The long road of drug development for endometriosis - Pains, gains, and hopes

DEC-Tec is a transformative tool in drug discovery and development, offering unprecedented efficiency, diversity and scalability in identifying potential drug-like compounds [ 107 ]. DNA-encoded chemical libraries (DELs) are assembled through both chemical and enzymatic synthesis of small molecules and encoded unique double-stranded linkers, respectively. The affinity selections of target protein against DELs allow rapid and precise identification of binding events, as well as binders’ structures. DEC-Tec has been increasingly adopted in hit identification and lead generation for researchers in both academic and commercial settings, yielding a broad range of bioactive molecules [ 108 ]. Compared to traditional labor-intensive, cost-inefficient, high-throughput screening methods, DEC-Tec shines in its ability to accelerate the process of screening and testing billions of compounds in single reactions; it offers additional advantages such as easily multiplexed targets and focused library selections. We will briefly summarize the framework of DEC-Tec from the development of DELs to hit identification. Since 2004, molecules generated from DEL screening have been reported to become promising probes and therapeutic candidates [ 107 , 109 – 111 ]. Deviated from the traditional small-molecule library synthesis, one key component of DEL synthesis is maintaining the precise sequence-structure linkage. Early strategies to establish DELs include (a) DNA-templated synthesis, which uses a modified DNA oligo as the template for multistep molecular synthesis [ 112 ]; (b) encoded self-assembling chemical libraries, which utilizes established libraries of organic molecules attached to individual oligonucleotides to mediate the self-assembly of the library [ 110 ]; and (c) DNA routing, which relies on partitioning nanomole levels of oligonucleotides into physically distinct sub-pools and facilitating a complex network of DNA-programmed chemical synthesis [ 113 ]. The most recent and widely used DEL synthesis method is the split-and-pool approach ( Fig. 1A ). As a part of combinatorial chemistry, split-and-pool is applied in a repeated stepwise procedure. After dividing the starting compound mixtures, which have unique covalently attached DNA sequences, into different reaction portions, each portion is coupled with a unique chemical building block. Each added building block is then barcoded by another unique DNA identifier that can be ligated onto the DNA tags on the starting compound mixtures. After re-pooling and thoroughly mixing of all the portions, the procedure is repeated for each additional building block [ 107 , 114 , 115 ]. Advances in DNA-compatible reactions combined with the availability of a variety of panels of building blocks provide great chemical diversity and complexity for the generation of more drug-like libraries. This split-and-pool approach makes it possible to prepare billions of compound mixtures to be used for subsequent selections in a versatile, fast, and cost-effective manner. Screening of DELs for small molecule discovery commonly involves binding-based selection assays where the library pools are exposed to immobilized target protein or other biological molecules. The selection workflow starts with pooling DELs into a single solution system and incubating with a target of interest. Target proteins can be immobilized on solid supports, including magnetic beads or resin-filled tips, by non-covalent binding of suitable tags (i.e., His-tag or biotin) or by covalent modification of lysine residues [ 116 ]. Subsequent washes will separate the molecules bound to the target and the non-binding members of the pool. The unique DNA sequences of binding moieties will be amplified and quantified by reverse transcription-quantitative polymerase chain reaction. After the sequencing library preparation and next-generation sequencing, DNA sequences of the target-binding molecules will be identified and then decoded into their original structures. Researchers have also developed different data analysis platforms to process the many unique sequences isolated from DEL selections [ 117 – 119 ]. The output results will highlight the enrichment of individual members against their abundance in the control groups (e.g., pre-selection library or resin-only selections). The highly sensitive nature of affinity selection and DEL sequencing can identify even low-affinity binders, providing additional information for compound testing. Upon the decoding of the enriched molecule structures, hit compounds will be resynthesized in the DNA-free form, followed by medicinal chemistry optimization for advanced efficacy assays. DELs screening is a powerful hit-identification platform; steps following hit identification center around the lead compound generation, validation, and optimization. The hit-to-lead process that changes partial physicochemical properties and adds structural modifications post DEL screens are similar to traditional high throughput screens; however, due to the combinatorial synthetic methods used for the generation of DELs, the linker effect at the covalent DNA tagging point requires updated strategies for lead optimization. Hit-to-lead optimizations from DEL hits show a trend of emphasizing structural truncations and pursuing the minimum pharmacophore of targets [ 120 ]. This aligns with the need for molecular weight reduction to achieve oral bioavailability, given hits from DEL screens usually encompass high molecular weight [ 121 ]. Other than truncation, reducing lipophilicity is also a key optimization strategy