. The cells were incubated with compound at room temperature (23°C) for 30 minutes then the assay plate was placed on a FLIPR Tetra or Penta high-throughput cellular screening system to measure sample fluorescence intensity. Fluorescence readings were recorded every second for 10 seconds before the addition of thallium sulfate solution and an additional 60 seconds after the addition of thallium sulfate solution. Data were imported into Genedata for analysis and IC 50 generation. Trypanosome assay methods Trypanosoma brucei brucei were obtained from ATCC and grown in ATCC medium 2834 modified HMI-9 medium. Trypanosomes in phenol red/FBS free media containing Serum Plus medium supplement (Sigma, 14008C) were plated at a concentration of 120,000 parasites/well in 384-well plates containing compound and recombinant APOL1. The final concentration of APOL1 used in this assay was sufficient to kill approximately 90% of the parasites. AlamarBlueÔ was added to the plates following 16 h incubation at 37°C with APOL1 and compound, and then returned to the incubator for an additional 2.5 h. The plate was then read on a SpectraMax plate reader with excitation/emission wavelengths of 555/585 nm. Data was imported into Genedata for analysis and dose response curves generated using the Smart Fit algorithm. Multitox assay methods HEK293 APOL1-inducible cells were plated in DMEM with 2% (v/v) tet‑free FBS with 50 ng/mL tet to induce APOL1 expression for 24 h. Cells were plated into a 384-well Poly-D-Lysine coated plate at a density of 23,400 cells/well. Compounds were added to the plates for the full 24 h with a 10 point 3-fold dilution curve (top concentration, 10 μM) run in duplicate. The next day, assay buffer containing 2x MultiTox reagent from the MultiTox Multiplex cytotoxicity assay was added to each well and incubated an additional 30 min at 37°C. The plates were read on an EnVision plate reader for viability (excitation: 400 nm, emission: 486 nm) and cytotoxicity (excitation: 485 nm, emission: 535 nm). A ratio of dead (cytotoxicity) to live (viability) cells was used to normalize data. The data was exported and analyzed in Genedata. Data was normalized using two controls, no tet treatment (100% viability) and 50 ng/mL tet treatment (0% viability) and dose response curves generated using the Smart Fit algorithm. NMDG media (109.50 mM N-Methyl-D-Glucamine, 5.4 mM NaCl, 1.8 mM CaCl 2 , 0.81 mM Mg 2 SO 4 , 25 mM glucose, 4.5 nM Ferric Nitrate, 10 mM HEPES) or High K + media (115 mM KCl, 1.8 mM CaCl 2 , 0.8 mM Mg 2 SO 4 , 25 mM glucose, 4.5 nM Ferric Nitrate, 10 mM HEPES) were used to isolate the ionic flux to assess the critical ion for cell death. The MultiTox Multiplex cytotoxicity assay was performed as described above with standard media. Mouse model methods To evaluate the pharmacodynamic effects of APOL1 inhibitors in vivo, we established a transgenic mouse using a 104 kb portion of a bacterial artificial chromosome (BAC) containing APOL1 in its native human genomic context using human BAC clone CTD-2333M18 (BACPAC Resource Center at the Children's Hospital Oakland Research Institute), which includes the entire human APOL1 gene, the human promoter, introns, and all other cis regulatory regions including untranslated regions. The APOL1 G2 defining variant was incorporated by recombineering into an APOL1 G0 BAC construct. The transgenes were linearized by Notl digestion, purified, and microinjected into zygotes from the Friend leukemia virus B (FVB) mouse strain. Experiments were conducted in transgenic male and female FVB APOL1 multi-copy G2-expressing homozygous mice ( APOL1 G2 mc ) (8 to 15 weeks old, Charles River, Wilmington, MA, USA). The mice were bred to homozygosity as hemizygous mice do not have a phenotype. FVB APOL1 G2 single copy ( APOL1 G2 sc ) homozygous transgenic mice (with glutamate at amino acid 150) were generated at the Beth Israel Deaconess Medical Center (BIDMC) transgenic core facility. The G2 defining variant was derived from a G1 mouse line using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) was incorporated by recombineering (Yale O’Brien Center transgenic core facility) into an APOL1 G0 BAC construct. Mice were group-housed in a temperature-controlled environment (22 ± 1.5°C, 30 to 70% relative humidity, 12-hour light/dark) and were acclimatized in the animal facility for at least 3 days prior to use. Studies were conducted under a protocol that has been approved by the Vertex Animal Care and Use Committee. Experiments were performed at the Vertex Boston site, which is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC). Prophylactic assessment of Compound 3 In APOL1 G2 hom multicopy mice, increased proteinuria (mean urinary albumin-to-creatinine ratio, UACR) was induced by a single intraperitoneal (IP) injection of 150 µg/kg of murine IFNγ (R&D Systems, Catalog# 485-MI/CF) in a volume of 10 mL/kg. Urine was collected at various time points throughout the experiment, which lasted no more than 72 h. For proteinuria analysis, urine was diluted appropriately, and urine albumin and creatinine were measured using a mouse albumin ELISA and creatinine companion kit (Bethyl Laboratories, Catalog# E99-134 and Ethos Biosciences, Catalog# 1012). Compound 3 (30 mg/kg, dosed as a crystalline suspension) or vehicle, which is composed of 1% Hydroxypropyl methylcellulose acetate succinate, type H, 0.25% of Polyvinylpyrrolidone K 30 and 2% of d-α-Tocopheryl polyethylene glycol 1000 succinate (1%HPMCAS-H/0.25%PVP-K30/2%TPGS, Shin-Etsu, MP Biomedicals and Antares manufacturers, respectively) was administered orally twice daily (bid) at 12 h interval in a volume of 10 mL/kg. Compound 3 was administered 1.5 h before IFNγ injection. UACR was calculated by dividing the average of albumin replicate values (expressed in ng/mL) to the average of creatinine replicate values (expressed to mg/dL) and then converted to the unit µg/mg. Area under the curve (AUC) was calculated per animal for UACR values from 0 to 48 h or 72 h, using GraphPad Prism, with baseline set to the mean of all animals of the same strain at baseline (day 0). Percent reduction of UACR AUC was calculated using the following equation: ((Mean vehicle - Mean compound 3 )/(Mean Vehicle )) * 100. Statistical analysis was performed using GraphPad Prism. UACR AUC data was log-transformed and analyzed with two tailed t-test comparing Vehicle vs IFNγ within a mouse strain or to compound 3. Statistical significance was set at p≤0.05. Therapeutic assessment of Compound 3 in plasmid chronic G2 single copy mice Murine IFNγ cDNA was cloned in a CpG-free plasmid (Catalog #pcpgf-mcs) at Vertex laboratories. The day before the hydro dynamic injection (HDI) individual body weight was collected. The day of the injection, CpG-free IFNγ plasmid was thawed on wet ice and diluted in sterile saline to administer a total of 0.3 μg in a total volume corresponding to 10% body weight in grams. Mice were left in their cage under a heat lamp for 2 min, transferred to a restrainer where the area of injection was cleaned with ethanol wipes. Syringes (3 mL) with a 25-gauge needle were used for the tail vein injection. The full volume was injected in a continuous motion over approximately 5 to 10 seconds. Mice were placed in a cage on a heating pad and monitored for 30 minutes immediately following injection. Serum IFNγ levels were quantified using the ProQuantum Mouse IFNγ Immunoassay (Invitrogen, Catalog# A41150), to confirm successful administration of IFNγ plasmid in the chronic therapeutic mouse model. Blood (20 µL) was collected on days 1, 5, 9, 14 to evaluate serum IFNγ levels. Blood was allowed to clot for 30 minutes at room temperature and then centrifuged at 1500 relative centrifugal force (RCF) for 15 minutes. The serum fraction was collected and used to monitor IFNγ levels. For the ProQuantum Mouse IFNγ Immunoassay, the manufacturer’s protocol to quantify IFNγ was followed except for the following modifications to sample dilution: for day 1 dilution was 1:1000, for day 5 dilution was 1:50, for day 9 and 14 dilution was at 1:20. The standard curve was fit using a 4-parameter fit and sample values were interpolated using GraphPad Prism. UACR and UACR AUC Day6-14 data were graphed as mean ± standard error of the mean (SEM). UACR AUC day6-14 data was log-transformed and analyzed with One-way ANOVA followed by Tukey’s comparison test. Statistical significance was set at p≤0.05. Histopathology and immunohistochemistry At the end of the study, animals were euthanized as per the IACUC-approved protocol. Euthanized animals were necropsied immediately, the left kidney was resected and fixed in 10% neutral buffered formalin (48 h), followed by transfer to 70% histology grade ethanol (Cat # R3154-1GA, Millipore Sigma) and submitted to the pathology core laboratory. Formalin-fixed kidneys were longitudinally bisected through the mid-sagittal plane to include cortex, medulla, and pelvis. Bisected kidney samples were routinely processed (Tissue-Tek tissue processing system, Sakura-Fintek), and paraffin-embedded. Serial sections from paraffin-embedded blocks were stained for a) hematoxylin and eosin (H&E), and b) periodic acid Schiff’s (PAS) using standard staining protocols as per Ventana Discovery Ultra autostainer protocols (Ventana Medical Systems, Roche). Specific protocols for APOL1 and nephrin immunohistochemistry are described below and were performed using Ventana Discovery Ultra autostainer (Ventana Medical Systems, Roche). APOL1 and nephrin immunohistochemistry Formalin-fixed paraffin-embedded (FFPE) tissue blocks were cut at 3 µm thickness, dried overnight at 37°C, and baked at 60°C for 1 hour before use. All subsequent steps were performed on the Ventana Discovery Ultra platform (Ventana Medical Systems, Roche). The protocol for nephrin and APOL1 immunohistochemistry were similar with minor differences. FFPE sections were deparaffinized at 69°C, followed by epitope retrieval (APOL1 – 91°C, CC2 buffer for 32 min; nephrin – 95°C, CC1 buffer for 64 min), peroxide block for 8 min (inhibitor conditioning medium), and primary antibody incubation for 1 hour (APOL1 – rabbit anti APOL1 monoclonal antibody, Cat # ab252218, 1:250, Abcam; nephrin – rabbit anti-nephrin monoclonal antibody, Cat# ab216341, 1:1000, Abcam). Signals were amplified using OmniMap anti-Rabbit HRP kit (Cat # 760-4311) for 16 min and detected using DAB chromogen (Chromomap DAB Kit; Cat #760-159) for 8 min. Slides were counterstained for 8 min (Hematoxylin II, Cat # 970-2208), followed by bluing for 4 min (Bluing reagent, Cat # 760-2037) and coverslipped. Rabbit monoclonal IgG (clone