A novel class of allosteric glucosylceramidase beta 1 correctors that reduce cellular stress and enhance lysosomal function

A novel class of allosteric glucosylceramidase beta 1 correctors that reduce cellular stress and enhance lysosomal function | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results A novel class of allosteric glucosylceramidase beta 1 correctors that reduce cellular stress and enhance lysosomal function Ilaria Fregno , Natalia Pérez-Carmona , Mikhail Rudinskiy , Tatiana Soldà , Timothy J. Bergmann , Ana Ruano , Aida Delgado , Elena Cubero , Manolo Bellotto , Ana María García-Collazo , Maurizio Molinari doi: https://doi.org/10.1101/2024.11.15.623779 Ilaria Fregno 1 Università della Svizzera italiana , CH-6900 Lugano, Switzerland ; 2 Institute for Research in Biomedicine , CH-6500 Bellinzona, Switzerland ; Find this author on Google Scholar Find this author on PubMed Search for this author on this site Natalia Pérez-Carmona 3 Gain Therapeutics, Sucursal en España, Parc Científic de Barcelona , 08028 Barcelona, Spain ; Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mikhail Rudinskiy 1 Università della Svizzera italiana , CH-6900 Lugano, Switzerland ; 2 Institute for Research in Biomedicine , CH-6500 Bellinzona, Switzerland ; 4 Department of Biology, Swiss Federal Institute of Technology; CH-8093 Zurich , Switzerland ; Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tatiana Soldà 1 Università della Svizzera italiana , CH-6900 Lugano, Switzerland ; 2 Institute for Research in Biomedicine , CH-6500 Bellinzona, Switzerland ; Find this author on Google Scholar Find this author on PubMed Search for this author on this site Timothy J. Bergmann 1 Università della Svizzera italiana , CH-6900 Lugano, Switzerland ; 2 Institute for Research in Biomedicine , CH-6500 Bellinzona, Switzerland ; Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ana Ruano 3 Gain Therapeutics, Sucursal en España, Parc Científic de Barcelona , 08028 Barcelona, Spain ; Find this author on Google Scholar Find this author on PubMed Search for this author on this site Aida Delgado 3 Gain Therapeutics, Sucursal en España, Parc Científic de Barcelona , 08028 Barcelona, Spain ; Find this author on Google Scholar Find this author on PubMed Search for this author on this site Elena Cubero 3 Gain Therapeutics, Sucursal en España, Parc Científic de Barcelona , 08028 Barcelona, Spain ; Find this author on Google Scholar Find this author on PubMed Search for this author on this site Manolo Bellotto 5 GT Gain Therapeutics SA , Lugano, Switzerland ; Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ana María García-Collazo 3 Gain Therapeutics, Sucursal en España, Parc Científic de Barcelona , 08028 Barcelona, Spain ; Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: maurizio.molinari{at}irb.usi.ch amgarcia{at}gaintherapeutics.com Maurizio Molinari 1 Università della Svizzera italiana , CH-6900 Lugano, Switzerland ; 2 Institute for Research in Biomedicine , CH-6500 Bellinzona, Switzerland ; 6 School of Life Sciences , École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: maurizio.molinari{at}irb.usi.ch amgarcia{at}gaintherapeutics.com Abstract Full Text Info/History Metrics Preview PDF Abstract Mutations in glucosylceramidase beta 1 (GCase) disrupt the protein’s conformational maturation in the endoplasmic reticulum (ER) and hinder its transport to the lysosome. The intralysosomal accumulation of glucocerebrosides, which are substrates of the GCase enzyme, impairs lysosomal function and is linked to Gaucher disease (GD). GCase mutations also increase the risk of Parkinson’s disease (PD) and Dementia with Lewy Bodies. We used Site-directed Enzyme Enhancement Therapy (SEE-Tx®) technology to design two structurally targeted allosteric regulators (STARs) of GCase. Administration of GT-02287 and GT-02329 to cultured GD patient-derived primary human fibroblasts enhances folding and protects the two most common disease-causing GCase variants, GCase Asn370Ser and GCase Leu444Pro , from proteasomal degradation. Mechanistically, these treatments facilitate the lysosomal delivery of enzymatically active forms of mutant GCase, leading to improved lysosomal function and reduced cellular stress in GD patient-derived fibroblasts. The findings suggest that the allosteric pharmacologic regulators GT-02287 and GT-02329 hold promise for further development as potential therapeutic agents for GCase-related disorders, including GD, PD and Dementia with Lewy Bodies. Introduction Glucosylceramidase beta 1 (GCase) is a lysosome-resident protein that cleaves the beta-glucosidic linkage of glycosylceramides, generating glucose and free ceramides. GCase mutations may hamper folding of the polypeptide chain and result in unwanted clearance of the protein and/or reduced transport from the endoplasmic reticulum (ER) to the lysosomal compartment. Defective lysosomal delivery of functional GCase causes toxic intralysosomal accumulation of GCase substrates. Mutations in GCase are linked to Gaucher disease (GD), a lysosomal storage disorder (LSD), and are also a risk factor for Parkinson’s disease (PD) and Dementia with Lewy Bodies ( Do, McKinney et al ., 2019 ). The most common mutation in GCase is the substitution of the asparagine residue (N in the single letter code) at position 370—or position 409 if the 39 residues of the leader peptide are included in the amino acid numbering—with a serine residue (S). The GCase Asn370Ser mutation is associated with type I GD, which typically presents a mild phenotype. Another common mutation is the replacement of the leucine residue (L) at position 444—or 483 if including the leader peptide—with a proline (P). This mutation often results in a more severe outcome, including the activation of unfolded protein responses (UPR) in both GD patients and a Drosophila model of the disease ( Maor, Rencus-Lazar et al ., 2013 ). In this study, we used the HaloTag (HT) reporter approach developed in our lab ( Rudinskiy, Bergmann et al ., 2022 , Rudinskiy and Molinari, 2022 , Rudinskiy, Pons-Vizcarra et al ., 2023 ), combined with an automated, deep learning method for segmenting and classifying fluorescence images, known as LysoQuant ( Morone, Marazza et al ., 2020 ). This approach captures cargo delivery within endolysosomes to monitor the impact of the disease-causing Asn370Ser and Leu444Pro mutations on the lysosomal delivery of GCase and the induction of cellular stresses. Moreover, we assessed the beneficial effects of structurally targeted allosteric regulators (STARs) of GCase, developed using the innovative drug discovery platform SEE-Tx ® ( Barroso, Puchwein-Schwepcke et al ., 2024 , Cubero, Ruano et al ., 2024 , Rudinskiy, Pons-Vizcarra et al ., 2023). Materials and methods Binding studies by surface plasmon resonance (SPR) Human full-length wild-type GCase protein (Cerezyme, Genzyme, Naarden, NL) was immobilized on an SPR CM5 sensor (GE Healthcare, #29149603) using standard amino coupling with a high protein concentration of 100 µg/mL. A nine-point, 2-fold serial dilution, starting