widely utilized for lead optimization from DEL hits [ 122 ]. Additionally, the structure-activity relationship (SAR) is a time-honored method for optimizing the potency, selectivity, and pharmacokinetic properties of lead compounds. SAR of the DNA attachment location can inform potential substituent structures [ 123 ]. Ultimately, the potency and efficacy of lead compounds need to be examined with biological-relevant assays, which will then grant possibilities for future clinical candidates and drugs. Detailed summary and comparison of lead optimization routes across published studies using DEL screening is beyond the scope of the current review; we encourage the readers to refer to this review by Reiher et al. [ 124 ] for an in-depth analysis. While DELs offer significant advantages, they also pose challenges. Like other screening hits, identified binders must be further validated to ensure specificity and minimize off-target effects. Also, given the solubility of the DNA barcodes, the affinity selections are usually carried out in aqueous solutions, which may limit their application on certain protein targets of interest. Also, some transcription-factor-like targets that harbor DNA binding domains in their structures may need additional optimization in generating the protein used for selection (i.e., truncation or mutation of original DNA binding pockets to avoid the enrichment of unspecific binders). The advancement of DEC-Tec marks a pivotal development for modern drug discovery pipelines. The combinatorial synthesis approach used to generate DELs yields billions of intrinsically diverse compounds with drug-like properties, thereby significantly shortening the time-frame for drug discovery and increasing the probability of identifying potent drug candidates. Preclinical and clinical studies demonstrated DELs are robust platforms for rapid and efficient hit generation of bioactive compounds [ 125 ]. We will review several molecules rooted from DEL screening in the upcoming sections. Endometrial diseases, specifically endometriosis, are challenging to recapitulate in model systems due to their tissue complexity and involvement of hormonal changes. Much effort has been made to establish both in vitro and in vivo models to mimic the disease. Culturing of the primary endometrial epithelial and stromal cells from the endometrial biopsy has been widely used since the 1990s [ 126 – 131 ]. One drawback of primary cultures is that the cultured cells lose their designated cell phenotypes after extensive passages, making it difficult to keep them for long-term cultures [ 132 ]. To overcome this, many groups established immortalized endometriotic cell lines by introducing human telomerase reverse transcriptase into the primary cells, and such immortalized cells tend to retain their ability to respond to ovarian hormones [ 133 – 137 ]. Researchers also developed co-culture methods, with the introduction of macrophages [ 138 – 140 ], mesothelial cells [ 141 – 143 ], and endothelial cells [ 144 , 145 ], to better mimic the microenvironment and cellular crosstalk during the establishment of endometriosis lesions. In addition to monolayer culturing, 3D culturing of the endometriotic tissues has also been heavily investigated over recent years. These in vitro culturing systems are termed endometrial organoids or assembloids, depending on the cell types included during the culture. The 3D culturing method allows the sampled tissues to self-assemble in the culturing media and develop into sphere-shaped structures. The organoids derived from the epithelial compartment of both eutopic and ectopic endometrium tissue from individuals with endometriosis can be stably expanded and also maintain the disease-associated traits and mutations, making them ideal for drug discovery and screening [ 146 ]. However, the caveat for organoids is that usually only one cell type is included in the culture, making it less optimal to understand the crosstalk between different endometrial compartments [ 147 ]. The establishment of assembloids benefited from the co-culture of the epithelial organoids and endometrial stromal cells, making it feasible to understand the epithelial-stromal crosstalk under their naïve environment [ 148 – 150 ]. Additional organ-on-a-chip devices [ 151 – 153 ] and bioprinting techniques [ 154 ] enabled the possibility of mimicking uterine physiology in a controlled manner. In vivo models of endometriosis can be characterized into non-human primate models, xenograft models and rodent models. Non-human primates are the only mammals reported to develop endometriosis spontaneously [ 155 , 156 ]. In baboons, endometriosis can be induced by inoculating the menstrual endometrial tissues in the peritoneal cavity [ 157 ]. Although the uterine physiology in non-human primates is closer to human compared to other models, considerations for ethics and cost make non-human primates a less commonly used option [ 158 ]. Similar to the cancer xenograft models, xenograft models for endometriosis require the transplantation of human endometrial tissues into immunodeficient mice [ 159 – 161 ]. However, using immunodeficient mice can also limit the investigations of other inflammatory processes concurrent with the lesion development [ 162 ]. Rodent models, namely mice and rats, are the most used in exploring the etiology and therapeutic options given their potential to be genetically manipulated and colonies being maintained in a cost-effective manner. The endometriosis lesions are induced through either surgical placement or intraperitoneal injection of mice/rat uterine horn fragments into the peritoneal cavity [ 163 – 166 ].