DA1E, Cat # 3900, Cell Signaling Technologies) was used as isotype control for both APOL1 and nephrin primary antibodies. All reagents unless specified otherwise were provided by the manufacturer (Ventana Medical Systems). Coverslipped and immunolabeled glass slides were scanned using Aperio AT2 whole slide scanner (Leica Biosystems) at 40x objective magnification and were visualized using image scope/web scope software (Leica Biosystems). Immunolabeled glass slides were scanned using Aperio AT2 whole slide scanner (Leica Biosystems) at 40x objective magnification and were visualized using image scope/web scope software (Leica Biosystems). All antibodies were diluted using the antibody diluent (Cat# ADB250, Ventana Medical Systems). All reagents unless specified otherwise were provided by the manufacturer (Ventana Medical Systems). Morphometric analysis nephrin expression in glomeruli Immunolabelled Stained glass slides were scanned at 40x magnification (Aperio AT2, Leica Biosystems) and quality checked for any artifacts (i.e., damaged tissue, wrinkles, particles, scanning lines). Scanned whole slide images (WSI) were imported into HALO v3.4 (Indica Labs), where images were manually annotated to include the renal cortex and exclude artifacts. An Artificial Intelligence- Convolution Neural Network I - CNN (DenseNet from HALO, Indica Labs V3.2) based classifier (“Kidney_Glomerulus_APOL1 DenseNet”), was trained to automatically identify glomeruli within kidney sections. The images were manually curated to remove any abnormalities or non-glomerular structures from further analysis. Morphometric image analysis was performed using an Area Quantification (v1.0) module in HALO (“APOL1- Kidney_Glomerulus_APOL1 DenseNet”). Intensity thresholds were set to detect weak, moderate, and strong intensity of DAB chromogen (brown) staining for APOL1 and nephrin with pathologist’s guidance. All the available glomeruli detected by the algorithm in a one longitudinal section of the kidney per animal were analyzed from each sample and a cumulative output (H-score) was obtained. H-score was calculated as the following: Statistical analysis was performed in GraphPad Prism 9 using the Kruskall-Wallis test with multiple comparisons. Graphs were generated in Microsoft Excel and Prism. Podocyte exact morphology measurement procedure A subset of mice was selected for a morphological assessment of the glomerular slit diaphragm. Mice were injected with a cocktail of 40 mg/mL ketamine and 2 mg/mL Xylazine. Level of anesthesia was then assessed with firm toe pinch. Once the subject was fully anesthetized, it was transferred to the working station for the perfusion. Area of incision was cleaned with an alcohol pad and tweezers were used to cut through the skin and expose the chest cavity. The diaphragm and the ribcage were cut to expose the heart. The ribcage was lifted, and a 25-gauge needle connected was inserted in the apex of the left ventricle. The right atrium was then cut, and the subject was perfused with 1X PBS/15,000-unit heparin at the rate of 20 mL/min for 3 min to washout the blood from the organs using a standard programmable infusion pump (Harvard Apparatus, Cat # 70-4500 ). To restrict fixation to the right kidney, the left kidney renal artery was clamped. Right kidney was perfused with 4% PFA at the rate of 20 mL/min for 5 min using an infusion pump (New Era Pump Systems, Inc. Cat # NE-9000). Right kidneys were then cut in half and left rotating at room temperature for 1 h inside a 15 mL conical tube. Sample were transferred to 4°C before shipment to NIPOKA GmbH. After deparaffinization and rehydration, formalin-fixed and paraffin-embedded kidney sections (3 µm) were boiled in Tris-EDTA buffer (10 mmol/l Tris, 1 mmol/l EDTA, pH 9) in a pressure cooker for 5 min, followed by the blocking step (1% FBS, 1% BSA, 0.1% fish gelatin, 1% normal goat serum) for 1 hour. The following primary antibodies were incubated overnight at 4°C: rabbit anti‐podocin 1:150 (IBL International, Hamburg, Germany, JP-29040) and mouse anti‐integrin alpha3 1:500 (Santacruz, Dallas, Texas, USA, sc-374242). After three washing steps in PBS, secondary antibodies were incubated for 1 hour at room temperature (anti-rabbit Alexa Fluor 488‐conjugated IgG 1:600 (ChromoTek, Planegg, Germany, srbAF488-1-100) and anti-mouse Cy3‐conjugated IgG antibody 1:600 (Jackson ImmunoResearch, West Grove, PA, 115‑166-006) for 1 hour at room temperature. DAPI (1:100) was added to the slides for 5 min, followed by a washing step in PBS. Finally, the slides were incubated in H2O and mounted in Mowiol (Carl Roth, Karlsruhe, Germany) using high precision coverslips (Paul Marienfeld, Lauda-Königshofen, Germany). The evaluation of the filtration slit density (FSD) was performed using a recently established super-resolution microscopy-based methodology termed podocyte exact morphology measurement procedure (PEMP) 31 . For three-dimensional structured illumination microscopy (3D-SIM), z-stacks of 19 planes of both channels (488 and 561 nm) were acquired from the stained kidney section using an N-SIM super-resolution microscope (Nikon, Tokyo, Japan) equipped with a 100x silicone objective. The images were reconstructed into 3D-SIM images using NIS-Elements AR 5.30 (Nikon, Tokyo, Japan). The z-stacks were converted into a maximum intensity projection followed by the automatized identification of podocyte foot process areas that were selected based on the foot process positive antibody. Inside these areas, the filtration slit diaphragm length was automatically determined. The FSD was expressed as the ratio of the total filtration slit diaphragm length per podocyte foot process area. The FSD of 20 randomly selected glomeruli was determined for every animal. Synthesis and characterization of chemical compounds: The chemical compounds ( 2 - 4 ) in this manuscript were prepared according to the synthetic procedures detailed in the published patent, WO2020131807, titled “Inhibitors of APOL1 and methods of using same”. Purity assessment for final compounds was based on analytical UPLC: 2.1 mm × 50 mm Waters CSH C18 column, 1.7 μM, 130 Å. Mobile phases were as follows: A, H 2 O with 0.1% trifluoroacetic acid; B, acetonitrile with 0.1% trifluoroacetic acid; gradient, 10–60% B in 0.6 min with a 1.4 min run time. The flow rate was 0.6 mL/min. Mass samples were analyzed on a Waters 3100 single quad mass spectrometer operated in positive MS mode with electrospray ionization. The mobile phase for all mass analysis consisted of acetonitrile–water mixtures with either 0.1% trifluoroacetic acid or ammonium formate. 1 H NMR spectra were recorded using either a Bruker Avance 400 (400 MHz) or a Bruker Avance II-300 (300 MHz) instrument. The screening hit, 1 , is commercially available and was purchased from an external vendor. Compound 1 has a Chemical Abstracts Service Registry Number of 687574-42-1. References Friedman DJ, Pollak MR (2021) APOL1 Nephropathy: From Genetics to Clinical Applications. Clin J Am Soc Nephrol 16:294–303. https://doi.org:10.2215/CJN.15161219 Lipkowitz MS et al (2013) Apolipoprotein L1 gene variants associate with hypertension-attributed nephropathy and the rate of kidney function decline in African Americans. Kidney Int 83:114–120. https://doi.org:10.1038/ki.2012.263 Kopp JB et al (2011) APOL1 genetic variants in focal segmental glomerulosclerosis and HIV-associated nephropathy. 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J Clin Invest. https://doi.org:10.1172/jci172262 Hung AM et al (2023) Genetic Inhibition of APOL1 Pore-Forming Function Prevents APOL1-Mediated Kidney Disease. J Am Soc Nephrol. https://doi.org:10.1681/ASN.0000000000000219 Table Table 1: Medicinal chemistry progression of the screening hit 1 to VX-147. Reversal of the amide and installation of the aminolactam afforded compound 2 with increased potency and metabolic stability. Oxidation of the lactam to compound 3 further increased potency and metabolic stability. Further Structure-Activity-Relationship (SAR) investigation of the indole core gave VX-147 with optimized properties. V d : volume of distribution, T 1/2 : half-life, Cl: clearance Additional Declarations Yes there is potential Competing Interest. The authors declare the following competing financial interest(s): All authors listed are current or past employees of Vertex Pharmaceuticals, Incorporated and may hold stock in the company. 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Pharmaceuticals","correspondingAuthor":false,"prefix":"","firstName":"Francois","middleName":"","lastName":"Denis","suffix":""},{"id":287809646,"identity":"da361e0b-e8fc-4428-a7f0-affc65de0f0a","order_by":26,"name":"Olivier Nicolas","email":"","orcid":"","institution":"Vertex Pharmaceuticals","correspondingAuthor":false,"prefix":"","firstName":"Olivier","middleName":"","lastName":"Nicolas","suffix":""},{"id":287809647,"identity":"86179a50-0381-4695-bec3-31dddae6aee6","order_by":27,"name":"Niresh Hariparsad","email":"","orcid":"","institution":"Vertex Pharmaceuticals","correspondingAuthor":false,"prefix":"","firstName":"Niresh","middleName":"","lastName":"Hariparsad","suffix":""},{"id":287809648,"identity":"0869127b-9f43-4783-a150-565271cac7c1","order_by":28,"name":"Shyamesh Kumar","email":"","orcid":"","institution":"Vertex Pharmaceuticals","correspondingAuthor":false,"prefix":"","firstName":"Shyamesh","middleName":"","lastName":"Kumar","suffix":""},{"id":287809649,"identity":"ceac09ea-53da-4a1a-8f75-609da5f8920c","order_by":29,"name":"Jennifer Proctor","email":"","orcid":"","institution":"Vertex Pharmaceuticals","correspondingAuthor":false,"prefix":"","firstName":"Jennifer","middleName":"","lastName":"Proctor","suffix":""},{"id":287809650,"identity":"fdb2a285-358f-442c-82d4-1269744f4ebb","order_by":30,"name":"Timothy Senter","email":"","orcid":"https://orcid.org/0000-0003-2766-8353","institution":"Vertex Pharmaceuticals","correspondingAuthor":false,"prefix":"","firstName":"Timothy","middleName":"","lastName":"Senter","suffix":""},{"id":287809651,"identity":"58d53f84-b187-43f0-835d-6f4385afdb3d","order_by":31,"name":"Brinley Furey","email":"","orcid":"","institution":"Vertex Pharmaceuticals Incorporated","correspondingAuthor":false,"prefix":"","firstName":"Brinley","middleName":"","lastName":"Furey","suffix":""},{"id":287809652,"identity":"9af9f306-04cf-4a89-84a2-7edde109890c","order_by":32,"name":"Mark Bunnage","email":"","orcid":"","institution":"Vertex","correspondingAuthor":false,"prefix":"","firstName":"Mark","middleName":"","lastName":"Bunnage","suffix":""}],"badges":[],"createdAt":"2024-02-02 