from 100 µmol/L GT-02287 and GT-02329 (prepared from a 10 mmol/L stock solution in DMSO), was measured by SPR at two pH conditions: pH 7.4 in 10 mmol/L HEPES, 5 mmol/L EDTA, 150 mmol/L NaCl and 0.01% Tween-20; and pH 5.0 in 20 mmol/L Na phosphate, 2.7 mmol/L KCl, 137 mmol/L NaCl, 5 mmol/L tartrate, 0.01% Tween-20. Reference channels on the SPR sensor included empty, activated, and deactivated parallel channels. Raw SPR signals from the active channel (with immobilized GCase protein) were double-referenced by subtracting signals from the reference channel (empty sensor surface) and the running buffer, with corrections for DMSO signal mismatch between the sample and the buffer. Binding affinity values were extracted by fitting the plotted SPR data with a four-parameter logarithmic dose-response equation using GraphPad Prism version 10.2.3. To assess the mode of action of the analyzed compounds, competition experiments were conducted with isofagomine (IFG), an inhibitory reference compound, present in the running buffer at saturating concentrations under both pH conditions (pH 7.4 and 5.0). These competition experiments were performed as single measurements and repeated twice. GCase biochemical and competition assay in wild-type lysates Lysates were prepared from wild-type fibroblasts using a lysis buffer containing 0.9 % NaCl and 0.01 %Triton-X100. The protein concentration was determined using a BCA protein assay kit. To inhibit 50% of GCase activity, cell lysates were pre-incubated with 40 µM conduritol-β-epoxide (CBE) for 15 minutes. Subsequently, GT-02287 or GT-02329 was added for another 15 minutes. The assay was conducted in a reaction buffer containing 5 mM 4-methylumbelliferyl-beta-D-glucopyranoside in 0.1 M citrate-phosphate buffer at pH 5.6. Reactions were incubated at 37°C for one hour. To stop the reaction, 140 µL of 100 mM Glycine-NaOH buffer at pH 10.7 was added. The released 4-MU was measured using a Glomax® Discover microplate reader with excitation at 340 nm and emission at 460 nm. Cell culture, transient transfection, and use of compounds Mouse embryonic fibroblasts (MEF) and Human Embryonic Kidney 293 (HEK293) cell lines were cultured at 37°C and 5% CO 2 in Dulbecco’s Modified Eagle Medium (DMEM) with high glucose (GlutaMAX™, Gibco) supplemented with 10% Fetal bovine serum (FBS, Gibco). Fibroblasts from GD patients were obtained from the Coriell Institute for Medical Research and the Telethon Network of Genetic Biobanks. The specific genotypes used were as follows: p.N370S/ins (GD type I, Coriell GM00372), p.L444P/p.L444P (GD type II, Coriell GM08760), p.L444P/p.L444P (GD type III, Telethon 20526), p.L444P/p.L444P (GD type I, Coriell GM10915), p.L444P/p.F213I (GD type III, Telethon 21142), p.L444P/p.R496C (GD type III, Telethon 20624), and p.N188S/p.S107L (GD type III, Telethon 20843). Healthy fibroblasts (Coriell GM03377) were also included. For enzyme enhancement and substrate depletion studies, fibroblasts were cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin at 37°C under 5% CO 2 . Patient fibroblasts used in other studies were cultured in DMEM-GlutaMAX™ with 15% non-inactivated FBS under similar conditions. Transient transfections were conducted using JetPrime (Polypus) in DMEM 10% FBS supplemented with non-essential amino acids (NEAA, Gibco) following the manufacturer’s protocol. Pharmacological chaperones from Gain Therapeutics (GT), GT-02287 and GT-02329, were dissolved in dimethylsulfoxide (DMSO, Sigma) and used at concentrations specified in the figure legends. The GCase inhibitor, conduritol-β-epoxide (CBE), was acquired from Merck Millipore (Catalogue No: 234599). The proteasome inhibitor PS341 (Bortezomib, LubioScience) was used at a final concentration of 10 µM. Plasmids and cloning Plasmids encoding for GCase-HA (human influenza hemagglutinin), GCase-Halo Asn370Ser and Leu444Pro mutant polypeptides within the pcDNA3.1(+) backbone, flanked by HindIII and XhoI restriction sites were synthesized by GenScript. The transfer of HA and HaloTag tags between various plasmids was achieved through restriction digestion of NotI and XhoI sites flanking the tags. Ligation was conducted using T4 DNA ligase (NEB) at a 3:1 insert to vector ratio, following the manufacturer’s protocol. Plasmids were subsequently amplified and isolated from JM109 bacteria (Promega) using the GenElute™ HP plasmid MidiPrep kit (Sigma). Confocal laser scanning microscopy (CLSM) MEFs were seeded on alcian blue-treated (Sigma) glass coverslips (VWR) and transiently transfected using JetPrime (Polypus) in DMEM 10% FBS supplemented with non-essential amino acids (NEAA; Gibco) according to the manufacturer’s protocol. Thirty-two hours after transfection, bafilomycin A1 (BafA1; Calbiochem) was added to the cell medium at the final concentration of 50 nM for 17 h. Cells expressing HaloTag-fusion proteins were supplemented with 100 nM tetramethylrhodamine (TMR) HaloTag ligand (Promega). After treatment, MEFs were fixed at room temperature (RT) for 20 min in 3.7% formaldehyde (FA, v/v) in phosphate buffered saline (PBS). Coverslips were incubated for 20 min in permeabilization solution (PS, 10 mM HEPES, 15 mM glycine, 10% goat serum (v/v), 0.05% saponin (w/v)). Following permeabilization, primary antibodies diluted 1:100 in PS (unless otherwise specified in Table S1 ) were applied for 120 min, washed three times in PS, and then Alexa Fluor-conjugated secondary antibodies diluted 1:300 in PS were applied for 45 min. Cells were washed three times with PS and once with deionized water and mounted on a drop of Vectashield (Vector Laboratories) supplemented with 40,6-diamidino-2-phenylindole (DAPI). Coverslips were imaged using a Leica TCS SP5, microscope equipped with Leica HCX PL APO lambda blue 63.0 × 1.40 oil objective. Leica LAS X software was used for image acquisition, with excitation provided by 488-, 561-, and 633 nm lasers. Fluorescence light was collected within the ranges of 504-587 nm (AlexaFluor488), 557-663 nm (TMR) and 658-750 nm (AlexaFluor646), respectively, with a pinhole setting of 1 AU. The accumulation of GCase variants within LAMP1-positive endolysosomes was quantified with LysoQuant plugin within the FIJI/ImageJ software (Morone, Marazza et al ., 2020, Schindelin, Arganda-Carreras et al ., 2012 ). Image post-processing was performed with Adobe Photoshop. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), HaloTag cleavage assay, co-Immunoprecipitation and western blot (WB) HEK293 cells or patient fibroblasts were washed with ice-cold 1xPBS containing 20 mM N-ethylmaleimide (NEM), then lysed with RIPA (1% Triton X-100 (v/v), 0.1% SDS (w/v), 0.5% sodium deoxycholate in HBS, pH 7.4, (w/v)) or 2% CHAPS (in HEPES-buffered saline [HBS], pH 7.4, (w/v)) buffer containing 20 mM NEM and protease inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF), 16.5 mM Chymostatin, 23.4 mM Leupeptin, 16.6 mM Antipain, 14.6 mM Pepstatin) for 20 min on ice. The lysate was centrifuged to extract the post-nuclear supernatant (PNS) for 10 min at 4°C, 10,600 g. The PNS was denatured and reduced by adding 100 mM dithiothreitol (DTT, Roche) and heating the sample for 5 min at 95°C. Boiled samples were subjected to 12% Acrylamide SDS-PAGE. Lysates of cells expressing GCase-HT variants labeled with fluorescent TMR Halo ligand (Promega) were imaged in-gel with Typhoon™ FLA 9500 fluorescent scanner (GE Healthcare) with a 532 nm laser. The intensity of TMR bands was quantified using the ImageQuantTL software (Molecular Dynamics, GE Healthcare). Total protein content was quantified by staining the polyacrylamide gel with 0.25% Brilliant blue R 250 (Sigma) solution (50% MeOH (v/v), 10% acetic acid (v/v)) for 20 min at RT, and destaining in 20% MeOH (v/v), 7.5% acetic acid (v/v) solution. The protein bands were acquired on a Fusion FX7 system (Vilber) with transilluminator and quantified with FIJI/ImageJ software. For immunoprecipitation, the lysates of cells treated with DMSO or GT compounds were diluted with lysis buffer and incubated with the select bait antibodies and protein A-conjugated beads (1:10 w/v, swollen in PBS) at 4°C for 4 hours. The beads were centrifuged down and washed three times with 0.5% Triton X-100 (v/v) in HBS pH 7.4. After that, beads were denatured for 5 min at 95°C, and the liquid fraction was subjected to SDS-PAGE. For detection of protein band by WB protein bands were transferred from polyacrylamide gel to polyvinylidene fluoride (PVDF) membrane using a TransBlot Turbo device (BioRad). PVDF membrane was blocked for 10 min with 10% blocking milk (BioRad, w/v) in Tris-buffered saline, 0.1% Tween 20 (v/v) (TBS-T), rinsed quickly in TBS-T and incubated with primary antibodies ( Table S1 ) overnight at 4°C with shaking. After primary antibody washout with TBS-T, membranes were incubated with HRP-conjugated secondary antibody or protein A ( Table S1 ) for 45 min at RT with shaking. Protein bands were detected on a Fusion FX7 chemiluminescence detection system (Vilber) using the WesternBright™ Quantum (Advansta) system following the manufacturer’s protocol. Protein bands were quantified with FIJI/ImageJ software. Radioactive metabolic labelling Four days after the start of treatment with DMSO or GT compounds, patient fibroblasts were pulsed 4 h with 0.2 mCi [35S] methionine/cysteine mix and chased for 10 min with DMEM supplemented with 5 mM cold methionine and cysteine. Cells were solubilized in RIPA buffer, and radiolabeled proteins were revealed with Typhoon™ FLA 9500 fluorescent scanner (GE Healthcare). The intensity of radioactive bands was quantified using the ImageQuantTL software (Molecular Dynamics, GE Healthcare). RNA extraction and reverse transcription-quantitative polymerase chain reaction Patient fibroblasts treated with DMSO or GT compounds were detached with trypsin and then centrifuged for 5 min at 450 g . Total mRNA was extracted using the GenElute TM mammalian total RNA Miniprep kit (Sigma) according to manufacturer’s protocol. The isolated mRNA was reverse transcribed into cDNA for quantitative polymerase chain reactions (qPCRs) using the BioTool™ 2x SYBR Green qPCR Master Mix (BioTool) as per the manufacturer’s instructions. The master mix was combined with qPCR primers, which were obtained from Microsynth AG and used at a final concentration of 10 µM, on the reading plate. qPCRs were conducted using the QuantStudio™ 3 Real-Time PCR System (Applied Biosystems). Data analysis was performed using QuantStudio™ Design & Analysis Software v1.5.5 (Applied Biosystems). GCase enzyme enhancement assay Patient-derived fibroblasts were seeded at 5,000 cells per well in 96-well cell culture plates in DMEM supplemented with 10% FBS, 1% P/S (Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 37°C, 5% CO2 overnight for cell attachment. Subsequently, cells were incubated with or without the specified compound at the indicated concentrations for 4 days. After incubation, cells were washed with PBS and incubated with 5 mM of 4-methylumbelliferyl-beta-D-glucopyranoside (Apollo Scientific) in 0.1 M acetate buffer pH 4 for 1 h at 37°C. The reaction was stopped by adding 200 µL of 100 mM Glycine-NaOH buffer, pH 10.7. The liberated, 4-methylumbelliferyl (4-MU) was measured on a Glomax® Discover microplate reader with excitation at 340 nm and emission at 460 nm. GlcCer quantification in patient-derived fibroblasts Patient-derived fibroblasts were seeded at 750,000 cells in 75 cm 2 flasks in DMEM supplemented with 10% FBS, 1% P/S. The following day, fibroblasts were treated with compounds at the indicated concentrations. On day 3 and day 7 of the treatment, supernatant was discarded and new media with compound was added. On day 10, supernatant from the cell culture was discarded and fibroblasts were trypsinized. Growth cell media was then added to the detached cells and collected in tubes. Next, the pellets were centrifuged in cool PBS and after discarding the supernatants, and the pellets were stored at -80°C. GlcCer quantification was performed as follows: The pellets from the cell culture samples were transferred into Eppendorf tubes and rapidly weighed. The tubes were stored in an ice-water bath, and to each tube deionized water was pipetted in a 1:10 (weight/volume) ratio. Deuterium-labelled GlcCer-D5 was used as internal standard. The pellets were resuspended in water by use of an ultrasonic homogenizer VCX130 (Sonics & Materials, CT, USA) and 10 μl from each vial was pipetted for quantification of protein content. From the remaining volumes, 70 μl was pipetted into the separate vials and GlcCer was extracted from the resuspended cell debris using liquid-liquid extraction. The ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) system for quantification of glycosphingolipids included a Xevo TQ-S micro triple quadrupole mass spectrometer, an ACQUITY UPLC system consisting of a binary solvent manager, a sample manager operating at +8°C and equipped with a 10 μl loop, a high temperature column heater, and a solvent tray module (all from purchased from Waters Micromass, Milford, MA, USA). The mass spectrometer was supplied by nitrogen gas, generated by a Genius 3045 nitrogen/air generator (Peak Scientific Instruments, Inchinnan, Scotland, UK). The electrospray ionization (ESI) source operated in a positive mode with the following parameters: Capillary voltage 4500 V, sample cone voltage 30 V, source temperature 150°C, desolvation temperature 500°C, cone gas flow 0 L/hr, nebulizer gas flow 1000 L/hr. Acquisition parameters were: Centroid MRM scans of parent and daughter ions, 0.1 second dwell time. The divert valve was programmed as follows: 0.00 min waste, 1.30 min run, 3.2 min waste. The chiral separation of GlcCer from GalCer was achieved on a HALO HILIC column (2.1 x 150 mm, 2 μm particle size, purchased from Advanced Materials Technology, Wilmington, DE, USA) and maintained at +35°C. The mobile phase consisted of 95% ACN, 2.5% MeOH, and 2.5% water containing 5 mM ammonium formate and 0.5% formic acid; the flow rate was 0.240 ml/min. The protein content in 10 μl volumes of the resuspended samples was measured by Pierce Rapid Gold BCA protein