Patients with endometriosis are often associated with infertility [ 167 ]. However, for those patients with endometriosis who wish to become pregnant, the treatment options are very limited. Researchers have been actively expanding the drug identification and development for endometriosis in the light of circumventing the unfavorable effects of the hormonal-suppressing drugs currently prescribed. Here, we briefly summarize recent advances in identifying additional drug targets for endometriosis. Table 2 summarizes the discussed approaches. Other highly sought-after targets are kinases [ 168 , 169 ]. Compared with pain relievers and hormonal modulators, kinase inhibitors are attractive not only because of their pivotal roles in a broad range of biological activities but also because of their actionable structural properties with successful adaptations in other diseases, such as cancers [ 170 ]. Our team tested whether the KIT/CSF1R inhibitor pexidartinib can have therapeutic potential for endometriosis [ 171 ]. The KIT gene encodes mast/stem cell growth factor receptor; it has been reported to affect cell survival, proliferation and migration, which could play roles in the development of endometriosis [ 172 ]. Similarly, CSF1R encodes for macrophage colony-stimulating factor 1 receptor, which has a known role in the invasion process at the uteroplacental interface [ 173 , 174 ]. Such characteristics provide a potential pathologic role of CSF1R in the formation of endometriosis. We first validated the overexpression of KIT and CSF1R in endometriotic lesions, with a preference for epithelial deposition. We further tested the efficacy of inhibiting KIT/CSF1R kinase activities using pexidartinib, in the immortalized 12Z endometriotic epithelial cells. Treatment of pexidartinib in 12Z cells leads to the inhibition of key pro-inflammatory and survival pathways, namely JNK, STAT3, and AKT pathways. Pexidartinib also suppressed the gene expression of IL8 and CCND1 , which are pro-inflammatory and survival markers. Lastly, we also reported that pexidartinib could decrease endometriotic cell growth and viability. These results provided robust rationale that CSF1R and KIT are promising targets for nonhormonal treatment options for endometriosis. Our team subsequently reported that ephrin receptor kinases could be actionable targets for the treatment of endometriosis and developed potent and selective pan-ephrin receptor kinase inhibitors using DNA-encoded chemistry technology [ 175 ]. From transcriptomic profiling, we discovered that EPHA2 and EPHA4 are over-expressed in the endometriosis lesions compared to normal counterparts. Next, utilizing DEL screening techniques, we developed a series of small molecule inhibitors targeting both EPHA2 and EPHA4. Specifically, CDD-2693 and CDD-3167, the hit compound and its chemically optimized analog, exhibited picomolar/nanomolar kinase activity against EPHA2 (CDD-2693: K i : 4.0 nM; CDD-3167: Ki: 0.13 nM) and EPHA4 (CDD-2693: K i : 0.81 nM; CDD-3167: K i : 0.38 nM). Chemical structures of CDD-2693 and CDD-3167 are included in Fig. 1B . Efficacy testing in 12Z endometriotic epithelial cells demonstrated that both CDD-2693 and CDD-3167 can significantly decrease cell viability, gene expression of inflammatory marker PTGS2 , and ligand-induced phosphorylation of EPHA2/4. In addition, CDD-2693 and CDD-3167 can decrease the expansion of primary endometrial epithelial organoids derived from patients with endometriosis. We not only proposed novel therapeutic options for endometriosis but also reiterated the advantages of DEL screens in generating drug-like molecules with real-world examples. Mitogen-activated protein kinase (MAPK) signaling pathways have been reported to contribute to the inflammation and induction of cell proliferation in endometriosis [ 176 ]. Many studies have also been actively examining the possibility of targeting MAPK for the treatment of endometriosis. Yoshino et al. first demonstrated that using MAPK/JNK inhibitors, including SB202190, PD98059, and SP600125, can produce anti-inflammatory effects in primary endometriotic stromal cells [ 177 ]. Other compounds, including U0126, SB203580, SP600125, and Sorafenib, targeting MEK1/2, ERK1/2, JNK and p38-MAPK pathways have also been validated to inhibit the expressions of inflammatory and proliferative markers in both endometriotic cell cultures and in vivo mouse models [ 178 – 185 ]. Transforming growth factor β (TGFβ) superfamily signaling pathways are essential in maintaining the normal physiological functions in the female reproductive tract [ 186 ]. There is increasing evidence showing that the TGFβ superfamily plays a major role in the pathogenesis of endometriosis. In endometriosis, significantly increased levels of TGFβ1 have long been reported in the peritoneal fluid and serum of patients with endometriosis [ 187 – 190 ]. TGFβ pathway was reported to be over-activated in the sites of endometriosis lesions, suggesting altered TGFβ activity in the endometriotic lesions and their surrounding peritoneum may lead to the symptomatic effects of endometriosis [ 191 ]. A more recent study from our group mechanistically linked TGFβ pathways with the infertility phenotype observed in patients with endometriosis [ 150 ]. By transcriptomic profiling of stromal cells from endometriosis patients, the authors reported defective TGFβ signaling pathways and altered functions of key regulators of the TGFβ signaling pathways, namely SMAD4, in the affected individuals. Bone morphogenetic proteins, a subset of the TGFβ superfamily that controls the endometrial receptivity, were also observed