21:15:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3922092/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3922092/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-024-55408-2","type":"published","date":"2025-01-02T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54288415,"identity":"14eeb899-7eff-4c9a-9923-5c540b7a73fe","added_by":"auto","created_at":"2024-04-08 11:04:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":53063,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMedicinal chemistry progression of the screening hit to VX-147\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3922092/v1/f96a042e21db9e922ff6cd28.png"},{"id":54288416,"identity":"780761d6-b772-401d-8bf7-c3418c0a14ba","added_by":"auto","created_at":"2024-04-08 11:04:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":93532,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAPOL1 G1 and G2 toxicity is associated with enhanced outward currents on the cell surface. (A)\u003c/strong\u003e Average reversal potential of APOL1-G0, -G1, -G2 -mediated outward currents. \u003cstrong\u003e(B) \u003c/strong\u003eAverage peak current amplitude of APOL1 – G0, G1- and G2- mediated outward currents. \u003cstrong\u003e(C)\u003c/strong\u003e HEK293 cells stably expressing tetracycline-inducible APOL1 G0, G1 or G2 variants. APOL1 protein surface levels were measured by fluorescence activated cell sorting (FACS) analysis and the Median Fluorescence Intensity (MFI) of the AF647 channel was reported. MFI was analyzed by One-way ANOVA with post-hoc Dunnett’s multiple comparison test with APOL1 G0 as the control. Significance was set at p≤0.05. ****p\u0026lt;0.0001. Results are presented as arithmetic mean ± SD. \u003cstrong\u003e(D)\u003c/strong\u003eNormalized cell death of HEK293 cells expressing APOL1 G1 in different media compositions.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3922092/v1/d226193131467ba0742bfb6d.png"},{"id":54288418,"identity":"a0aff615-b5de-4ae3-b486-77860c237584","added_by":"auto","created_at":"2024-04-08 11:04:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":84727,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVX-147 inhibits APOL1-mediated flux and cell death.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e The graph shows baseline normalized APOL1-mediated ionic current measured for HEK293 cell lines expressing APOL1 G0, APOL1 G1, and APOL1 G2 variants, as a function of compound concentration. The first concentration point is no VX-147 and was artificially assigned as 2 orders of magnitude smaller than the lowest concentration tested for graphical representation. \u003cstrong\u003e(B)\u003c/strong\u003e Assessment of \u003cem\u003eTrypanosoma brucei brucei \u003c/em\u003ecell viability in the presence of recombinant APOL1 of the indicated variant using an alamarBlue™ assay. \u003cstrong\u003e(C)\u003c/strong\u003e Assessment of cell viability for HEK293 cell lines expressing APOL1 G0, APOL1 G1, and APOL1 G2 variants using the Promega MultiTox fluorescence assay. All results are presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3922092/v1/944baf73a84574de798fff9d.png"},{"id":54288529,"identity":"cd96baea-3d46-473c-ac43-1db5e6bb6877","added_by":"auto","created_at":"2024-04-08 11:12:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":84245,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of Compound 3 efficacy in therapeutic mouse model of APOL1-mediated kidney disease\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Assessment of urinary albumin-to-creatinine ratio (UACR) in response to hydrodynamic injection of IFNγ plasmid in \u003cem\u003eAPOL1 G2 \u003c/em\u003etransgenic mice over 14 days. n=11-12/group. \u003cstrong\u003e(B)\u003c/strong\u003eUACR area under the curve (AUC) showed reduced proteinuria in Compound 3-treated mice. Percent reduction in UACR AUC with compound 3 treatment was calculated. UACR AUC log transformed data were analyzed by One-way ANOVA with post-hoc Tukey’s multiple comparison test. Significance was set at p≤0.05. ****p\u0026lt;0.0001. Results are presented as arithmetic mean ± SEM.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3922092/v1/5357a4afeea8aef19ff77820.png"},{"id":54288420,"identity":"9c2ac61b-88f1-4304-a036-5deeeccfc0e0","added_by":"auto","created_at":"2024-04-08 11:04:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":816867,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCompound 3 prevents APOL1-mediated morphological and histopathological damage of kidney glomeruli.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A and D)\u003c/strong\u003e No microscopic findings were present in control group mice. \u003cstrong\u003e(B and E)\u003c/strong\u003e Glomerulopathy was frequent and severe in vehicle-treated mice, however all glomeruli were not affected. The affected glomerulus on left (\u003cstrong\u003eB\u003c/strong\u003e, black arrow) was characterized by collapsed capillary lumina, mesangial hyalinization and duplication of parietal epithelium with frequent synechiae and accumulation of PAS+ve \u003cstrong\u003e(E)\u003c/strong\u003e material in the mesangium. The adjacent glomerulus on right (B, white arrow) was morphologically normal. \u003cstrong\u003e(C and F)\u003c/strong\u003e No morphologic findings were noted in Compound 3-treated mice. \u003cstrong\u003e(G)\u003c/strong\u003e Quantitative image analysis of nephrin immunolabeling using H-score. Nephrin was significantly reduced in vehicle-treated mice compared to control and was statistically comparable to the Compound 3 treated group. H-score (label intensity * % area); weak (+1), moderate (+2) strong (+3). Statistical Analysis: Nephrin H-score data was analyzed by a Kruskal-Wallis test with post hoc Dunn’s multiple comparison test. Significance was set at p≤0.05. *p≤0.05. \u003cstrong\u003e(H-J) \u003c/strong\u003eMaximum intensity projection (MIP) of kidney filtration slit in control mice \u003cstrong\u003e(H)\u003c/strong\u003e, in mice treated with IFNγ and vehicle \u003cstrong\u003e(I)\u003c/strong\u003e, and in mice treated with IFNγ and 30 mg/kg Compound 3 \u003cstrong\u003e(J)\u003c/strong\u003e. Kidneys were immunolabeled for podocin and integrin α3. Images were acquired with 3D-Structured Illumination Microscopy. \u003cstrong\u003e(K) \u003c/strong\u003eThe filtration slit density (FSD) of n=3 mice/group. 20 glomeruli per mouse were randomly selected and analyzed for FSD. FSD was calculated as the ratio between filtration slit length and surface area. Vehicle-treated mice showed reduced FSD when compared to Compound 3‑treated and naïve mice. Data were analyzed with One-way ANOVA. The Benjamini-Hochberg procedure was applied post-hoc to control for false discovery rate. Significance was set at p≤0.05.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3922092/v1/e69d43e5ec09b76d7c87715e.png"},{"id":72875692,"identity":"a53a0170-7cf0-420c-889e-9b8b231f54e3","added_by":"auto","created_at":"2025-01-03 08:09:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1865692,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3922092/v1/77b64b87-189d-404f-b772-66c17ec69636.pdf"},{"id":54288527,"identity":"0e595992-5558-47e7-8f61-fd6b7a10a63b","added_by":"auto","created_at":"2024-04-08 11:12:12","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":872038,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-3922092/v1/ec8ced13a57f7a754ee0fd84.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nThe authors declare the following competing financial interest(s): All authors listed are current or past employees of Vertex Pharmaceuticals, Incorporated and may hold stock in the company.","formattedTitle":"Small Molecule APOL1 Inhibitors as a Precision Medicine Approach for APOL1-mediated Kidney Disease","fulltext":[{"header":"Main","content":"\u003cp\u003eChronic kidney disease (CKD) represents a significant global health burden, and the discovery of disease modifying treatments that address the underlying cause of kidney disease has proven difficult. \u0026nbsp;Genome-wide association studies identified that individuals with recent ancestry from sub-Saharan Africa and chronic kidney disease are more likely to be homozygous or compound heterozygous for the G1 (S342G and I384M) or G2 (N388del:Y389del) variants in the \u0026nbsp;\u003cem\u003ea\u003c/em\u003e\u003cem\u003epolipoprotein L1\u003c/em\u003e (\u003cem\u003eAPOL1)\u003c/em\u003e gene. Proteinuric kidney disease in the setting of two APOL1 alleles has been termed APOL1‑mediated kidney disease (AMKD)\u0026nbsp;\u003csup\u003e1,2\u003c/sup\u003e. Patients with AMKD progress more rapidly than people with similar clinical features who lack APOL1 genotype\u0026nbsp;\u003csup\u003e3-5\u003c/sup\u003e, and have higher rates of end stage renal disease, transplant, dialysis and death\u0026nbsp;\u003csup\u003e1,6\u003c/sup\u003e. \u0026nbsp;Moreover, inheriting two \u003cem\u003eAPOL1\u003c/em\u003e risk variants carries increased risk of proteinuric kidney disease both in the absence of any other condition, as well as in the setting of inflammatory conditions such as interferon treatment, systemic lupus erythematosus, sickle cell disease, HIV, and COVID-19\u0026nbsp;\u003csup\u003e7,8\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAPOL1\u003c/em\u003e is\u0026nbsp;expressed in multiple tissues, most predominantly the liver\u0026nbsp;\u003csup\u003e9\u003c/sup\u003e, where APOL1 is secreted into the bloodstream as part of high-density lipoprotein. Circulating APOL1 protects against Human African Trypanosomiasis, caused by \u003cem\u003eTrypanosoma brucei brucei\u003c/em\u003e (\u003cem\u003eT.b.b.\u003c/em\u003e). The mechanism whereby APOL1 confers resistance is through the formation of ion channels in the parasite membrane resulting in\u0026nbsp;swelling and\u0026nbsp;lysis of\u0026nbsp;\u003cem\u003eT.b.b.\u0026nbsp;\u003c/em\u003e\u003cem\u003e\u003csup\u003e1,10\u003c/sup\u003e\u003c/em\u003e. \u0026nbsp;The \u003cem\u003eAPOL1\u003c/em\u003e \u003cem\u003eG1\u003c/em\u003e and \u003cem\u003eAPOL1\u003c/em\u003e \u003cem\u003eG2\u003c/em\u003e variants evolved from the ancestral allele, \u003cem\u003eAPOL1\u003c/em\u003e \u003cem\u003eG0,\u0026nbsp;\u003c/em\u003eand rose to high frequency in\u0026nbsp;sub-Saharan Africa due to positive selection: they protect against evolved strains of \u003cem\u003eTrypanosoma brucei\u0026nbsp;\u003c/em\u003ethat evade APOL1 G0\u0026nbsp;\u003csup\u003e11\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAPOL1\u003c/em\u003e is also expressed in the podocyte\u0026nbsp;\u003csup\u003e12\u003c/sup\u003e, a specialized kidney cell involved in glomerular function\u0026nbsp;\u003csup\u003e13\u003c/sup\u003e. Cell-specific podocyte \u003cem\u003eAPOL1\u003c/em\u003e variant expression in mice leads to proteinuric nephropathy, providing evidence that renal APOL1 expression is intrinsic to the causal biology of disease progression\u0026nbsp;\u003csup\u003e14\u003c/sup\u003e. In response to inflammation, expression levels of APOL1 in the podocyte increase\u0026nbsp;\u003csup\u003e15\u003c/sup\u003e. The relationship between inflammation and APOL1 expression in the kidney offers a potential explanation as to both why not all people carrying two \u003cem\u003eAPOL1 G1/G2\u003c/em\u003e variants develop kidney disease, and why numerous inflammatory conditions are associated with AMKD.