Clinical Candidate Research Articles

Synergy of Prediction Rule and Total Synthesis in Solving the Stereochemical Puzzle of Valactamides.

Natural products with diverse functional groups and stereogenic centers have inspired therapeutics and underpin the modern drug discovery process. Their three-dimensional molecular structures need to be unambiguously determined in order to be realized as clinical candidates or to achieve further activity-guided structural optimization. Although recent advances in spectroscopic methods have made it possible for researchers to determine the structures of microgram samples of complex natural products, there is no universally accepted method for determining the relative and absolute configuration of a naturally occurring compound. We report the determination of the full stereostructure of valactamide A, an eight-stereogenic-center-containing fungal metabolite by the synergy of prediction rule-guided analysis and chemical synthesis. The expedient total synthesis resulted in unambiguous verification of the predicted stereochemistry for valactamide A.

Discovery of a peripheral 5HT2A antagonist as a clinical candidate for metabolic dysfunction-associated steatohepatitis

Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) is currently the leading cause of chronic liver disease worldwide. Metabolic Dysfunction-Associated Steatohepatitis (MASH), an advanced form of MASLD, can progress to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Based on recent findings by our team that liver 5HT2A knockout male mice suppressed steatosis and reduced fibrosis-related gene expression, we developed a peripheral 5HT2A antagonist, compound 11c for MASH. It shows good in vitro activity, stability, and in vivo pharmacokinetics (PK) in rats and dogs. Compound 11c also shows good in vivo efficacy in a diet-induced obesity (DIO) male mice model and in a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) male mice model, effectively improving histologic features of MASH and fibrosis. According to the tissue distribution study using [14C]-labeled 11c, the compound was determined to be a peripheral 5HT2A antagonist. Collectively, first-in-class compound 11c shows promise as a therapeutic agent for the treatment of MASLD and MASH.

Sudapyridine (WX-081) antibacterial activity against Mycobacterium avium, Mycobacterium abscessus, and Mycobacterium chelonae in vitro and in vivo.

Due to the rapidly increased cases globally, non-tuberculous mycobacteria (NTM) disease has become a significant public health problem. NTM accounted for 11.57% of all mycobacterial isolates in China, with a high detection rate of Mycobacterium abscessus, Mycobacterium avium, and Mycobacterium chelonae during 2000-2019. Treatment of NTM infection is often challenging, as natural resistance to most antibiotics is quite common among different NTM species. Hence, identifying highly active anti-NTM agents is a priority for potent regimen establishment. The pursuit of new drugs to treat multidrug-resistant tuberculosis may also identify some agents with strong activity against NTM. Sudapyridine (WX-081) is a structural analog of bedaquiline (BDQ), which was developed to retain the anti-tuberculosis efficacy but eliminates the severe side effects of BDQ. This study initially evaluated the antimicrobial activity of this novel compound against M. avium, M. abscessus, and M. chelonae in vitro, in macrophages and mice, respectively.

A miniaturized mode-of-action profiling platform enables high throughput characterization of the molecular and cellular dynamics of EZH2 inhibition

The market approval of Tazemetostat (TAZVERIK) for the treatment of follicular lymphoma and epithelioid sarcoma has established “enhancer of zeste homolog 2” (EZH2) as therapeutic target in oncology. Despite their structural similarities and common mode of inhibition, Tazemetostat and other EZH2 inhibitors display differentiated pharmacological profiles based on their target residence time. Here we established high throughput screening methods based on time-resolved fluorescence energy transfer, scintillation proximity and high content analysis microscopy to quantify the biochemical and cellular binding of a chemically diverse collection of EZH2 inhibitors. These assays allowed to further characterize the interplay between EZH2 allosteric modulation by methylated histone tails (H3K27me3) and inhibitor binding, and to evaluate the impact of EZH2’s clinically relevant mutant Y641N on drug target residence times. While all compounds in this study exhibited slower off-rates, those with clinical candidate status display significantly slower target residence times in wild type EZH2 and disease-related mutants. These inhibitors interact in a more entropy-driven fashion and show the most persistent effects in cellular washout and antiproliferative efficacy experiments. Our work provides mechanistic insights for the largest cohort of EZH2 inhibitors reported to date, demonstrating that—among several other binding parameters—target residence time is the best predictor of cellular efficacy.