assay (Thermo Fisher Scientific, USA) and an UV/VIS plate reader (Labrox, Finland). The results for GlcCer content in the pellets were presented as ng/mg protein. Data analysis was performed using GraphPad Prism 5.0 Software. All values show the mean +/- SD (standard deviation) from 3 wells per condition. Statistical differences vs. untreated GCase Leu444Pro were determined by one-way analysis of variance (ANOVA) followed by a Dunnett’s Multiple Comparison Test. Asterisks indicate statistically significant values from untreated GCase Leu444Pro **** p≤0.0001 and *** p≤0.001. Statistical analyses For figures 1 ‒ 4 : Data analysis was performed using GraphPad Prism 5.0 Software. For figures 5 ‒8 : Statistical comparisons and graphical plots were performed in GraphPad Prism 10 (GraphPad Software Inc.). An ordinary one-way ANOVA Dunnett’s multiple comparisons test was used to assess statistical significance (for Figs. 4A and 4B ). An adjusted p-value < 0.05 (for one-way ANOVA with Dunnett’s multiple comparison test) was considered statistically significant: *** p<0.001 and **** p<0.0001. For figures 5C , 5J, 6B, 7D and 7G , an unpaired t-test was used to assess statistical significance. An exact p-value <0.05 was considered significant; ns is not significant, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. All replicates represent biological replicates, and for all statistical comparisons the number of repetitions and the number of cells analyzed (n, for LysoQuant analyses) are indicated in the figure legends. Download figure Open in new tab Figure 1. GCase binding confirmation. Surface plasmon resonance direct binding measured for compounds GT-02287 and GT-02329 at (A) pH 7.4 and (B) pH 5.0. All values show the mean +/- SD (standard deviation) in duplicates. Results GT-02287 and GT-02329 bind to the GCase protein in an allosteric pocket SEE-Tx ® technology ( Alonso, Garcia et al ., Patent 2012 , Alvarez-Garcia and Barril, 2014 , Alvarez-Garcia, Schmidtke et al ., 2022 ) utilized the high-resolution structure of native human GCase (PDB: 2F3V) ( Shaaltiel, Bartfeld et al ., 2007 ) to identify an allosteric binding site and predict its druggability. This process guided subsequent steps: key binding hotspots informed a high-throughput virtual screening assay ( Ruiz-Carmona, Alvarez-Garcia et al ., 2014 ), leading to the identification of novel virtual hits that were purchased and tested for interaction with the GCase protein. Experimentally validated hits served as starting points for a medicinal chemistry program, resulting in the selection of GT-02287 and GT-02329. Notably, chemical structures remain undisclosed due to the sensitive nature of proprietary research in the drug development phase. The direct binding of GT-02287 and GT-02329 to the GCase target protein was confirmed by SPR. SPR provides real-time measurements of molecular interaction kinetics and affinity, making it an optimal technique for this investigation ( Schuck, 1997 ). These experiments were conducted at pH 7.4 and 5.0, mimicking the ER and the luminal environment of acidic organelles, respectively ( Patching, 2014 ). GT-02287 and GT-02329 showed a dose-dependent binding to GCase protein at neutral and acidic conditions, with comparable binding affinities at pH 7.4 and 5.0 ( Figs. 1A , 1B ). SPR competition experiments with IFG ( Park, Chen et al ., Shire Human Genetic Therapies, Inc. Patent 2019 ), a competitive inhibitor of GCase, confirmed that GT-02287 and GT-02329 bind to an allosteric site and do not block the active site on GCase. IFG binds to the active site of the GCase protein and acts as a pharmacological chaperone, stabilizing mutant enzymes to improve their function. Dissociation constant (K D ) values obtained with and without IFG were comparable at pH 7.4 and 5.0, indicating that these compounds bind to a pocket distinct from the active site occupied by IFG ( Table 1 ; see also supporting information). View this table: View inline View popup Download powerpoint Table 1. SPR direct binding affinities measured at pH 7.4 and 5.0, and competition data with isofagomine (IFG). K D , dissociation constant. GT-02287 and GT-02329 enhance GCase activity in fibroblast lysates by binding to a site on the enzyme different from the active site The effect of GT-02287 and GT-02329 on the enzymatic activity of GCase was assessed in wild-type fibroblast extracts at pH 5.6. Both compounds enhance GCase activity at concentrations above 10 µM, as evidenced using the substrate 4-methylumbelliferyl-β-D-glucopyranoside ( Figs. 2B and 2C ). These results support the notion that the compounds bind to a site that is different from the catalytic site. Otherwise, they would be expected to exhibit inhibitory behavior, as observed with known inhibitors such as IFG. Download figure Open in new tab Figure 2. Competitive inhibition assay with CBE in lysates. Wild-type fibroblast lysates were incubated with (A) CBE black), (B) GT-02287 (green) or (C) GT-02329 (purple) or for 15 minutes at the indicated doses. GCase activity was then measured using the 4-methylumbelliferyl β-D-glucopyranoside substrate. Where indicated, an additional preincubation with CBE at 40 µM ( C , D in grey) was performed to inhibit 50% of GCase activity. All values show the mean +/- SD (standard deviation) from 3 wells per condition. CBE, conduritol-β-epoxide. CBE, a potent, selective irreversible competitive inhibitor, covalently binds GCase in the active site with a IC 50 of 28.19 µM, as shown in Fig. 2A ( Premkumar, Sawkar et al ., 2005 ). When the activity of GT-02287 and GT-02329 was assessed in the presence of CBE at a concentration that irreversibly inhibits 50% of GCase activity, the effect of the GT compounds on GCase enzymatic activity was reduced by 50% ( Figs. 2B and 2C , respectively). This result demonstrates that the enhancement of GCase activity is mediated by binding to GCase, showing a clear relationship between GT-02287 and GT-02329’s activity enhancement and the availability of functional GCase in patient fibroblast extracts. GT-02287 and GT-02329 enhance GCase enzyme activity in primary human fibroblasts and from GD patients The ability of GT-02287 and GT-02329 to enhance the activity of GCase proteins with disease-causing mutations was assessed in primary human fibroblasts from a healthy donor ( Figs. 3A and 3A ’ ) and from six patients affected by GD type I, GD type II and GD type III ( Figs. 3B ‒3G and 3B’‒3G’ ), resulting from various mutations in the GBA1 gene. Briefly, fibroblasts were seeded, and the following day, the compounds were added. After a 4-day incubation, GCase activity was measured using the fluorogenic substrate 4-methylumbelliferyl-β-D-glucopyranoside. In all cases, incubation of the primary human fibroblasts with GT-02287 and GT-02329 resulted in a dose-dependent increase in GCase activity. Download figure Open in new tab Figure 3. Cellular GCase enhancing activity of GT-02287 and GT-02329. Patient-derived fibroblasts were treated with GT-02287 ( A‒G , green) or GT-02329 ( A’ ‒ G’ , purple) at the specified doses