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe cellular and molecular mechanisms whereby APOL1 G1/G2 expression leads to podocyte cytotoxicity remains unclear. Proposed mechanisms include effects on autophagy, lysosomal permeability, mitochondrial dysfunction, endoplasmic reticulum stress, cation channel formation,\u0026nbsp;\u003csup\u003e10,16-18\u003c/sup\u003e pyroptosis and activation of stress kinases\u0026nbsp;\u003csup\u003e15,19-21\u003c/sup\u003e. While these various cellular processes may be consequential for downstream pathophysiology, the key to addressing the underlying cause of AMKD is to define the root cause whereby the G1 and G2 variants trigger podocyte cell damage. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe hypothesized that APOL1 ion channel activity in the podocyte membrane was the source of APOL1-mediated podocyte toxicity and thus AMKD. \u0026nbsp;We based this hypothesis on\u0026nbsp;the high frequency of \u003cem\u003eG1\u003c/em\u003e and \u003cem\u003eG2\u0026nbsp;\u003c/em\u003evariants due to protection from\u0026nbsp;\u003cem\u003eTrypanosoma\u0026nbsp;\u003c/em\u003einfection, and that the biological mechanism of APOL1 protection is known to involve\u0026nbsp;formation of ion channels in the parasite membrane. \u0026nbsp;We further hypothesized that a highly specific APOL1 ion channel inhibitor would be effective in AMKD. We reasoned an APOL1 ion channel inhibitor would be likely to have limited on-target safety risk, because\u0026nbsp;there is a published report of an individual with no detectable APOL1 protein expression due to homozygous truncation mutations. This individual was healthy except for a sensitivity to trypanosome infection\u0026nbsp;\u003csup\u003e22\u003c/sup\u003e.\u0026nbsp;Further support for this hypothesis comes from the observation that the\u0026nbsp;\u003cem\u003eAPOL1\u0026nbsp;\u003c/em\u003egene is found only in certain higher primates and is absent in other species, suggesting that \u003cem\u003eAPOL1\u003c/em\u003e is not an essential gene in mammals\u0026nbsp;\u003csup\u003e23,24\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo test the hypothesis that APOL1 ion channel activity drives kidney disease in AMKD, we set out to discover and develop a specific inhibitor of channel function, and then to evaluate the impact of APOL1 inhibition in a clinical trial of patients with AMKD. \u0026nbsp;Here we present the development of assays and discovery of a novel series of oral, small molecule APOL1 inhibitors, exemplified by the clinical candidate inaxaplin (VX-147)\u0026nbsp;\u003csup\u003e25\u003c/sup\u003e. \u0026nbsp;These molecules are potent and specific inhibitors of APOL1 channel function in multiple cellular models. \u0026nbsp;They both prevent proteinuria in an acute mouse model of AMKD and resolve established proteinuria and glomerular histopathology in a chronic mouse model of AMKD. As recently published, a 13-week clinical trial of VX-147 in patients with focal segmental glomerulosclerosis demonstrated a 47.6% reduction in proteinuria\u0026nbsp;\u003csup\u003e25\u003c/sup\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDevelopment of assays to identify small molecule inhibitors of APOL1 channel activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify small molecule APOL1 channel inhibitors and characterize their properties and biological consequence, we generated a suite of \u003cem\u003ein vitro\u003c/em\u003e assays and \u003cem\u003ein vivo\u003c/em\u003e transgenic mouse models. \u0026nbsp;First, using tetracycline (tet)-inducible HEK293 cell lines expressing \u003cem\u003eAPOL1 G0\u003c/em\u003e, \u003cem\u003eG1\u003c/em\u003e or \u003cem\u003eG2,\u0026nbsp;\u003c/em\u003ewe assessed APOL1 channel function by monitoring APOL1-mediated ion flux in a fluorescence-based thallium flux assay. Second, to confirm that inhibition of ion flux seen in the thallium flux assay was directly related to APOL1 ion channel inhibition, we also established a patch clamp electrophysiology assay to directly monitor APOL1-mediated ionic current. Third, to assess the impact of APOL1 ion channel function on cell viability, we generated an APOL1-mediated cell death assay in the same tet-inducible cell lines. Fourth, we rationalized that an APOL1 inhibitor should be able to prevent the only known biological function for APOL1, namely trypanolysis, so we established a trypanosome cell death assay. Finally, to investigate the effect of APOL1 channel inhibition \u003cem\u003ein vivo\u003c/em\u003e, we generated transgenic mice homozygous for the \u003cem\u003eAPOL1 G2\u003c/em\u003e variant to assess the impact of APOL1 inhibition on APOL1‑dependent proteinuria and renal pathology. Our expectation was that an APOL1 ion channel inhibitor would show efficacy against all these preclinical endpoints.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiscovery of small molecule inhibitors of APOL1 channel activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing the tet-inducible \u003cem\u003eAPOL1 G1\u003c/em\u003e cell line, we initiated a high throughput screening campaign using a fluorescence-based thallium flux assay, which monitored APOL1-mediated passage of thallium (a surrogate for potassium ions [K\u003csup\u003e+\u003c/sup\u003e]) across the plasma membrane. Compound 1 was our initial hit from the screen, with modest potency but full efficacy at inhibiting APOL1-mediated flux. We embarked on a medicinal chemistry campaign, which brought us to the lead optimization candidate, Compound 2. Further optimization of this candidate gave rise to a series of compounds that included Compound 3 and the clinical candidate, VX-147 (Fig. 1). Characterization of Compounds 1-4 are described in the methods, in Table S1 and Fig. S1-4.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAPOL1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;risk variants lead to increased channel activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe hypothesized that the enhanced toxicity related to APOL1 G1 and G2 was the result of increased ion flux. Indeed, in the thallium flux assay, we saw increased flux activity in cells expressing risk variants relative to APOL1 G0. Using these APOL1-inducible cell lines, we assessed the ability of APOL1 to function as an ion channel by performing automated whole-cell patch clamp recordings. We found that all forms of APOL1 are permeable to monovalent cations (Na\u003csup\u003e+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e, Cs\u003csup\u003e+\u003c/sup\u003e) and had a negative reversal potential when isolating outward currents (Fig. 2A). In addition, we discovered that the outward current amplitude generated by APOL1 G1 and APOL1 G2 was at least two-fold greater, compared to APOL1 G0-mediated currents (Fig. 2B). To ensure the increased current was not due to differential cell surface APOL1 expression, we monitored expression by flow cytometry. Cells expressing APOL1 G0 showed the highest cell surface expression, while cells expressing APOL1 G1 and G2 had significantly lower levels (~50% or less) on the surface (Fig. 2C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, we were interested to understand whether K\u003csup\u003e+\u003c/sup\u003e efflux or Na\u003csup\u003e+\u003c/sup\u003e influx is the initiating event of APOL1‑mediated toxicity. Therefore, we performed a cell death rescue assay in three different media conditions. We saw robust cell death in standard assay media allowing for Na\u003csup\u003e+\u003c/sup\u003e and K\u003csup\u003e+\u003c/sup\u003e flux as well as media only allowing K\u003csup\u003e+\u003c/sup\u003e efflux, however, when in media where K\u003csup\u003e+\u003c/sup\u003e efflux was inhibited, cell death was almost completely abrogated (Fig. 2D). Taken together, these experiments support the hypothesis that the cellular toxicity of APOL1 risk variants is due to increased channel activity of the APOL1 protein and associated increases in K\u003csup\u003e+\u003c/sup\u003e efflux.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;characterization of VX-147\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo confirm direct inhibition of APOL1 ion channel activity by APOL1 inhibitors, we performed automated whole-cell patch recordings as above\u0026nbsp;with increasing concentrations of VX-147.\u0026nbsp;Using the average peak outward current before and after application, we determined VX-147 potently inhibited the APOL1 ion channel with IC\u003csub\u003e50\u003c/sub\u003e values of 2.3 nM for G0, 1.3 nM for APOL1 G1, and 1.1 nM for APOL1 G2\u0026nbsp;(Fig. 3A). \u0026nbsp; Next, we evaluated the functional consequence of APOL1 inhibition by the ability of APOL1 to lyse \u003cem\u003eT.b.b..\u003c/em\u003e We found that VX‑147 prevented APOL1-mediated \u003cem\u003eT.b.b.\u003c/em\u003e death with essentially equivalent potencies to ion channel inhibition, with EC\u003csub\u003e50\u003c/sub\u003e values of 2.0 nM for APOL1 G0, 2.1 nM for APOL1 G1, and 1.2 nM for APOL1 G2 (Fig. 3B). We further explored whether APOL1 inhibition could rescue cell death in the same HEK293 cells. We saw complete inhibition of APOL1-dependent cell death with EC\u003csub\u003e50\u003c/sub\u003e values of 4.3 nM for APOL1 G0, 2.0 nM for APOL1 G1, and 2.2 nM for APOL1 G2 (Fig. 3C). In conclusion, the fact that VX-147 is equipotent across all of these \u003cem\u003ein vitro\u003c/em\u003e functional assays underscores the importance of channel activity as the mechanism of APOL1-mediated cell death.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;characterization of APOL1 channel inhibitors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the robust inhibition of APOL1 \u003cem\u003ein vitro\u003c/em\u003e, we sought to explore the functional effect of APOL1 inhibition \u003cem\u003ein vivo.