Discovery of tricyclic PARP7 inhibitors with high potency, selectivity, and oral bioavailability

PARP7 has been recently identified as an effective drug target due to its specific role in tumor generation and immune function recovery. Herin, we report the discovery of compound 8, which contained a tricyclic fused ring, as a highly selective PARP7 inhibitor against other PARPs. In particular, compound 8 strongly inhibits PARP7 with an IC50 of 0.11 nM, and suppresses the proliferation of NCI–H1373 lung cancer cells with an IC50 of 2.5 nM. Compound 8 exhibits a favorable pharmacokinetic profile with a bioavailability of 104 % in mice, and 78 % in dogs. Importantly, daily treatment of 30 mg/kg of 8 induced 81.6 % tumor suppression in NCI–H1373 lung xenograft mice tumor models, which is significantly better than the clinical candidate, RBN-2397. These intriguing features highlight the promising advantages of 8 as an antitumor agent.

Discovery of IRAK4 Inhibitors BAY1834845 (Zabedosertib) and BAY1830839.

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in innate inflammatory processes. Here, we describe the discovery of two clinical candidate IRAK4 inhibitors, BAY1834845 (zabedosertib) and BAY1830839, starting from a high-throughput screening hit derived from Bayer's compound library. By exploiting binding site features distinct to IRAK4 using an in-house docking model, liabilities of the original hit could surprisingly be overcome to confer both candidates with a unique combination of good potency and selectivity. Favorable DMPK profiles and activity in animal inflammation models led to the selection of these two compounds for clinical development in patients.

TAAR1 agonists improve glycemic control, reduce body weight and modulate neurocircuits governing energy balance and feeding

ObjectiveMetabolic Syndrome, which can be induced or exacerbated by current antipsychotic drugs (APDs), is highly prevalent in schizophrenia patients. Recent preclinical and clinical evidence suggest that agonists at trace amine-associated receptor 1 (TAAR1) have potential as a new treatment option for schizophrenia. Intriguingly, preclinical tudies have also identified TAAR1 as a novel regulator of metabolic control. Here we evaluated the effects of three TAAR1 agonists, including the clinical development candidate ulotaront, on body weight, metabolic parameters and modulation of neurocircuits implicated in homeostatic and hedonic feeding. MethodsEffects of TAAR1 agonists (ulotaront, RO5166017 and/or RO5263397) on body weight, food intake and/or metabolic parameters were investigated in rats fed a high-fat diet (HFD) and in a mouse model of diet-induced obesity (DIO). Body weight effects were also determined in a rat and mouse model of olanzapine-, and corticosterone-induced body weight gain, respectively. Glucose tolerance was assessed in lean and diabetic db/db mice and fasting plasma glucose and insulin examined in DIO mice. Effects on gastric emptying were evaluated in lean mice and rats. Drug-induced neurocircuit modulation was evaluated in mice using whole-brain imaging of c-fos protein expression. ResultsTAAR1 agonists improved oral glucose tolerance by inhibiting gastric emptying. Sub-chronic administration of ulotaront in rats fed a HFD produced a dose-dependent reduction in body weight, food intake and liver triglycerides compared to vehicle controls. In addition, a more rapid reversal of olanzapine-induced weight gain and food intake was observed in HFD rats switched to ulotaront or RO5263397 treatment compared to those switched to vehicle. Chronic ulotaront administration also reduced body weight and improved glycemic control in DIO mice, and normalized corticosterone-induced body weight gain in mice. TAAR1 activation increased neuronal activity in discrete homeostatic and hedonic feeding centers located in the dorsal vagal complex and hypothalamus with concurrent activation of several limbic structures. ConclusionThe current data demonstrate that TAAR1 agonists, as a class, not only lack APD-induced metabolic liabilities but can reduce body weight and improve glycemic control in rodent models. The underlying mechanisms likely include TAAR1-mediated peripheral effects on glucose homeostasis and gastric emptying as well as central regulation of energy balance and food intake.