for 4 days. GCase activity was measured using the 4-methylumbelliferyl β-D-glucopyranoside substrate. The dose-response effect is shown as the percentage increase in activity relative to untreated cells. Mean +/- standard deviation (SD) from at least three wells per condition are shown. GT-02287 and GT-02329 treatment reduces endogenous substrate accumulation in primary human fibroblasts Reduced GCase activity in patient-derived p.L444P/p.L444P primary fibroblasts leads to an accumulation of glucosylceramide (GlcCer), a primary physiological substrate of GCase ( Fig. 4 ). To determine whether enhancing GCase activity in patient cells by treating with GT-02287 or GT-02329 reduces the intracellular accumulation of GlcCer, fibroblasts were treated for 10-days, starting one day after seeding, after which, cells were harvested and processed as described in the methods section. Quantification by UHPLC-MS/MS revealed that treatment of patient fibroblasts with increasing concentrations of GT-02287 ( Fig. 4A ) or GT-02329 ( Fig. 4B ) effectively mitigates GlcCer accumulation. Download figure Open in new tab Figure 4. GlcCer substrate depletion following treatment with GT-02287 and GT-02329. p.L444P/p.L444P fibroblasts (Coriell GM08760) were treated with GT-02287 ( A ) or GT-02329 ( B ) at the specified doses for 10 days. GlcCer levels were quantified by ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). The mean +/- standard deviation (SD) is shown from 3 wells per condition. A one-way analysis of variance (ANOVA) followed by Dunnett’s Multiple Comparison Test was performed. Asterisks indicate statistically significant values from untreated GCase Leu444Pro **** p≤0.0001, *** p≤0.001. HaloTagged chimeras to monitor defective lysosomal transport of disease-causing GCase mutations by CLSM To quantitatively assess the performance and the mode-of-action of GT-2287 and GT-2329, we developed a biochemical protocol. This protocol has been instrumental in evaluating STARS that enhance the activity of the galactosidase beta 1 (GLB1) in GLB1-related LSDs, including GM1 gangliosidosis (Rudinskiy, Bergmann et al ., 2022, Rudinskiy and Molinari, 2022 , Rudinskiy, Pons-Vizcarra et al ., 2023). Briefly, GCase and two disease-causing variants (GCase Asn370Ser and GCase Leu444Pro ) were tagged with a HaloTag polypeptide to create GCase-HaloTag (GCase-HT) chimeras. HaloTag is an engineered bacterial dehalogenase, whose active site covalently binds cell-permeable haloalkanes modified with fluorescent ligands ( England, Luo et al ., 2015 , Los, Encell et al ., 2008 ). Chimeric polypeptides modified with HaloTag can be fluorescently labeled by adding cell-permeable fluorescent Halo ligands, such as tetramethylrhodamine (TMR), to the cell culture media. The intracellular distribution of HaloTagged proteins can be assessed by CLSM. Given that HaloTag (297 residues) is much larger than conventional tags such as the HA-tag (9 residues) ( Kimple, Brill et al ., 2013 ), we first checked whether HaloTag perturbs the lysosomal delivery of GCase, the mature form of which is composed of 497 residues ( Fig. 5A ). GCase, GCase Asn370Ser and GCase Leu444Pro were tagged at their C-terminus with HA or HaloTag epitopes and transiently expressed in MEFs. The delivery of the GCase variants, immunostained with anti-HA antibody (red) in lysosomes marked with an anti-LAMP1 antibody (green), was assessed by CLSM ( Fig. 5B ) and quantified by Lysoquant to monitor the accumulation of the HA-tagged GCase in lysosomes inactivated by 50 nM BafA1 (Morone, Marazza et al ., 2020) ( Fig. 5C ). Micrographs show that GCase-HA ( Fig. 5B , upper panels) and GCase Asn370Ser -HA ( Fig. 5B , middle panels) are efficiently transported to LAMP1-positive lysosomes (LysoQuant quantifications, Fig. 5C ). In contrast, transport of the GCase Leu444Pro -HA mutant is highly defective ( Figs. 5B lower panels and 5C ). This correlates with the GD phenotype, which is mild for GCase Asn370Ser and severe for GCase Leu444Pro patients (Do, McKinney et al ., 2019). Download figure Open in new tab Figure 5. Lysosomal delivery of wild type and disease-linked GCase variants. (A) Sequence of GCase. The signal sequence is underlined. The sites of Asn 370 Ser and Leu 444 Pro mutations in the disease-causing gene products are shown in bold italics. ( B) Lysosomal delivery of HA-tagged GCase variants. Representative CLSM images of MEF cells expressing GCase variants for 48 h and exposed for 17h to 50 nM bafilomycin A1 (BafA1). Immunostaining with anti-HA (red) anti-LAMP1 antibody (endolysosomes, green). Nuclei (blue) are stained with DAPI. Scale bars 10 µm (merge) and 1 µm (inset). ( C) Quantification of ( B) as a percentage of LAMP1-positive lysosomes containing GCase variants. Images analyzed with LysoQuant (Morone, Marazza et al ., 2020). Mean -/+ SEM is shown, n=3 independent experiments. Statistical analysis: One-way ANOVA followed by Dunnett’s multiple comparison test, non-significant (ns) p>0.05, ****p<0.0001. ( D) Same as ( B) for Halotagged (HT) GCase variants. MEF cells expressing GCase variants for 48 h were exposed 17h to 50 nM BafA1 and 100 nM TMR. Staining for GCase variants with TMR (red) and lysosomes with anti-LAMP1 antibody (green). Scale bars 10 µm (merge) and 1 µm (inset). ( E) Quantification of ( D) as a percentage of LAMP1-positive lysosomes containing the TMR signal within their lumen. Images analyzed with LysoQuant (Morone, Marazza et al ., 2020). Mean -/+ SEM is shown, n=2 independent experiments. ( F) Co-immunoprecipitation of GCase-HA variants (WB: HA, lower panel and CNX, upper panel) and BiP (middle panel) in HEK293 cells. ( G) Quantification of CNX association with the three GCase variants. ( H) Same as (G) for BiP association. Mean is shown, n=2 independent experiments. ( I) Fluorescent gel showing the TMR signal of GCase-HT variants expressed for 4 days in HEK293 cells in the presence or absence of 50 nM BafA1 for the last 17h. NT, non-transfected; FL, full-length GCase-HT. ( J) Quantification of ( I) . Mean -/+ SEM is shown, n=5 independent experiments. Statistical analysis: One-way ANOVA followed by Dunnett’s multiple comparison test, *p<0.05, ****p<0.0001. GCase glucocerebrosidase; HA hemagglutinin tag; CLSM, confocal laser scanning microscopy; MEF, mouse embryonic fibroblasts; BafA1, bafilomycin A1; LAMP1, lysosomal-associated membrane protein 1; DAPI, 4’,6-diamidino-2-phenylindole; EL, endolysosomes; SEM, standard error of the mean; HT, HaloTag; TMR, tetramethylrhodamine; CNX, calnexin; BiP, binding immunoglobulin protein; HEK293, human embryonic kidney 293 cells; WB, western blot; NT, non-transfected; FL, full length. To assess intracellular trafficking of the same GCase variants when tagged with HaloTag, MEF cells were transiently transfected with the expression plasmids and the media supplemented with 100 nM TMR to label GCase-HaloTag variants. Their delivery to the LAMP1-positive compartment was assessed with CLSM ( Fig. 5D ) and quantified with LysoQuant ( Fig. 5E ). Results mirror those with HA-tagged polypeptides, i.e., GCase-HT