\u003c/em\u003e Since model organisms lack endogenous \u003cem\u003eAPOL1\u0026nbsp;\u003c/em\u003eexpression, we established transgenic mice expressing human \u003cem\u003eAPOL1 G2\u003c/em\u003e. We determined the \u003cem\u003eAPOL1 G2\u003c/em\u003e transgene inserted multiple times in tandem, and therefore classify these mice as \u003cem\u003eAPOL1 G2\u003c/em\u003e multicopy mice (APOL1 G2\u003csub\u003emc\u003c/sub\u003e). We found that homozygous APOL1 G2\u003csub\u003emc\u003c/sub\u003ewere viable, healthy, lived a normal lifespan, and had no baseline phenotype. However, a single injection of interferon-γ\u0026nbsp;(IFNγ), which induces APOL1 kidneyexpression (Fig. S5A), led to significant APOL1‑dependent proteinuria in APOL1 G2\u003csub\u003emc\u003c/sub\u003ecompared to control Friend leukemia B virus (FVB) mice (Fig. S5B and 5C). We recently reported prophylactic treatment with VX-147 reduced APOL1-dependent proteinuria in APOL1 G2\u003csub\u003emc\u0026nbsp;\u003c/sub\u003e\u003csup\u003e25\u003c/sup\u003e. Here we show that a close structural analog of VX‑147, Compound 3, with comparable potency (Table 1), was able to prevent APOL1‑mediated proteinuria in this model at 30 mg/kg bid (Fig. S5D, S5E and S5F).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur prophylactic model demonstrated that APOL1 inhibitors could prevent proteinuria, but their impact on pre-established proteinuria and kidney histology was unknown.\u0026nbsp;Therefore, we utilized a more aggressive published proteinuria model to assess the effect of APOL1 inhibition on established proteinuria and kidney pathology. This model entails the hydrodynamic injection of an IFNγ‑expressing plasmid into single copy, homozygous \u003cem\u003eAPOL1 G2\u003c/em\u003e mice (APOL1 G2\u003csub\u003esc\u003c/sub\u003e)\u0026nbsp;\u003csup\u003e26\u003c/sup\u003e. These mice develop proteinuria approximately 5 days after plasmid injection, peaking around day 8 and remaining elevated through the study time course (Fig. 4A). Mice were randomized into two groups based on day 5 urinary albumin-to-creatinine ratio (UACR), and treatment with vehicle or 30 mg/kg bid Compound 3 began on day 6. Compound 3 had a rapid effect at lowering the UACR (Fig. 4A). Analysis of the UACR AUC following compound intervention revealed a 97% reduction over the 8 days of compound administration (Fig. 4B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMicroscopic evaluations of kidney tissues showed that mice not injected with the IFNγ\u0026nbsp;plasmid (Fig. 5A and 5D) had no evidence of glomerular or tubular damage. In vehicle-treatedAPOL1 G2\u003csub\u003esc\u003c/sub\u003e, approximately 60% of glomeruli exhibited histomorphologic changes (Fig. 5B, black arrow) while remaining glomeruli were morphologically normal (Fig 5B, white arrow). Glomerulopathy was characterized by segmental to global collapse of capillary lumina (Figure 5B), mesangial hyalinization (Fig. 5E), synechiae between capillary basement membranes and parietal epithelium, and vacuolation and reduplication of parietal epithelial cells (PEC). Tubules were often dilated, lined by attenuated epithelium, and contained eosinophilic proteinaceous fluid (Fig 5B and 5E). In contrast, no findings were noted in the animals treated with Compound 3 (Fig. 5C and 5F).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSerial sections of kidneys were immunolabeled for nephrin\u0026nbsp;\u003csup\u003e27,28\u003c/sup\u003e to investigate the effect of systemic inflammation (IFNγ)\u0026nbsp;on glomerulal filtration barrier function. Whole slide imaging and an artificial intelligence (AI) based classifier\u0026nbsp;\u003csup\u003e29,30\u003c/sup\u003e were used to quantify glomerular expression of nephrin. The quantitative scoring was expressed as an H-score, a cumulative assessment of area and intensity. The nephrin H-scores were comparable between the no IFNγ\u0026nbsp;control and Compound 3-treated groups, while the vehicle-treated group had significantly lower nephrin H-scores (Fig. 5G).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBased on the microscopic data, we employed a novel quantitative analysis of kidney health using the podocyte exact morphology measurement procedure (PEMP)\u0026nbsp;\u003csup\u003e31\u003c/sup\u003e. This technique uses fluorescence immunolabeling for podocin and integrin α3 and super-resolution microscopy to assess podocyte health based on the filtration slit density (FSD). The FSD represents the ratio of length of the filtration slit for a given glomeruli (podocin staining) relative to its surface area (integrin staining) with high FSD values indicating healthy, intact glomeruli. Representative maximum intensity projections of the kidney filtration slit are shown for naïve, vehicle-treated and Compound 3-treated mice (Fig. 5H, 5I, 5J). Vehicle-treated mice showed significantly reduced FSD when compared to Compound 3-treated mice or naïve mice as summarized in Fig. 5K.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIndividuals with proteinuric CKD and two APOL1 risk variants (G1 or G2) have a genetically defined form of kidney disease referred to as AMKD. AMKD occurs in the presence of inflammatory conditions such as interferon treatment, lupus, SCD, HIV and COVID, but may also be observed in the absence of an overt inflammatory comorbidity. People with CKD and two \u003cem\u003eAPOL1\u003c/em\u003e risk variants progress more rapidly to end-stage kidney disease relative to those without two \u003cem\u003eAPOL1\u003c/em\u003e risk variants. While the genetic evidence demonstrates a key role of \u003cem\u003eAPOL1 G1\u003c/em\u003e and \u003cem\u003eG2\u003c/em\u003e genetic variants in the human causal biology of proteinuric non-diabetic kidney disease, the molecular and cellular mechanism whereby APOL1 variants lead to kidney damage has been less clear. APOL1 is expressed in multiple tissues, and various cellular and molecular mechanisms have been proposed as relevant to AMKD.\u003c/p\u003e \u003cp\u003eBased on the known mechanism of human biology whereby APOL1 variants protect from \u003cem\u003eTrypanosoma Brucei\u003c/em\u003e infection, we hypothesized that APOL1 ion channel function leads to kidney damage. To test this hypothesis we developed multiple assays, conducted a high throughput screen to identify small molecule inhibitors of APOL1-mediated ion flux, and optimized those molecules to identify compounds with the potency, specificity, and drug-like properties suitable to evaluate the impact of APOL1 inhibition in cells, animal models, and, ultimately, AMKD patients.\u003c/p\u003e \u003cp\u003eHere we present data that show the highly specific channel blocker, VX-147, directly inhibits APOL1mediated ion flux and prevents both APOL1-dependent trypanosome and mammalian cell death. VX-147 was shown to bind directly to APOL1 with high affinity and broad profiling across a large panel of potential off-targets did not identify any significant activity, indicating that the mechanism of action is due to direct inhibition of the APOL1 ion channel \u003csup\u003e25\u003c/sup\u003e. We report that the mechanism by which APOL1 G1 and G2 variants cause cell death is likely a result of their enhanced channel activity in human cells. We demonstrate that efflux of K\u003csup\u003e+\u003c/sup\u003e, and not influx of Na\u003csup\u003e+\u003c/sup\u003e, is responsible for APOL1-mediated cell death. There seems to be many downstream mechanisms by which APOL1-mediated ion flux leads to cell death \u003csup\u003e10,15\u0026ndash;21\u003c/sup\u003e, but the root cause, and thus ideal therapeutic target, appears to be the ion channel function of APOL1. In fact, a recent publication highlighted the many proximal and distal events caused by APOL1-mediated ion flux and demonstrated that VX-147 treatment prevented or reversed these downstream events \u003csup\u003e32\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn transgenic \u003cem\u003eAPOL1\u003c/em\u003e mouse models, we used Compound 3, a close analog of VX-147 with comparable potency, to show that blockage of the APOL1 channel can both prevent and reverse APOL1-dependent proteinuria as well as preserve glomerular integrity. This is the first reported evidence demonstrating that functional inhibition of APOL1 can significantly reduce established proteinuria in an \u003cem\u003ein vivo\u003c/em\u003e preclinical model.\u003c/p\u003e \u003cp\u003eFurther evidence in support of the ion channel hypothesis comes from a recent human genetics study demonstrating that the N264K substitution in the protein sequence of APOL1 both decreased APOL1 ion flux and reduced kidney disease incidence in \u003cem\u003eAPOL1 G2\u003c/em\u003e carriers \u003csup\u003e33\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe most important evaluation of the ion channel hypothesis is to perform clinical trials of potent and specific APOL1 inhibitors in AMKD patients. We recently reported Phase 2a proof-of-concept data with VX-147 in patients with two \u003cem\u003eAPOL1\u003c/em\u003e risk alleles and proteinuric nondiabetic kidney disease \u003csup\u003e25\u003c/sup\u003e. This 13-week study in 16 individuals demonstrated a 47.6% reduction in proteinuria with stable GFR. An ongoing Phase 2b/3 clinical trial of inaxaplin in patients with two \u003cem\u003eAPOL1\u003c/em\u003e risk variants and proteinuric non-diabetic kidney disease will further define the clinical potential of APOL1 inhibition for patients with AMKD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThank you to all the APOL1 project team members, past and present, who helped further our understanding around APOL1 biology and contributed to the Vertex APOL1 program. We would also like to thank Dr. David Altshuler for advice on the project and comments on the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConceptualization: B.Z, L.A.D., A.F., TS, BF, MEB.\u003c/p\u003e\n\u003cp\u003eMethodology: BZ, LAD, AF, EN, AB, NH, SK, JP, TS, BF.\u003c/p\u003e\n\u003cp\u003eInvestigation: BZ, LAD, EN, AB, JM, HM, MF, TW, SN, GM, CM, PM, GW, PS, KD, HS, KS, FB, AP, FL, SP, SKP, LH, MH, FD, ON, NH, SK, JP, TS\u003c/p\u003e\n\u003cp\u003eWriting – original draft: BZ, EN, BF, MEB.\u003c/p\u003e\n\u003cp\u003eWriting – review \u0026amp; editing: BZ, LAD, EN, JP, BF, MEB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare the following competing financial interest(s): All authors listed are current or past employees of Vertex Pharmaceuticals, Incorporated and may hold stock in the company.