to be defective in patients with endometriosis. Additionally, the authors demonstrated exogenous supplementation of BMP2 can improve decidual marker expressions in the 3-dimensional cultures of endometrial stromal and epithelial cells. These results provided evidence as well as potential therapeutic directions for decidualization defects and subsequent pregnancy complications observed in patients with endometriosis. One of the hallmarks of endometriosis is its chronic inflammatory conditions [ 2 ]. Proinflammatory cytokines in the microenvironment of endometriosis lesions contribute to common endometriosis symptoms, including pain and infertility [ 2 ]. Thus, immunotherapies are in consideration for future treatments of endometriosis. Polylactic- co -glycolic acid that encapsulates anti-CTLA4 antibody was able to inhibit ectopic endometrial cell proliferation and invasion [ 192 ]. Another study tested the possibility of adoptive cell therapy in the mouse model of endometriosis, showing that autologous transplantation of human immune cells was beneficial for disease relief, demonstrated by the reduction of endometriotic lesion volume and micro-vessel density in mice [ 193 ]. More recently, the use of an IL-8 antibody (AMY109) showed efficacy in a surgically induced endometriosis primate model, showing the promise of an immune-modifying drug for the treatment of endometriosis [ 194 ]. Another study proposed that targeting IL-1/IL-33 family can be promising for endometriosis treatment. By using monoclonal antibodies targeting the ligands or inhibitors targeting the downstream effectors of the IL-1/IL-33 signaling pathway, the endometriosis lesions exhibit a volume decrease in the mouse model of endometriosis [ 195 ]. Epigenetically targeted therapeutics have also attracted attention, given recent progress in understanding the epigenetic role in the pathogenesis progress in endometriosis. Dysregulated epigenetic regulators have been reported in samples from patients with endometriosis, including DNA methyltransferases [ 196 – 198 ], DNA demethylases [ 199 – 201 ], histone deacetylases and acetyltransferases [ 202 – 204 ], and histone methyltransferases and demethylases [ 205 – 209 ]. Inhibition of Enhancer of zeste homolog 2 (EZH2), a known histone methyltransferase up-regulated in endometriotic lesions, leads to reduced migration and invasiveness as well as lesion weights both in vitro and in vivo [ 207 , 210 ]. Similarly, inhibition of Sirtuin 1 (SIRT1), a histone deacetylase up-regulated in endometriotic lesions [ 208 , 211 ], results in a decreased number of endometriotic lesions in a mouse endometriosis model [ 211 ].

With an overall 10 % incidence rate, endometriosis is a multifactorial disease that presents a wide variety of symptoms and different responses toward medicinal management. There are around 190 million people affected by the disease worldwide [ 2 ], and the average delay in diagnosis of endometriosis ranges from 3 to 11 years [ 212 ]. Such delay can cause significant financial, psychological, and physical stress for the affected women and communities [ 213 ]. Both clinicians and researchers have been advocating for more resources devoted to basic research, diagnostic and therapeutic innovations for endometriosis [ 214 – 216 ]. Theories analyzing the origin of endometriosis provide different angles of how to interpret the disease, neuronal, hormonal, inflammatory or genetic. However, endometriosis is a multifaceted disease with an array of clinical symptoms. Current knowledge of the etiology and pathophysiology of endometriosis and its related co-morbidities can only partially explain the diverse symptoms, and there are no definitive diagnostic tools other than laparoscopic surgery [ 2 ]. This gap in knowledge warranted further research endeavors to uncover the potential biomarkers for diagnosis, as well as an advanced understanding of molecular basis during lesion establishment and disease progression. Current treatment plans for endometriosis can be characterized by surgical management and pharmacological management. A comprehensive and personalized treatment plan needs to be tailored to each individual’s need based on the disease severity and other clinical features (i.e., infertility, pain, prior health conditions) or personal preferences (i.e., daily medicine intake or route of intake). Although the current management is sufficient in halting symptoms for certain patients during treatment, both surgery and medicinal management can pose considerable drawbacks, such as disease recurrence, surgery-related complications, or the undesired effects of hormonal suppression [ 215 ]. Thus, scientific research in developing and evaluating new targets for endometriosis is in dire need. Here, we reviewed the technological advancement in drug discovery for endometriosis with a focus on the DEC-Tec platform, from the screening library construction to lead compound optimization, we acknowledge and emphasize the revolutionized DEC-Tec can be of great use to enable the drug discovery for these new targets and streamlines this process in a rapid yet precise manner. In conclusion, endometriosis is still a challenging disease to manage due to its complex nature in women. Future studies expanding and validating new targets for non-hormonal therapies will greatly increase the quality of life for women with endometriosis. In addition to scientific advancement in understanding the disease, there is also an urgent need to call for public awareness, enhance affordable access to care, and implement comprehensive and personalized care structures in global public health policies.