and GCase Asn370Ser -HT are efficiently transported to LAMP1-positive lysosomes while GCase Leu444Pro -HT transport is highly defective ( Figs. 5D and 5E ). Thus, both the HA and the HaloTag chimeric proteins report on GCase transport efficiency from the ER to the lysosomes. Notably, co-precipitation assays reveal that, unlike wild-type GCase ( Fig. 5F , lane 2, and quantification in Fig. 5G ) and the GCase Asn370Ser mutant ( Fig. 5F , lane 3, and quantification in Fig. 5G ), the GCase Leu444Pro variant associates much stronger with the ER chaperone calnexin (CNX, Fig. 5F , upper panel, lane 4, and quantification in Fig. 5G ) and binding immunoglobulin protein (BiP; Fig. 5F , middle panel, lane 4, and quantification in Fig. 5H ). Thus, the defective delivery to the endolysosomal compartment derives from GCase Leu444Pro variant persistent association with ER-resident chaperones ( Figs. 5F - 5H ), indicating the mutant GCase’s inability to attain its native conformation efficiently ( Molinari, 2007 ). Halotagged chimeras to monitor defective lysosomal transport of disease-causing GCase mutations by gel electrophoresis Upon delivery of the chimeric proteins to lysosomal compartments, the HaloTag is cleaved off, generating acid- and protease-resistant fluorescent Halo fragments of about 33 kDa. This allows for direct visualization and quantification of lysosomal delivery of the protein under investigation by monitoring the formation of the 33 kDa fluorescent Halo fragment in gel (Fregno, Fasana et al ., 2018, Fregno, Fasana et al ., 2021 , Fumagalli, Noack et al ., 2016 , Loi, Raimondi et al ., 2019 , Rudinskiy, Bergmann et al ., 2022, Rudinskiy and Molinari, 2022 , Rudinskiy, Pons-Vizcarra et al ., 2023). Cells expressing GCase-HT, GCase Asn370Ser -HT or GCase Leu444Pro -HT were grown in the presence of 100 nM TMR. Cells were detergent-solubilized, and PNS-separated by SDS-PAGE. TMR-labeled polypeptides were directly visualized in gel with a 532 nm wavelength laser. The PNS of cells expressing GCase-HT contains two fluorescent polypeptides. The upper band corresponds to the 97 kDa full-length GCase-HT ( Fig. 5I , upper panel, lane 2, FL). The lower band is the 33 kDa Halo fragment, generated upon arrival of the GCase-HT chimera within the lysosomes, where lysosomal enzymes cleave the linker between the GCase protein and the HaloTag ( Fig. 5J , upper panel, lane 2, HaloTag fragment, quantification in Fig. 5J ) (Rudinskiy, Bergmann et al ., 2022, Rudinskiy and Molinari, 2022 ). The generation of this polypeptide is blocked when cells are exposed to 50 nM BafA1, which inactivates lysosomal enzymes ( Fig. 5I , upper panel, lane 3). The fluorescent 33 kDa Halo fragment, indicating transport of the chimeric polypeptide to lysosomal compartments, is also generated in cells expressing the GCase Asn370Ser mutant ( Fig. 5I , upper panel, lane 4, Halo fragment, Fig. 5J ) but not, or only negligibly, in cells expressing the GCase Leu444Pro mutant ( Fig. 5I , upper panel, lane 6, Halo fragment, Fig. 5J ). Overall, both the imaging ( Figs. 5A ‒5E ) and the biochemical assays ( Figs. 5I ‒ 5J ) accurately reflect the defective delivery of the GCase Leu444Pro protein, which is associated with severe disease phenotypes linked to GBA1 mutations. GT-02287 and GT-02329 (STARS) restore lysosomal transport of GCase Leu444Pro in cellulo Next, we quantitatively assessed the performance of GT-02287 and GT-02329 by monitoring variations in the lysosomal generation of the Halo fragment in cells expressing the transport-deficient GCase Leu444Pro -HT chimera, using the previously validated quantitative biochemical assay. Cells expressing GCase Leu444Pro -HT were grown in the presence of increasing concentrations of GT-02287 ( Fig. 6A , lanes 4-7) or GT-02329 (lanes 9-12) for 4 days and exposed to 100 nM TMR for 17h. The cells were detergent-solubilized, and TMR-labeled polypeptides present in the PNS were directly visualized in gel with a 532 nm wavelength laser. Inhibition of lysosomal hydrolases with BafA1 prevents the generation of the Halo fragment ( Fig. 6A , lane 2 vs. lane 3 and Fig. 6B , BafA1 vs. DMSO). The intensity of the fluorescence of the polypeptide corresponding to the Halo fragment generated in mock-treated cells ( Fig. 6A , lanes 3, 8 and Fig. 6B , DMSO) indicates the basal level of GCase Leu444Pro -HT delivered to the lysosomal compartments in these cells, which is much lower than the amount of GCase and GCase Asn370Ser ( Figs. 5I and 5J ). Notably, cell exposure to GT-02287 ( Fig. 6A , lanes 4-7) or GT-02329 (lanes 9-12) increases the generation of the Halo fragment in a dose-dependent manner ( Fig. 6B ). Thus, both GT-02287 and GT-02329 substantially increase the transport of the GCase Leu444Pro protein from the ER to the lysosome, its site of activity. Download figure Open in new tab Figure 6. STARS promote lysosomal delivery of the ER-retained variant GCase Leu444Pro and reduce toxic accumulation of GCase endogenous substrate in patient fibroblasts. (A) TMR fluorescence of GCase Leu444Pro -HT expressed in HEK293 cells treated for 4 days with DMSO or GT compounds (GT-02287 or GT-02329) at concentrations ranging from 3.1 µM to 25 µM. The cells were incubated with 100 nM TMR for the last 17 h. Control cells were treated for 17 h with 50 nM bafilomycin A1 (BafA1; lane 2). After exposure, the proteins were transferred onto a PVDF membrane and immunoblotted against GAPDH. ( B) Quantification of the Halo fragment, corrected for GAPDH signal and normalized to DMSO. The mean -/+ SEM is shown, with n=4 independent experiments for 25 µM concentrations, n=2 for 6.25 µM and 12.5 µM, and n=3 for BafA1. Statistical analysis was conducted using an unpaired t-test, with ***p<0.001 and ****p<0.0001. TMR, tetramethylrhodamine; HEK293, human embryonic kidney 293 cells; DMSO, dimethyl sulfoxide; BafA1, bafilomycin A1; PVDF, polyvinylidene fluoride; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SEM, standard error of the mean. GT compounds increase intracellular levels of GCase Leu444Pro , by promoting its release from ER resident chaperones and by inhibiting its proteasomal clearance from patient-derived primary human fibroblasts The performance of STARs was assessed in patient fibroblasts containing the homozygous 1448T>C mutation in the GBA1 gene corresponding to the Leu444Pro substitution. Exposure of the cells to increasing concentrations of GT-02329 for 4 days progressively reduced the co-precipitation of CNX ( Figs. 7A and 7B ) and substantially raised the level of intracellular, endogenous GCase Leu444Pro ( Fig. 7A , lanes 2-5). An increase in the intracellular GCase Leu444Pro protein in patient cells was also observed upon exposure to a second STAR, GT-02287, as shown by comparing the GCase Leu444Pro level in mock-treated cells ( Fig. 7C , lane 1) with those exposed to GT-02287 and GT-02329 (lanes 2 and 3, respectively, Fig. 7D ). Download figure Open in new tab Figure 7. GT compounds promote CNX release and prevent proteasomal degradation of GCase Leu444Pro . (A) Co-immunoprecipitation