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials \u0026amp; Correspondence:\u0026nbsp;\u003c/strong\u003eCorrespondence or material requests should be directed to Brandon Zimmerman or Mark E. Bunnage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll data are available in the manuscript or the supplementary material.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eCell line generation and growth\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman embryonic kidney (HEK) cell lines containing a tetracycline (tet)-inducible expression system (T‑REx\u0026trade;; Invitrogen, Carlsbad, CA) and pAAVS1‑Puro‑APOL1 G0, pAAVS1‑Puro‑APOL1 G1 or pAAVS1‑Puro‑APOL1 G2 were generated. Individual clones of each cell line were generated and clones G0 DC2.13, G1 DC3.25, and G2 DC4.44 were used in this study. Cell growth medium was Dulbecco\u0026apos;s modified Eagle\u0026rsquo;s medium (DMEM; Gibco, Gaithersburg, MD; Catalog # 11960‑077) supplemented with 10% (v/v) tet‑free fetal bovine serum (FBS; Takara Bio USA, Mountain View, CA; Catalog # 631368), 2 mM L‑glutamine (Gibco; Catalog # 25030‑081), 5 \u0026micro;g/mL Blasticidin S HCl (Gibco; Catalog # \u0026nbsp;A11139‑03), 1 \u0026micro;g/mL puromycin dihydrochloride (Gibco; Catalog # A11138‑03), and 100 U/mL penicillin-streptomycin (Gibco; Catalog # 15140‑122).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElectrophysiology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK293 APOL1-inducible cells were induced with 15 ng/mL of tet for 16‑20 hours. Automated electrophysiology was performed by Qpatch48 (Sophion Biosciences). Cells were perfused with extracellular solution containing: 137 mM NMDG, 5.4 mM KCl, 2 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 mM Glucose, 10 mM HEPES. The pH was adjusted to 7.2 with NaOH and adjusted to 300-305 mOsm with sucrose. Ouabain (100 \u0026mu;M) was added in the extracellular solution for blockade of Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eATP-ase. NMDG replaced Na\u003csup\u003e+\u003c/sup\u003e in the extracellular solution to isolate K\u003csup\u003e+\u003c/sup\u003e efflux. The intracellular solution contained: 124 mM CsF, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 11 mM EGTA, 10 mM HEPES, 1 mM ATP pH 7.2, 285-290 mOsm. We used Cs\u003csup\u003e+\u003c/sup\u003e in the internal solution because APOL1 channels are permeable to Cs\u003csup\u003e+\u003c/sup\u003e but Cs\u003csup\u003e+\u003c/sup\u003e blocks endogenous potassium voltage-gated channels. Resistances of the QPatch plate chambers (holes) was 1.5-2.5 M\u0026Omega;. Steady state inhibition of APOL1 current was measured using a ramp protocol in response to six consecutive compound concentrations (100 pM, 1 nM, 3 nM, 10 nM, 100 nM, 1 \u0026mu;M) with an exposure duration of 5 min to each concentration, after an initial 5 min current recording (control). Each solution exchange of 5\u0026nbsp;mL volume was performed twice. The voltage protocol applied to the cells was as follows: Resting step:\u0026nbsp;‑80 mV for 100 ms; test pulse: -100 mV to 80 mV for 200 ms; resting step: -80 mV for 100 ms repeated at 10 s intervals, from a holding potential of -90 mV. Peak outward current was measured at the end of the 200 s test pulse at +80 mV.\u0026nbsp;Data acquisition and analysis was performed using the Sophion QPatch Assay Software (Sophion Biosciences). \u0026nbsp;We continuously monitored the series resistance (M\u0026Omega;) and the capacitance (pF). Cells were removed from analysis if the series resistance exceeded 15 M\u0026Omega;, if the peak amplitude current was smaller than 100 pA, and if the reversal potential was smaller than\u0026nbsp;‑20 mV. The\u0026nbsp;average peak outward currents before and after test compound application were used to calculate the percentage of current inhibited at each concentration. Outward currents in response to each concentration of VX-147 were normalized to its initial control current without VX-147. Concentration-response data were fit by GraphPad Prism using non-linear regression analysis with variable slope (four parameters).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK293 APOL1-inducible cells were grown in DMEM\u0026nbsp;with 2% (v/v) doxycycline (dox)-free FBS in 96 well plates (Corning, 3904) at a seeding density of 50,000 cells/well in 100 \u0026mu;L of media and treated +/-\u0026nbsp;15 ng/mL doxycycline to induce APOL1 expression for 16-24 h. Cells were washed with 100\u0026nbsp;\u0026mu;L DPBS and detached with 100\u0026nbsp;\u0026mu;l of 1x Versene (Gibco, 15040066) at 37\u0026deg;C for 15 min. Cells were then resuspended in complete media and transferred to a 96-well plate round bottom plate (Corning, 3799) and pelleted in a tabletop centrifuge at 300 x g for 5 mins. All staining reactions were performed in FACS buffer (DPBS supplemented with 2% FBS and 2mM EDTA). \u0026nbsp;Cells were stained for surface APOL1 expression in 50\u0026nbsp;\u0026mu;L of a 1:100 dilution of anti‑APOL1 rabbit monoclonal antibody (Abcam, ab252218) in FACS buffer on ice for 30 mins. Cells were washed in 200\u0026nbsp;\u0026mu;L of FACS buffer twice. Secondary staining was performed in 50\u0026nbsp;\u0026mu;L of a 1:2000 dilution of secondary goat anti-rabbit Alexa Fluor 647 antibody (Thermofisher,\u0026nbsp;A32733) on ice for 20 mins. Cells were washed in 200\u0026nbsp;\u0026mu;L of FACS buffer twice. Cells were fixed in 50\u0026nbsp;\u0026mu;l of 4% paraformaldehyde for 15 min at room temperature. 4% PFA was washed off and replaced with 200\u0026nbsp;\u0026mu;L of FACS buffer for FACS analysis. Cells were immediately analyzed on a Beckman Coulter CytoFLEX instrument. The Alexa Fluor 647 signal was excited using the 638 nm laser and 660/10 nm BP filter. Cells were analyzed using FACS Diva and FlowJo Softwares.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThallium assay methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK293 APOL1-inducible cells were plated in DMEM\u0026nbsp;with 2% (v/v) tet-free FBS\u0026nbsp;with 15 ng/mL tet to induce APOL1 expression for 17-19 h. Cells were seeded into 384 well BioCoat\u0026trade; Poly-D-Lysine coated, black/clear, flat-bottom, tissue culture-treated microplates at 2.6 x 10\u003csup\u003e4\u003c/sup\u003e cell/well.\u0026nbsp;Loading buffer (HBSS, 20 mM HEPES, 5 mM Probenecid, 0.2 mM Ouabain, and components A and C from the FLIPR potassium assay bulk kit (Molecular Devices; Catalog # R8223) was added to the wells of the cell-containing assay plate. The cells were incubated for 30 minutes at room temperature (23\u0026deg;C) and then compounds were added to the assay plate wells. Compounds were tested with a top concentration of 10 \u0026mu;M followed by a quarter-log dilution series with 20 total points down to 0.312 nM. An active control was used in each assay and the DMSO concentration was kept fixed at 0.2%. The cells were incubated with compound at room temperature (23\u0026deg;C) for 30 minutes then the assay plate was placed on a FLIPR Tetra or Penta high-throughput cellular screening system to measure sample fluorescence intensity. Fluorescence readings were recorded every second for 10 seconds before the addition of thallium sulfate solution and an additional 60 seconds after the addition of thallium sulfate solution. Data were imported into Genedata for analysis and IC\u003csub\u003e50\u003c/sub\u003e generation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrypanosome assay methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTrypanosoma brucei brucei\u003c/em\u003e were obtained from ATCC and\u0026nbsp;grown in ATCC medium 2834 modified HMI-9 medium. Trypanosomes in phenol red/FBS free media containing Serum Plus medium supplement (Sigma, 14008C) were plated at a concentration of\u0026nbsp;120,000 parasites/well in 384-well plates containing compound and recombinant APOL1. The final concentration of APOL1 used in this assay was sufficient to kill approximately 90% of the parasites. AlamarBlue\u0026Ocirc;\u0026nbsp;was\u0026nbsp;added to the plates following 16 h incubation at 37\u0026deg;C\u0026nbsp;with APOL1 and compound, and then returned to the incubator for an\u0026nbsp;additional\u0026nbsp;2.5 h. The plate\u0026nbsp;was then read on a SpectraMax plate reader with excitation/emission wavelengths of 555/585 nm. Data was imported into Genedata for analysis and dose response curves generated using the Smart Fit algorithm.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMultitox assay methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK293 APOL1-inducible cells were plated in DMEM\u0026nbsp;with 2% (v/v) tet‑free FBS\u0026nbsp;with 50 ng/mL tet to induce APOL1 expression for 24 h. Cells were plated into a\u0026nbsp;384-well Poly-D-Lysine coated plate at a density of 23,400 cells/well. Compounds were added to the plates for the full 24 h with a 10 point 3-fold dilution curve (top concentration, 10 \u0026mu;M) run in duplicate. The next day, assay buffer containing 2x MultiTox reagent from the MultiTox Multiplex cytotoxicity assay was added to each well and incubated an additional 30 min at 37\u0026deg;C. The plates were read on an EnVision plate reader for viability (excitation: 400 nm, emission: 486 nm) and cytotoxicity (excitation: 485 nm, emission: 535 nm). \u0026nbsp;A ratio of dead (cytotoxicity) to live (viability) cells was used to normalize data. The data was exported and analyzed in Genedata. Data was normalized using two controls, no tet treatment (100% viability) and 50 ng/mL tet treatment (0% viability) and dose response curves generated using the Smart Fit\u0026nbsp;algorithm. NMDG media (109.50 mM N-Methyl-D-Glucamine, 5.4 mM NaCl, 1.8 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 0.81 mM Mg\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 25 mM glucose, 4.5 nM Ferric Nitrate, 10 mM HEPES) or High K\u003csup\u003e+\u003c/sup\u003e media (115 mM KCl, 1.8 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 0.8 mM Mg\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 25 mM glucose, 4.5 nM Ferric Nitrate, 10 mM HEPES) were used to isolate the ionic flux to assess the critical ion for cell death. The MultiTox Multiplex cytotoxicity assay was performed as described above with standard media.