Treatment approaches for endometriosis can be characterized by pharmacological and surgical interventions. The pharmacological management includes symptomatic pain relievers (i.e., nonsteroidal anti-inflammatory drugs (NSAIDs), hormonal therapies (i.e., oral contraceptives), gonadotropin-releasing hormone (GnRH) modulators, and aromatase inhibitors. In surgical management, conservative surgery is the first-line option, aiming to perform excision or ablation of endometriotic lesions. A multidisciplinary approach is often needed to comprehensively address endometriosis disease, with the goal of improving the overall quality of life for affected women. Here, we mainly discuss the non-surgical management of endometriosis. Table 1 summarizes the drugs discussed in this section. NSAIDs, including propionic acids, fenamates, acetic acids, salicylates, and oxicams, are the first-line treatment for pelvic pain and can effectively treat the symptom of dysmenorrhea [ 63 ]. However, no robust trial data has confirmed that NSAIDs are effective in managing pain specifically caused by endometriosis [ 64 ]. OCs are recognized as the first-line treatment for women with pelvic pain related to endometriosis [ 65 ]. The advantage of these medicines is that they are generally well-tolerated and economically friendly. The efficacy of OCs for endometriosis-associated pain relief is confirmed by randomized clinical trials [ 66 ]. Cyclically usage of OCs allows monthly menses, which may not completely subside the dysmenorrhea caused by endometriosis [ 67 , 68 ], however, continued use of OCs is likely to be beneficial for these patients [ 69 ]. Once amenorrhea is achieved through medications, the pain relief effect will be long-term and sustainable [ 70 ]. Intake of OCs inhibits the synthesis and release of GnRH, which leads to decreased levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). As a result, follicular development and ovulation are halted. This provides effective contraception and can reduce the risk of endometriosis-related ovarian cancers [ 71 ]. However, women seeking spontaneous conception need to consider alternative approaches, such as conservative surgery, when choosing management plans. Another downside is that there are still around 15 % of patients who do not respond to OC treatments [ 72 ]. And even when used in postoperative endometriosis patients, 49 % of the patients prescribed with OCs require further surgical procedures or second-line treatments [ 73 ]. Additionally, the discontinuation rate of OCs spans between 8 % and 25 % due to adverse events or lack of efficacy in patients with endometriosis [ 72 , 74 ]. One of the most common side effects of OC is psychological changes [ 75 – 77 ]. It has long been recognized that depressive symptoms and decreased libido are observed more frequently in women taking OCs. In the 1970s, a longitudinal study following forty-six thousand women reported a 30 % increase in undesired psychological changes [ 78 ]. More recently, Skovlund and the group reported the positive associations between OC usage and mood disturbances in a large cohort of 1,061,997 women from Denmark in 2016 [ 79 ]. A follow-up prospective study from this group also reported that OC usage was associated with a higher risk for suicide attempts as well as completed suicide [ 80 ]. However, such a strong conclusion could be controversial [ 81 – 83 ]. Schaffir et al., underscored the controversies in linking OC usage to adverse psychological changes; including the limitations in methods to define mood changes, and the lack of distinguishment in different prescription regimes of OCs [ 84 ]. Also, other confounding factors, such as patient expectations, can affect the survey results [ 81 ]. Another genre of hormonal therapies for endometriosis is the GnRH modulators. GnRH is produced in the hypothalamus and is released into the hypophyseal-portal capillary circulation. After binding to the receptors on the pituitary gonadotrophic cells, GnRH initiates the production and release of FSH and LH [ 85 ]. Both GnRH agonists and antagonists have been shown to be effective in the treatment of endometriosis-related pain. GnRH agonists (e.g. leuprolide and nafarelin) have been demonstrated to be as effective as OCs in pain relief [ 86 ]. Such effect is achieved by the downregulation of the pituitary-ovarian axis and eventually leads to amenorrhea. Similarly, GnRH antagonists (e.g., relugolix and elagolix) are approved by the U.S. Food and Drug Administration to treat endometriosis, achieving the hypoestrogenic state by directly inhibiting the gonadotropin production [ 87 , 88 ]. GnRH antagonists are more easily accessible in an oral form and do not experience the initial surge effect of the gonadotropin hormone production. However, GnRH modulators lead to adverse effects, such as hot flushes, emotional lability and a decrease in bone mineral density, due to low level of estrogen. The usage of AIs in the treatment of endometriosis can be exemplified in unusually aggressive cases of postmenopausal patients [ 89 ]. Characterized by their inhibition effect on the CYP450 enzyme, AIs can limit the conversion of androgen to estrogen [ 90 ]. It was originally developed to treat hormone-sensitive breast cancers in post-menopausal women; however, given its effect on estrogen deprivation, it was adapted for the treatment of endometriosis [ 89 ]. Several studies tested the effects of combined AI (anastrozole or letrozole) and OCs in premenopausal