with CNX (upper panel) of endogenous GCase Leu444Pro (lower panel) in patient fibroblasts treated for 4 days with DMSO or GT compound GT-02329 at concentrations ranging from 3.1 µM to 25 µM. Protein content in the total cell extracts (TCE) is shown. * unspecific band. ( B) Quantification of ( A) . The mean is shown, based on n=2 independent experiments. ( C) Immunoisolated GCase Leu444Pro from patient fibroblasts treated for 4 days with DMSO or GT compounds (GT-02287 or GT-02329). ( D) Quantification of ( C) . The mean ± SEM is shown, based on n=4 independent experiments, using an unpaired t-test with **p<0.01 and ****p<0.0001. ( E) Radiolabeled protein content in the TCE is shown. ( F) Radiolabeled GCase Leu444Pro immunoisolate from patient fibroblasts. ( G) Quantification of ( F) . The mean ± SEM is shown, based on n=4 independent experiments and using an unpaired t test, with ns p>0.05 and *p<0.05. ( H) GCase Leu444Pro level in patient fibroblasts treated for 4 days with DMSO or GT compounds (GT-02287 or GT-02329) and, for the last 3 h, with the proteasome inhibitor PS341. * : unspecific band. CNX, calnexin; DMSO, dimethyl sulfoxide; TCE, total cell extracts; SEM, standard error of the mean; PS341, proteasome inhibitor, bortezomib; ns, not significant. To assess whether the elevation of GCase Leu444Pro in the patient fibroblasts treated with STARS relies on enhanced synthesis of the endogenous polypeptide, cells were metabolically labeled for 10 minutes with 35 S-methionine and -cysteine. Analyses of radiolabeled polypeptides ( Fig. 7E ) and radiolabeled GCase Leu444Pro ( Fig. 7F ) in lysates from mock-treated patient fibroblasts (lanes 1) or patient fibroblasts exposed to GT-02287 or GT-02329 (lanes 2 and 3, respectively) show that the STARS do not enhance total protein synthesis or the synthesis of endogenous GCase Leu444Pro . Rather, STARs substantially stabilize endogenous GCase Leu444Pro by preventing its proteasomal clearance. Consistently, in mock-treated cells, the GCase Leu444Pro level is substantially increased when patient cells are exposed to PS341, a selective inhibitor of 26S proteasomes ( Fig. 7H , lanes 1 vs. 2) ( Adams, Palombella et al ., 1999 ). Exposure to GT-02287 (lane 3) or GT-02329 (lane 5) raises the level of endogenous GCase Leu444Pro (compare Fig. 7H , lane 1 with lanes 3 and 5, Figs. 7A ‒7D ). Incubation with PS341 does not further increase the level, indicating that the turnover of GCase Leu444Pro has already been substantially delayed by the STARS ( Fig. 7H , lanes 3 vs. 4 and 5 vs. 6). GT compounds mitigate UPR in p.L444P/p.L444P patient-derived fibroblasts The engagement of ER-resident chaperones by folding-defective polypeptides induces low levels of unfolded protein responses, characterized by the transcriptional and translational induction of a very restricted number of ER stress markers ( Bergmann, Fregno et al ., 2018 ). The persistent engagement of the ER chaperone BiP and CNX by the GCase Leu444Pro mutant ( Fig. 5F ) is mirrored by the constitutively elevated levels of BiP transcripts in p.L444P/p.L444P mutant GD patient fibroblasts as established by real-time qPCR ( Fig. 8A ). This indicates that p.L444P/p.L444P mutant GD patient fibroblasts have higher basal levels of ER stress compared to fibroblasts from healthy patients and from patients expressing the GCase Asn370Ser mutant protein ( Fig. 8A ). Notably, existing literature reports elevated levels of ER stress in mammalian and insect cells expressing mutant forms of GCase (Do, McKinney et al ., 2019). In sharp contrast, our analyses show that p.N370S/ins patient-derived fibroblasts have normal levels of BiP, consistent with normal ER homeostasis ( Fig. 8A ). Download figure Open in new tab Figure 8. GT compounds alleviate UPR in p.N370S/ins and in p.L444P/p.L444P patient fibroblasts. (A) BiP mRNA levels as determined by RT-PCR in healthy donors (lane 1) and patient-derived fibroblasts (lanes 2 and 3). (B) BiP mRNA levels monitored by qPCR in GCase Leu444Pro patient fibroblasts (Coriell GM08760) treated for 4 days with DMSO or the STAR GT-02287 at concentrations ranging from 1.5 µM to 25 µM. (C) Same as (B) for patient fibroblasts treated with GT-02329. (D) mRNA levels of selected UPR markers (Bergmann, Fregno et al ., 2018 ) monitored by qPCR in GCase Leu444Pro patient fibroblasts (Coriell GM10915) treated for 10 days with DMSO or 12.5 µM GT-02287. (E) Same as (D) for cells treated with 6.25 µM GT-02329. (F) BiP and GRP94 protein levels monitored by WB analysis in GCaseLeu444Pro patient fibroblasts (Coriell GM08760) treated for 4 days with 25 µM GT-02287 and 12.5 µM GT-02329. ( G and H) Quantification of (F) . The mean -/+ SEM is shown. UPR, unfolded protein response; BiP, binding immunoglobulin protein; mRNA, messenger ribonucleic acid; RT-PCR, reverse transcription polymerase chain reaction; qPCR, quantitative polymerase chain reaction; DMSO: dimethyl sulfoxide; STAR, structurally targeted allosteric regulators; GRP94: glucose-regulated protein 94; WB, western blot; SEM, standard error of the mean. The STARS of GCase substantially reduce chaperone engagement and ER retention of GCase Leu444Pro ( Figs. 8A ‒8B ), allowing the delivery of the mutant polypeptide to the lysosomal compartment ( Fig. 6 ). This has positive effects by diminishing the lysosomal accumulation of GCase substrates. To verify if this also alleviates the constitutive ER stress found in patient cells, we monitored the transcript and protein levels of the ER stress markers upregulated in cells expressing misfolded polypeptides (Bergmann, Fregno et al ., 2018 ). Our experiments show that ER stress levels in patient fibroblasts exposed for 4 days to various concentrations of GT-02287 ( Fig. 8B ) or GT-02329 ( Fig. 8C ), as monitored by quantification of BiP transcripts via real-time qPCR ( Figs. 8B and 8C ) and by monitoring the levels of transcripts of other ER stress markers ( Figs. 8D ‒8E ), are reduced by 20-40%. More importantly, the reduction of ER stress is also demonstrated by lower levels of BiP and GRP94 proteins in cells treated with the two STARS of GCase ( Figs. 8F ‒8H ). Discussion In the present study, we used the innovative SEE-Tx® drug discovery platform to identify an allosteric druggable binding site on the lysosomal protein GCase. Based on our findings, we designed two STARs of GCase (GT-02287 and GT-02329), which bind the protein outside the catalytic site. GT-02287 and GT-02329 enhance the enzymatic activity of GCase in cell lysates, as well as in primary human fibroblasts from a healthy donor and from GD type I, II and III patients characterized by the expression of the Asn370Ser or Leu444Pro mutant forms of GCase. By reducing the lysosomal accumulation of the GCase’s endogenous substrates, GT-02287 and GT-02329 enhance lysosomal performance, alleviate lysosomal toxicity and reduce cellular stress. To understand the impact of the disease-causing Asn370Ser and Leu444Pro amino acid substitutions on the fate of the GCase protein, and to characterize the mode of action of the two STARs, we employed