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMouse model methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the pharmacodynamic effects of APOL1 inhibitors in vivo, we established a transgenic mouse using a 104 kb portion of a bacterial artificial chromosome (BAC) containing \u003cem\u003eAPOL1\u003c/em\u003e in its native human genomic context using human BAC clone CTD-2333M18 (BACPAC Resource Center at the Children\u0026apos;s Hospital Oakland Research Institute), which includes the entire human \u003cem\u003eAPOL1\u003c/em\u003e gene, the human promoter, introns, and all other cis regulatory regions including untranslated regions. The \u003cem\u003eAPOL1\u003c/em\u003e \u003cem\u003eG2\u003c/em\u003e defining variant was incorporated by recombineering into an \u003cem\u003eAPOL1 G0\u003c/em\u003e BAC construct. The transgenes were linearized by Notl digestion, purified, and microinjected into zygotes from the Friend leukemia virus B (FVB) mouse strain. Experiments were conducted in transgenic male and female FVB APOL1 multi-copy G2-expressing homozygous mice (\u003cem\u003eAPOL1 G2\u003csub\u003emc\u003c/sub\u003e\u003c/em\u003e) (8 to 15 weeks old, Charles River, Wilmington, MA, USA). The mice were bred to homozygosity as hemizygous mice do not have a phenotype.\u0026nbsp;FVB \u003cem\u003eAPOL1 G2\u003c/em\u003e single copy (\u003cem\u003eAPOL1 G2\u003csub\u003esc\u003c/sub\u003e\u003c/em\u003e) homozygous transgenic mice (with glutamate at amino acid 150) were generated at the Beth Israel Deaconess Medical Center (BIDMC) transgenic core facility. The G2 defining variant was derived from a G1 mouse line using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) was incorporated by recombineering (Yale O\u0026rsquo;Brien Center transgenic core facility) into an \u003cem\u003eAPOL1 G0\u003c/em\u003e BAC construct. Mice were group-housed in a temperature-controlled environment (22 \u0026plusmn; 1.5\u0026deg;C, 30 to 70% relative humidity, 12-hour light/dark) and were acclimatized in the animal facility for at least 3 days prior to use. Studies were conducted under a protocol that has been approved by the Vertex Animal Care and Use Committee. Experiments were performed at the Vertex Boston site, which is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eProphylactic assessment of Compound 3\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003eAPOL1 G2\u003c/em\u003e\u003cem\u003e\u003csub\u003ehom\u003c/sub\u003e\u003c/em\u003e multicopy mice, increased proteinuria (mean urinary albumin-to-creatinine ratio, UACR) was induced by a single intraperitoneal (IP) injection of 150 \u0026micro;g/kg of murine IFN\u0026gamma;\u0026nbsp;(R\u0026amp;D Systems, Catalog# 485-MI/CF) in a volume of 10 mL/kg. Urine was collected at various time points throughout the experiment, which lasted no more than 72 h. For proteinuria analysis, urine was diluted appropriately, and urine albumin and creatinine were measured using a mouse albumin ELISA and creatinine companion kit (Bethyl Laboratories, Catalog# E99-134 and Ethos Biosciences, Catalog# 1012). Compound 3 (30 mg/kg,\u0026nbsp;dosed as a crystalline suspension) or vehicle, which is composed of 1% Hydroxypropyl methylcellulose acetate succinate, type H, 0.25% of Polyvinylpyrrolidone K 30 and 2% of d-\u0026alpha;-Tocopheryl polyethylene glycol 1000 succinate (1%HPMCAS-H/0.25%PVP-K30/2%TPGS, Shin-Etsu, MP Biomedicals and Antares manufacturers, respectively) was administered orally twice daily (bid) at 12 h interval in a volume of 10 mL/kg. Compound 3 was administered 1.5 h before IFN\u0026gamma;\u0026nbsp;injection. UACR was calculated by dividing the average of albumin replicate values (expressed in ng/mL) to the average of creatinine replicate values (expressed to mg/dL) and then converted to the unit \u0026micro;g/mg. Area under the curve (AUC) was calculated per animal for UACR values from 0 to 48 h or 72 h, using GraphPad Prism, with baseline set to the mean of all animals of the same strain at baseline (day 0). Percent reduction of UACR AUC was calculated using the following equation:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e((Mean\u003csub\u003evehicle\u003c/sub\u003e - Mean\u003csub\u003ecompound 3\u003c/sub\u003e)/(Mean\u003csub\u003eVehicle\u003c/sub\u003e)) * 100. \u0026nbsp;Statistical analysis was performed using GraphPad Prism. UACR AUC data was log-transformed and analyzed with two tailed t-test comparing Vehicle vs IFN\u0026gamma;\u0026nbsp;within a mouse strain or to compound 3. Statistical significance was set at p\u0026le;0.05.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTherapeutic assessment of Compound 3\u003c/em\u003e \u003cem\u003ein\u0026nbsp;plasmid chronic G2 single copy mice\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMurine\u0026nbsp;IFN\u0026gamma; cDNA was cloned in a\u0026nbsp;CpG-free plasmid (Catalog #pcpgf-mcs) at Vertex laboratories. The\u0026nbsp;day before the hydro dynamic injection (HDI) individual body weight was collected. The day of the injection, CpG-free IFN\u0026gamma; plasmid was thawed on wet ice and diluted in sterile saline to administer a total of 0.3 \u0026mu;g in a total volume corresponding to 10% body weight in grams. Mice were left in their cage under a heat lamp for 2 min, transferred to a restrainer where the area of injection was cleaned with ethanol wipes. Syringes (3 mL) with a 25-gauge needle were used for the tail vein injection. The full volume was injected in a continuous motion over approximately 5 to 10 seconds. Mice were placed in a cage on a heating pad and monitored for 30 minutes immediately following injection.\u003c/p\u003e\n\u003cp\u003eSerum IFN\u0026gamma; levels were quantified using the ProQuantum Mouse IFN\u0026gamma; Immunoassay (Invitrogen, Catalog# A41150), to confirm successful administration of IFN\u0026gamma; plasmid in the chronic therapeutic mouse model. Blood (20 \u0026micro;L) was collected on days 1, 5, 9, 14 to evaluate serum IFN\u0026gamma; levels. Blood was allowed to clot for 30 minutes at room temperature and then centrifuged at 1500 relative centrifugal force (RCF) for 15 minutes. The serum fraction was collected and used to monitor IFN\u0026gamma; levels. For the ProQuantum Mouse IFN\u0026gamma; Immunoassay, the manufacturer\u0026rsquo;s protocol to quantify IFN\u0026gamma; was followed except for the following modifications to sample dilution: for day 1 dilution was 1:1000, for day 5 dilution was 1:50, for day 9 and 14 dilution was at 1:20. The standard curve was fit using a 4-parameter fit and sample values were interpolated using GraphPad Prism.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUACR and UACR AUC\u003csub\u003eDay6-14\u003c/sub\u003e data were graphed as mean \u0026plusmn; standard error of the mean (SEM). UACR AUC\u003csub\u003eday6-14\u003c/sub\u003e data was log-transformed and analyzed with One-way ANOVA followed by Tukey\u0026rsquo;s comparison test. Statistical significance was set at p\u0026le;0.05.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathology and immunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the end of the study, animals were euthanized as per the IACUC-approved protocol. Euthanized animals were necropsied immediately, the left kidney was resected and fixed in 10% neutral buffered formalin (48 h), followed by transfer to 70% histology grade ethanol (Cat # R3154-1GA, Millipore Sigma) and submitted to the pathology core laboratory. Formalin-fixed kidneys were longitudinally bisected through the mid-sagittal plane to include cortex, medulla, and pelvis. Bisected kidney samples were routinely processed (Tissue-Tek tissue processing system, Sakura-Fintek), and paraffin-embedded. Serial sections from paraffin-embedded blocks were stained for a) hematoxylin and eosin (H\u0026amp;E), and b) periodic acid Schiff\u0026rsquo;s (PAS) using standard staining protocols as per Ventana Discovery Ultra autostainer protocols (Ventana Medical Systems, Roche). Specific protocols for APOL1 and nephrin immunohistochemistry are described below and were performed using Ventana Discovery Ultra autostainer (Ventana Medical Systems, Roche).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAPOL1 and nephrin immunohistochemistry\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFormalin-fixed paraffin-embedded (FFPE) tissue blocks were cut at 3 \u0026micro;m thickness, dried overnight at 37\u0026deg;C, and baked at 60\u0026deg;C for 1 hour before use. All subsequent steps were performed on the Ventana Discovery Ultra platform (Ventana Medical Systems, Roche). The protocol for nephrin and APOL1 immunohistochemistry were similar with minor differences. FFPE sections were deparaffinized at 69\u0026deg;C, followed by epitope retrieval (APOL1 \u0026ndash; 91\u0026deg;C, CC2 buffer for 32 min; nephrin \u0026ndash; 95\u0026deg;C, CC1 buffer for 64 min), peroxide block for 8 min (inhibitor conditioning medium), and primary antibody incubation for 1 hour (APOL1 \u0026ndash; rabbit anti APOL1 monoclonal antibody, Cat # ab252218, 1:250, Abcam; nephrin \u0026ndash; rabbit anti-nephrin monoclonal antibody, Cat# ab216341, 1:1000, Abcam). Signals were amplified using OmniMap anti-Rabbit HRP kit (Cat # 760-4311) for 16 min and detected using DAB chromogen (Chromomap DAB Kit; Cat #760-159) for 8 min. Slides were counterstained for 8 min (Hematoxylin II, Cat # 970-2208), followed by bluing for 4 min (Bluing reagent, Cat # 760-2037) and coverslipped. Rabbit monoclonal IgG (clone DA1E, Cat # 3900, Cell Signaling Technologies) was used as isotype control for both APOL1 and nephrin primary antibodies. All reagents unless specified otherwise were provided by the manufacturer (Ventana Medical Systems). Coverslipped and immunolabeled glass slides were scanned using Aperio AT2 whole slide scanner (Leica Biosystems) at 40x objective magnification and were visualized using image scope/web scope software (Leica Biosystems).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImmunolabeled glass slides were scanned using Aperio AT2 whole slide scanner (Leica Biosystems) at 40x objective magnification and were visualized using image scope/web scope software (Leica Biosystems). All antibodies were diluted using the antibody diluent (Cat# ADB250, Ventana Medical Systems). All reagents unless specified otherwise were provided by the manufacturer (Ventana Medical Systems).