endometriosis patients. The combined treatment can significantly reduce the pain level in these patients with refractory endometriosis [ 91 , 92 ]. In another clinical trial studying the combined effects of AI with GnRH agonist, after six months of treatment with anastrozole and a GnRH agonist, patients acquired significantly longer pain-free intervals compared to GnRH agonist alone [ 93 ]. The most common route of delivery for OCs is oral ingestion; however, the oral route has a stronger first-pass effect in the liver than parenteral administration [ 94 ]. Apart from the oral route, other delivery methods such as tissue implants or transdermal patches have also been investigated. Etonogestrel subdermal implants and levonorgestrel intrauterine device placement have also been demonstrated to be beneficial for endometriosis-related pain symptoms [ 95 – 97 ]. Additionally, transdermal patches and vaginal rings containing derivatives of progestin and estrogen are also being actively explored as delivery methods for contraceptives [ 98 ]. GnRH agonists can be formulated into long-term-acting injectables or short-term-acting nasal spray formulas. For example, GnRH agonist leuprolide is formulated in a monthly injection regime by either intramuscular or subcutaneous injection [ 99 ]. The commercialized GnRH agonists are usually represented by the depot formulation consisting of biocompatible polymers, which create a depot of the drugs after the injection and initiate the drug release in a controlled manner [ 99 ]. Another delivery route is through the nasal mucosa; the advantages of nasal formulation include convenient patient access and extensive vasculature with easily permeable surface membrane, leading to rapid systematic incorporation [ 100 ]. For GnRH antagonists approved for clinical use; oral administration can achieve satisfying bioavailability [ 88 , 101 ]. In summary, current management of endometriosis is usually tailored toward the severity of the symptoms, as well as patient preferences and reproductive goals. Guidelines emphasize the use of a combination of pharmacological and surgical treatments to provide comprehensive care standards [ 102 – 104 ]. However, the efficacy of different treatment options usually varies, and the effect wears off quickly after discontinuation of treatment [ 105 ]. Non-surgical treatments often lead to hormonal suppression, thus limiting the fertility needs of a great subset of the patients, whereas around 45 % of patients suffer from recurrence after surgical operations [ 106 ]. Novel drug targets and non-hormonal therapy opportunities are in dire need. In the following sections, we will discuss the efforts made to identify novel drug targets and develop preliminary drug-like molecules using high throughput screening approaches.

Endometriosis, a chronic inflammatory gynecological disorder that is characterized by the presence of endometrial-like tissue outside the uterus, affects around 10 % of reproductive-age women worldwide [ 1 ]. Endometriosis not only causes chronic pelvic pain physically but also leads to a substantial economic burden for patients, with lifetime costs amounting to $27,855 per patient annually and the total annual costs are estimated at $22 billion in the United States alone [ 2 ]. Meanwhile, in the United Kingdom, this number has reached £12.5 billion, covering the cost of direct medical treatment, work loss, and healthcare expenses [ 2 ]. Another survey from the Global Burden of Disease Study demonstrated that both the incident rate and years of life lived with disability in regions with high sociodemographic index increased significantly from 1990 to 2017 [ 3 ]. These ectopic endometriotic lesions can respond to hormonal changes, causing inflammation, scarring, and tissue adhesions. Patients with endometriosis often present with chronic pelvic pain, dysmenorrhea, dyspareunia, and infertility, which drastically decrease patient work efficiency [ 4 , 5 ] and increase total medical costs [ 6 ]. Standard diagnosis is through direct laparoscopic surgical procedures and histological visualization, whilst some imaging techniques, such as transvaginal ultrasound and magnetic resonance imaging, may be helpful for initial assessment. However, an average delay of seven years was reported for the diagnosis of endometriosis due to the nature of the disease [ 7 ]. Current treatment options include symptomatic pain management, hormonal therapies, and surgical removal of the lesions. However, not all patients respond to these managements, and the recurrence rate is high in patients who undergo surgery [ 1 ]. Endometriosis remains a challenging condition that significantly impacts patients’ quality of life and increases healthcare burden. In this review, we will provide an overview of the current knowledge of endometriosis, focusing on recent advances in drug discovery and development for its treatment. Endometriosis has been a long-standing burden for women’s health. Organ damage due to tissue invasion and inflammation, together with other systematic symptoms, including chronic pain and dysmenorrhea, are currently associated with endometriosis and can be traced back almost 300 years ago [ 8 ]. More recent discussions also consider the historical perspectives of “suffocation of the womb”, which describes acute attacks of pain, was in fact, likely to be endometriosis for most patients back then [ 9 ]. However, although the descriptions of endometriosis have been documented for many years, the