a series of experimental readouts based on monitoring the fate of HaloTag chimeras of the proteins under investigation. This approach offers quantitative information on protein trafficking to endolysosomal compartments using biochemical and imaging techniques (Rudinskiy, Bergmann et al ., 2022, Rudinskiy and Molinari, 2022 , Rudinskiy, Pons-Vizcarra et al ., 2023) and can be implemented with LysoQuant an automated deep learning approach for the quantifying cargo delivery within endolysosomes (Morone, Marazza et al ., 2020). We established that the Leu444Pro mutation causes tight ER retention due to engagement with ER-resident molecular chaperones of the lectin (CNX) and the HSP70 families (BiP). The prolonged engagement of these chaperones indicates a significant delay in the mutant polypeptide’s folding process, preventing its release from the biosynthetic compartment and hampering its transport to the lysosomal compartments. Our experiments reveal that the consequences of the Asn370Ser mutation are much milder than those of the Leu444Pro mutation, consistent with the more severe disease progression of patients carrying the latter mutation. The prolonged engagement of ER chaperones by the GCase Leu444Pro polypeptide eventually triggers a restricted UPR, characterized by the induction of a class of chaperones including BiP, GRP70, ERdj3, GRP94, ERp72, HERP, MANF and CRELD2, recently associated with a transcriptional/translational response to intraluminal accumulation of folding-defective polypeptides (Bergmann, Fregno et al ., 2018 , Bergmann and Molinari, 2018 ). Notably, GT-02287 and GT-02329 reduce polypeptide retention by the ER chaperone system and protect mutant forms of GCase from unwanted proteasomal degradation, indicating enhanced folding capacity. This has an immediate positive effect, as shown by reduced stress levels in patient cell lines and enhanced delivery of mutant polypeptides to lysosomal compartments. GT-02287 and GT-02329 have been designed as STARS to bind the target polypeptide outside the active site, as confirmed in our tests by SPR competition experiments with IFG. Consistently, their enhanced delivery to the lysosomal compartments results in the clearance of GCase substrates, whose accumulation interferes with the proper lysosomal functions and is a hallmark of GD. The findings of this study suggest that GT-02287 and GT-02329 have the potential to be developed further as potential therapeutic agents for GCase-related disorders, including GD, PD and Dementia with Lewy Bodies. In conclusion, our findings indicate that GT-02287 and GT-02329 promote the conformational maturation of disease-causing mutant forms of GCase, facilitate their release from ER-resident molecular chaperones, protect them from unwanted proteasomal clearance, restore lysosomal activity and alleviate misfolded-protein-induced ER stress in cultured cells and in GD patient-derived primary human fibroblasts. Supporting information S1 Table. Antibodies used. (PDF) S2 Table. Primers for quantitative polymerase chain reactions (qPCR). (PDF) S1 File. SPR competition with IFG. (PDF) Funding This study was funded by the Eurostars-2 joint program with co-funding from the European Union Horizon 2020 research and Innosuisse – Swiss Innovation Agency [E!113321_CHAPERONE]. MM is supported by Swiss National Science Foundation grants 310030-214903 and 320030-227541. The funders had no role in study design, data collection and analysis, publication decisions or manuscript preparation. Author contributions Conceptualization: NPC, EC, MB, AMGC, MM; Methodology: NPC, AR, AD, EC, AMGC, IF, MR, TS, TJB and MM; Investigation: NPC, AR, AD, EC, IF, MR, TS and TJB; Visualization: NPC, AR, AD, EC, MB, AMGC, IF, MR, TS, TJB and MM; Funding acquisition: MB and MM; Project administration: MB and MM; Supervision: NPC, EC, AMGC and MM; Writing – original draft: NPC, EC, AMGC and MM; Writing – review & editing: NPC, AR, AD, EC, MB, AMGC, IF, MR and MM. Competing interests NPC, AR, AD, EC, MB, and AMGC are employees of Gain Therapeutics Sucursal en España or GT Gain Therapeutics SA, and their research and authorship of this article were completed within the scope of their employment. IF, MR, TS, TJB and MM declare that they have no competing interests. Data and materials availability All data and materials used in the analysis are available, without restriction, upon reasonable request to reproduce or extend the analyses. View this table: View inline View popup Download powerpoint S1 Table. Antibodies used in study View this table: View inline View popup Download powerpoint S2 Table. Primers for quantitative polymerase chain reactions (qPCR) S1 File. SPR competition with IFG Surface plasmon resonance (SPR) competition experiments with isofagomine (IFG) confirmed Gain Therapeutic (GT) compounds bind to Cerezyme at both pHs; however, not at the active site but at a different pocket. Download figure Open in new tab S1 Fig. SPR dose-response for GT compounds binding to immobilized GCase monitored at neutral (7.4) and acidic (5.0) pH with and without the presence of the inhibitory chaperone isofagomine (IFG). Acknowledgments We thank the members of Molinari’s laboratory for their discussions and critical reading of the manuscript. We also thank Dr Joanne Taylor for her helpful suggestions. Klara J. Belzar, Ph.D., Med-writeplus Ltd., UK, provided medical writing and editorial assistance in preparing the manuscript. Additionally, we appreciate the support and advice on the SPR technique provided by the Technological Centers (CCiTUB) at the University of Barcelona, Barcelona, Spain. References ↵ Adams , J , Palombella , VJ , Sausville , EA , et al. 1999 . Proteasome inhibitors: a novel class of potent and effective antitumor agents . Cancer Res , 59 , 2615 – 2622 . OpenUrl Abstract / FREE Full Text ↵ Alonso , XB , Garcia , DA & Schmidtke , P. Patent 2012 . Method of binding site and binding energy determination by mixed explicit solvent simulations . ↵ Alvarez-Garcia , D & Barril , X 2014 . Molecular simulations with solvent competition quantify water displaceability and provide accurate interaction maps of protein binding sites . J Med Chem , 57 , 8530 – 8539 . doi: 10.1021/jm5010418 . OpenUrl CrossRef PubMed ↵ Alvarez-Garcia , D , Schmidtke , P , Cubero , E , et al. 2022 . 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Share A novel class of allosteric glucosylceramidase beta 1 correctors that reduce cellular stress and enhance lysosomal function Ilaria Fregno , Natalia Pérez-Carmona , Mikhail Rudinskiy , Tatiana Soldà , Timothy J. Bergmann , Ana Ruano , Aida Delgado , Elena Cubero , Manolo Bellotto , Ana María García-Collazo , Maurizio Molinari bioRxiv 2024.11.15.623779; doi: https://doi.org/10.1101/2024.11.15.623779 Share This Article: Copy Citation Tools A novel class of allosteric glucosylceramidase beta 1 correctors that reduce cellular stress and enhance lysosomal function Ilaria Fregno , Natalia Pérez-Carmona , Mikhail Rudinskiy , Tatiana Soldà , Timothy J. 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