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMorphometric analysis nephrin expression in glomeruli\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eImmunolabelled Stained glass slides were scanned at 40x magnification (Aperio AT2, Leica Biosystems) and quality checked for any artifacts (i.e., damaged tissue, wrinkles, particles, scanning lines). Scanned whole slide images (WSI) were imported into HALO v3.4 (Indica Labs), where images were manually annotated to include the renal cortex and exclude artifacts. An Artificial Intelligence- Convolution Neural Network I - CNN (DenseNet from HALO, Indica Labs V3.2) based classifier (\u0026ldquo;Kidney_Glomerulus_APOL1 DenseNet\u0026rdquo;), was trained to automatically identify glomeruli within kidney sections. The images were manually curated to remove any abnormalities or non-glomerular structures from further analysis. Morphometric image analysis was performed using an Area Quantification (v1.0) module in HALO (\u0026ldquo;APOL1- Kidney_Glomerulus_APOL1 DenseNet\u0026rdquo;). Intensity thresholds were set to detect weak, moderate, and strong intensity of DAB chromogen (brown) staining for APOL1 and nephrin with pathologist\u0026rsquo;s guidance. All the available glomeruli detected by the algorithm in a one longitudinal section of the kidney per animal were analyzed from each sample and a cumulative output (H-score) was obtained. H-score was calculated as the following:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was performed in GraphPad Prism 9 using the Kruskall-Wallis test with multiple comparisons. Graphs were generated in Microsoft Excel and Prism.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePodocyte exact morphology measurement procedure\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA subset of mice was selected for a morphological assessment of the glomerular slit diaphragm. Mice were injected with a cocktail of 40 mg/mL ketamine and 2 mg/mL Xylazine. Level of anesthesia was then assessed with firm toe pinch. Once the subject was fully anesthetized, it was transferred to the working station for the perfusion. Area of incision was cleaned with an alcohol pad and tweezers were used to cut through the skin and expose the chest cavity. The diaphragm and the ribcage were cut to expose the heart. The ribcage was lifted, and a 25-gauge needle connected was inserted in the apex of the left ventricle. The right atrium was then cut, and the subject was perfused with 1X PBS/15,000-unit heparin at the rate of 20 mL/min for 3 min to washout the blood from the organs using a standard programmable infusion pump (Harvard Apparatus, Cat # \u003cstrong\u003e70-4500\u003c/strong\u003e). To restrict fixation to the right kidney, the left kidney renal artery was clamped. Right kidney was perfused with\u0026nbsp;4% PFA at the rate of 20 mL/min for 5 min using an infusion pump (New Era Pump Systems, Inc. Cat # NE-9000). Right kidneys were then cut in half and left rotating at room temperature for 1 h inside a 15 mL conical tube. Sample were transferred to 4\u0026deg;C\u0026nbsp;before shipment to NIPOKA GmbH.\u003c/p\u003e\n\u003cp\u003eAfter deparaffinization and rehydration, formalin-fixed and paraffin-embedded kidney sections (3 \u0026micro;m) were boiled in Tris-EDTA buffer (10 mmol/l Tris, 1 mmol/l EDTA, pH 9) in a pressure cooker for 5 min, followed by the blocking step (1% FBS, 1% BSA, 0.1% fish gelatin, 1% normal goat serum) for 1 hour. The following primary antibodies were incubated overnight at 4\u0026deg;C: rabbit anti‐podocin 1:150 (IBL International, Hamburg, Germany, JP-29040) and mouse anti‐integrin alpha3 1:500 (Santacruz, Dallas, Texas, USA, sc-374242). After three washing steps in PBS, secondary antibodies were incubated for 1 hour at room temperature (anti-rabbit Alexa Fluor 488‐conjugated IgG 1:600 (ChromoTek, Planegg, Germany, srbAF488-1-100) and anti-mouse Cy3‐conjugated IgG antibody 1:600 (Jackson ImmunoResearch, West Grove, PA, 115‑166-006) for 1 hour at room temperature. DAPI (1:100) was added to the slides for 5 min, followed by a washing step in PBS. Finally, the slides were incubated in H2O and mounted in Mowiol (Carl Roth, Karlsruhe, Germany) using high precision coverslips (Paul Marienfeld, Lauda-K\u0026ouml;nigshofen, Germany). The evaluation of the filtration slit density (FSD) was performed using a recently established super-resolution microscopy-based methodology termed podocyte exact morphology measurement procedure (PEMP)\u003csup\u003e31\u003c/sup\u003e. For three-dimensional structured illumination microscopy (3D-SIM), z-stacks of 19 planes of both channels (488 and 561 nm) were acquired from the stained kidney section using an N-SIM super-resolution microscope (Nikon, Tokyo, Japan) equipped with a 100x silicone objective. The images were reconstructed into 3D-SIM images using NIS-Elements AR 5.30 (Nikon, Tokyo, Japan). The z-stacks were converted into a maximum intensity projection followed by the automatized identification of podocyte foot process areas that were selected based on the foot process positive antibody. Inside these areas, the filtration slit diaphragm length was automatically determined. The FSD was expressed as the ratio of the total filtration slit diaphragm length per podocyte foot process area. The FSD of 20 randomly selected glomeruli was determined for every animal.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSynthesis and characterization of chemical compounds:\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe chemical compounds (\u003cstrong\u003e2\u0026nbsp;\u003c/strong\u003e- \u003cstrong\u003e4\u003c/strong\u003e) in this manuscript were prepared according to the synthetic procedures detailed in the published patent, WO2020131807, titled \u0026ldquo;Inhibitors of APOL1 and methods of using same\u0026rdquo;. \u0026nbsp;Purity assessment for final compounds was based on analytical UPLC: 2.1 mm \u0026times; 50 mm Waters CSH C18 column, 1.7 \u0026mu;M, 130 \u0026Aring;. Mobile phases were as follows: A, H\u003csub\u003e2\u003c/sub\u003eO with 0.1% trifluoroacetic acid; B, acetonitrile with 0.1% trifluoroacetic acid; gradient, 10\u0026ndash;60% B in 0.6 min with a 1.4 min run time. The flow rate was 0.6 mL/min. Mass samples were analyzed on a Waters 3100 single quad mass spectrometer operated in positive MS mode with electrospray ionization. The mobile phase for all mass analysis consisted of acetonitrile\u0026ndash;water mixtures with either 0.1% trifluoroacetic acid or ammonium formate. \u003csup\u003e1\u003c/sup\u003eH NMR spectra were recorded using either a Bruker Avance 400 (400 MHz) or a Bruker Avance II-300 (300 MHz) instrument. The screening hit, \u003cstrong\u003e1\u003c/strong\u003e, is commercially available and was purchased from an external vendor. \u0026nbsp;Compound \u003cstrong\u003e1\u003c/strong\u003e has a Chemical Abstracts Service Registry Number of 687574-42-1. \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFriedman DJ, Pollak MR (2021) APOL1 Nephropathy: From Genetics to Clinical Applications. 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Sci Rep 7:11473. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/s41598-017-11553-x\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/s41598-017-11553-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDatta S et al (2024) APOL1-mediated monovalent cation transport contributes to APOL1-mediated podocytopathy in kidney disease. J Clin Invest. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1172/jci172262\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1172/jci172262\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHung AM et al (2023) Genetic Inhibition of APOL1 Pore-Forming Function Prevents APOL1-Mediated Kidney Disease. J Am Soc Nephrol. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1681/ASN.0000000000000219\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1681/ASN.0000000000000219\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003e\u003cstrong\u003eTable 1: Medicinal chemistry progression of the screening hit 1 to VX-147.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cimg 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of the amide and installation of the aminolactam afforded compound \u003cstrong\u003e2\u003c/strong\u003e with increased potency and metabolic stability. \u0026nbsp;Oxidation of the lactam to compound \u003cstrong\u003e3\u003c/strong\u003e further increased potency and metabolic stability. \u0026nbsp;Further Structure-Activity-Relationship (SAR) investigation of the indole core gave VX-147 with optimized properties. V\u003csub\u003ed\u003c/sub\u003e: volume of distribution, T\u003csub\u003e1/2\u003c/sub\u003e: half-life, Cl: clearance\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3922092/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3922092/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChronic kidney disease affects ~10% of people worldwide and there are no disease modifying therapeutics that address the underlying cause of any form of kidney disease. Genome wide association studies have identified the G1 and G2 variants in the \u003cem\u003eapolipoprotein L1 \u003c/em\u003e(\u003cem\u003eAPOL1\u003c/em\u003e)\u003cem\u003e \u003c/em\u003egene as major contributors to a subtype of proteinuric kidney disease now referred to as APOL1‑mediated kidney disease (AMKD). We hypothesized that inhibition of APOL1 could have therapeutic potential for this genetically-defined form of kidney disease. Here we describe the development of preclinical assays and the discovery of highly potent and specific APOL1 inhibitors with drug-like properties. We provide evidence that APOL1 channel activity drives podocyte injury and that inhibition of this activity stops APOL1-mediated cell death and kidney damage in a transgenic mouse model. These preclinical data, combined with recent clinical data, support the potential of APOL1 channel inhibition for the treatment of AMKD.\u003c/p\u003e","manuscriptTitle":"Small Molecule APOL1 Inhibitors as a Precision Medicine Approach for APOL1-mediated Kidney Disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-08 11:04:05","doi":"10.21203/rs.3.rs-3922092/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"38af1279-01ba-4dc2-8f5a-e4db6d460d52","owner":[],"postedDate":"April 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":30300651,"name":"Health sciences/Nephrology/Kidney diseases/Glomerular diseases/Focal segmental glomerulosclerosis"},{"id":30300652,"name":"Health sciences/Nephrology/Kidney diseases/Chronic kidney disease/End-stage renal disease"},{"id":30300653,"name":"Biological sciences/Drug discovery/Target validation"},{"id":30300654,"name":"Biological sciences/Cell biology/Mechanisms of disease"}],"tags":[],"updatedAt":"2025-01-03T08:09:42+00:00","versionOfRecord":{"articleIdentity":"rs-3922092","link":"https://doi.org/10.1038/s41467-024-55408-2","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-01-02 05:00:00","publishedOnDateReadable":"January 2nd, 2025"},"versionCreatedAt":"2024-04-08 11:04:05","video":"","vorDoi":"10.1038/s41467-024-55408-2","vorDoiUrl":"https://doi.org/10.1038/s41467-024-55408-2","workflowStages":[]},"version":"v1","identity":"rs-3922092","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3922092","identity":"rs-3922092","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}