cause of the disease has not been pin-pointed, partially due to its heterogeneous nature; presenting with a broad spectrum of symptoms, onset ages, histopathological lesion types and responses to therapies. While many theories have been proposed to explain the development of endometriosis, here, we briefly summarize the theories of retrograde menstruation, Müllerian remnants, coelomic metaplasia, and genomic susceptibility. The retrograde menstruation theory for endometriosis development was first proposed by Sampson in 1927 [ 10 ]. It was hypothesized that the establishment of endometriosis lesions involves the reflux of menstrual materials into the pelvic cavity through fallopian tubes, and endometrial tissues can reside adjacent to peritoneal organs, such as the ovary, bladder and bowel, leading to lesion formation. In support of this theory, evidence of retrograde menstruation (i.e. bloody fluids in the peritoneal cavity during the perimenstrual time) was observed in more than 90 % of patients with endometriosis undergoing gynecological surgery [ 11 ]. Recent studies have extended the retrograde menstruation theory by adding the roles of stem cells. Endometrial stem/progenitor cells were found in both menstrual blood and the peritoneal fluid of women with endometriosis. In the same study, it was reported that more clonogenic cells were observed to persist through the menstrual phase in the peritoneal fluid of women with endometriosis compared to control samples [ 12 ]. Human endometrial stem/progenitor cells can be characterized into epithelial progenitor cells and mesenchymal stem cells, both of which demonstrate hallmark characteristics, including self-renewal, potential to differentiate, and high proliferative capability [ 13 , 14 ]. Additionally, markers were proposed to distinguish the two populations. In endometrial mesenchymal stem cells, SUSD2 [ 15 ], PDGFRβ , and CD146 [ 16 ] can be used to isolate mesenchymal stem cells by fluorescence activated cell sorting; while CDH2 (N-cadherin) [ 17 ], SSEA1 [ 18 ], AXIN2 [ 19 ], and SOX9 [ 18 ] were indicated to be relatively selective for epithelial cells with progenitor properties. These stem/progenitor cells were found in the peritoneal fluid from both women with and without endometriosis [ 12 ], suggesting a potential role of refluxed menstrual fluid in the endometriosis lesion initiation and survival. However, one caveat is that while 76 % to 90 % of women may experience retrograde menstruation [ 20 ], the prevalence of endometriosis is around 10 % [ 1 , 21 ], indicating other factors need to be considered in the development of endometriosis. Another hypothesis that explains the pathogenesis of endometriosis is the Müllerian remnants theory. It proposes that endometriosis lesions are derived from the proliferation and differentiation of misplaced primitive endometrial cells along the migratory route of the Müllerian duct [ 22 – 24 ]. In the meantime, given that the mesothelium and the epithelium lining of the Müllerian duct both derive from coelomic epithelium, Mayer first proposed the coelomic metaplasia theory in 1924. This theory was further refined into the hypothesis [ 25 ] that peritoneal mesothelium or germinal ovarian epithelium transforms into responsive endometrium spontaneously or after stimuli such as hormonal influences or inflammation [ 26 – 28 ]. However, some argue that the coelomic metaplasia theory lacks robustness for the etiology of superficial peritoneal lesions due to the high co-occurrences of different lesion types [ 28 ]. Recent technological advances in genetic research fueled the exploration of the pathogenesis of endometriosis. Population studies and genetic studies offer important evidence for the genetic contributions in the development of the disease [ 29 – 31 ]. The familial aggregation phenomenon of endometriosis was quantified by a twin study showing that 51 % of the variance in the endometriosis risk may be attributed to genetic influences [ 32 ]. Many genetic variants or genomic loci have been correlated with endometriosis [ 33 , 34 ]. A recent meta-analysis of the genome-wide association studies (GWASs) on endometriosis reported new genes implicated with hormone regulation ( FN1 , CCDC170 , ESR1 , SYNE1 , and FSHB ) are significantly associated with the disease [ 35 ]. Nine previously reported loci associated with WNT4 , GREB1 , ETAA1 , IL1A , KDR , ID4 , CDKN2B-AS1, and VEZT were also reported to be significant in the GWAS meta-analysis [ 35 ]. In addition to germline mutations, somatic mutations have also been heavily investigated for patients with endometriosis. Many mutations in cancer driver genes, such as ARID1A [ 36 ], PTEN [ 37 ], KRAS [ 36 , 38 – 41 ], PIK3CA [ 38 , 41 ], and ERBB2 [ 37 , 40 ], have been identified through unbiased sequencing methods in patients with endometriosis, suggesting the somatic mutations can contribute to the establishment of the lesions by promoting cell growth, attachment and survival. However, questions remain about understanding the genetic aspect of the disease. Although cancer driver mutations are detected in endometriosis lesions, the factors limiting the progression of a benign disease, endometriosis, into malignancies and cancer are still unknown. Together, both retrograde movement and genetic changes provide the opportunity for the transformation and attachment of endometrial progenitor/stem cells outside the uterine cavity, which then likely give rise to the establishment of endometriosis lesions.

One of the most prominent clinical symptoms in individuals with endometriosis is pelvic pain [ 42 ]. Clinical manifestations can vary and include both menstruation-related and non-menstruation-related pain, such as dysmenorrhea, dysuria, and dyspareunia. The painful sensation can span through the pelvis and abdomen and sometimes radiate to the back and legs [ 43 ]. The activation of the peripheral pain pathways is believed to be involved with the establishment of endometriosis lesions, which provided new blood supplies and initiated neuroangiogenesis in the affected site [ 44 ]. Another key factor that leads to pain symptoms is the inflammatory response in endometriosis patients. The endometriotic lesion itself or through the paracrine feedback mechanism can increase the levels of pro-inflammatory cytokines and growth factors in the peritoneal fluids. Such factors include prostaglandins, tumor necrosis factor α (TNFα), and interleukins (i.e., IL-1β, IL-8), which are known to lead to chronic inflammation [ 45 ]. These immune modulators can exacerbate a neuroinflammatory cascade, contributing to neuropathic pain. Of note, the extent of pain does not necessarily correlate with the severity of the lesions’ number or location [ 46 ]. In addition to the pain caused by the inflammatory stimuli, the tissue adhesions caused by the endometriosis lesions can also contribute to pelvic pain syndrome [ 47 , 48 ]. Local inflammation promotes adhesion formation, which connects normally separated organs together via the connective tissue. These adhesions will interfere with the organ functions and cause anatomic distortion, leading to pain and even restricted pelvic movement [ 48 ]. The persistent pain in patients with endometriosis can also lead to changes in the central nervous system, specifically central sensitization. It is usually demonstrated as increased sensitivity to noxious stimuli and an expanded peripheral receptive field [ 49 , 50 ]. A study from As-Sanie et al. confirmed that women with endometriosis-associated chronic pelvic pain have identifiable changes in the brain, showing decreased volume of grey matter regions in the brain areas involved in pain perception [ 51 ]. The central sensitization theory can partially explain the disproportionate pain symptoms to the severity of the lesions and how the pain signals can be widespread beyond the pelvic region [ 52 ]. Endometriosis is a frequent cause of infertility. Up to 50 % of infertile women are found to have endometriosis [ 42 , 53 ]. Endometriosis may affect female fertility by creating tissue adhesions or anatomical distortions in the pelvic region. Endometriosis patients with pelvic adhesions that ablate the recto-uterine pouch may suffer infertility due to tubal occlusion and sperm passage interference [ 54 ]. In addition, endometriomas around the ovary can cause endocrine dysfunctions by impeding the follicular environment and decreasing oocyte competence [ 55 ]. Another mechanism that contributes to infertility in patients with endometriosis is chronic inflammation. The elevated levels of pro-inflammatory cytokines, such as TNFα and interleukins, are linked to reduced ovarian response and sperm motility; they can also cause additional sperm DNA damage [ 56 – 58 ]. The chronic inflammatory micro-environment may also disrupt oocyte-sperm interactions, delaying embryo development and impairing implantation [ 59 , 60 ]. Assisted reproductive technology (ART) is frequently used to treat infertility in patients with endometriosis. For women with minimal or mild peritoneal endometriosis, both intrauterine insemination (IUI) and in vitro fertilization (IVF) are effective options, with success rates comparable to those seen in other causes of infertility. However, women with more advanced endometriosis tend to have lower success rates with IVF [ 61 , 62 ].