Abstract C047: Improving the efficacy of dual ERK-MAPK and autophagy inhibition as a therapeutic strategy for pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS- and autophagy-dependent growth. We and others recently demonstrated that inhibition of KRAS signaling through targeting the RAF-MEK-ERK kinase cascade resulted in further reliance on autophagy. We found that targeting this increased reliance on autophagy with the autophagy inhibitor hydroxychloroquine (HCQ)/chloroquine (CQ) together with MEK or ERK inhibition (MEKi, ERKi) synergistically blocked PDAC growth. These findings provided rationale for our initiation of Phase I/II clinical trials evaluating the combination of MEKi (binimetinib; NCT04132505) or ERKi (LY3214996; NCT04386057) with HCQ in PDAC. However, a limitation of this approach is that HCQ/CQ are not specific or potent autophagy inhibitors. Thus, we sought to identify a more efficacious autophagy inhibition strategy. To this end, we performed a CRISPR-Cas9 mediated genetic loss-of-function screen in PDAC cells to identify other targetable mediators of autophagy. We identified PIKfyve, a lipid kinase critical for the recycling dynamics of lysosomes, as an essential autophagy-related gene in PDAC cells. PIKfyve inhibition by the clinical candidate inhibitor apilimod resulted in potent reduction of autophagic flux and growth. Additionally, PIKfyve inhibition induced intracellular vacuolization. These PIKfyve-regulated vacuoles stained positive for the lysosomal marker, LAMP1, and exhibited reduced acidity and impaired cargo degradation. Importantly, PIKfyve inhibition prevented the increased autophagic flux we observed when we inhibited MEK with the clinical stage MEKi, mirdametinib. As a result, co-targeting MEK and PIKfyve led to synergistic impairment of PDAC cell proliferation. Similar results were observed following dual inhibition of KRAS and PIKfyve. We found the synergistic growth inhibition was due in part to a substantial induction of apoptosis unique to combination treatment. We then tested the combination of MEKi and PIKfyvei in a panel of patient derived PDAC organoids and observed a robust synergistic reduction in viability, suggesting this may be an efficacious therapeutic strategy for PDAC treatment. Future work includes in vivo studies, currently underway, to determine the efficacy of single agent PIKfyve inhibition, as well as efficacy studies assessing combined MEKi and PIKfyvei in orthotopic mouse models of PDAC. Taken together, these findings implicate PIKfyve as an effective anti-autophagy target when paired with RAS or ERK-MAPK pathway inhibition in pre-clinical models of pancreatic cancer. Citation Format: Jonathan M. DeLiberty, Mallory K. Roach, Noah L. Pieper, Kristina Drizyte-Miller, Elyse G. Schechter, Runying Yang, Channing J. Der, Adrienne D. Cox, Clint A. Stalnecker, Kirsten L. Bryant. Improving the efficacy of dual ERK-MAPK and autophagy inhibition as a therapeutic strategy for pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C047.

Abstract A080: Target genes in pancreatic cancer cells of the pan-quadruplex clinical candidate compound QN-302 revealed by comparative transcriptome profiling

Abstract QN-302 is a tetra-substituted naphthalene derivative designed to have high affinity to quadruplexes in cancer-related genes, which have multiple quadruplex occurrence in their promoter sequences. QN-302 has ca 1 nM in vitro activity against a panel of pancreatic ductal adenocarcinoma (PDAC) cell lines and shows high activity against four distinct PDAC in vivo models, including the KPC genetic model. QN-302 is currently being developed for clinical trials in solid tumors including PDAC. It has also recently been granted orphan drug drug (ODD) status by the FDA. We have previously reported on whole-genome transcriptome studies for QN-302, which show that it selectively targets quadruplex-rich genes in PDAC-related pathways, and thus acts as a pan-quadruplex agent. We have recently extended our analysis to include two closely related analogs of QN-302 as well as data from a gemcitabine-resistant line. QN-302 is consistently ca 10-fold more potent in PDAC cells and in vivo than the second-best analog. RNA-seq whole-cell and comparative bioinformatics analyses have been used to probe these differences. This has resulted in the identification of those target genes that the naphthalene diimide family have in common, many of which are involved in PDAC pathways such as hedgehog, axon guidance, WNT/β-catenin and hippo. It has also been possible to identify a small number of quadruplex target genes that are highly and selectively down-regulated by QN-302, and which are also highly up-regulated in human PDAC. We suggest that these contribute to the exceptional in vivo potency of this compound. Citation Format: Ahmed Ahmed, Tariq Arshad, Maria Roman-Escorza, Dan M. Neidle, Stephen Neidle. Target genes in pancreatic cancer cells of the pan-quadruplex clinical candidate compound QN-302 revealed by comparative transcriptome profiling [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A080.

Improving brain penetration using elacridara P-glycoprotein and BCRP inhibitor

The central nervous system (CNS) is a site for a myriad of disorders and diseases, such as schizophrenia, Alzheimers disease, and Parkinsons disease. Many medications targeting these illnesses remain challenged due to efflux transporters forming a blood-brain barrier (BBB). To combat such challenges, elacridar shown promise at inhibiting such transporters, such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), therefore improving brain penetration. However, as an early clinical candidate, many remain unknown about elacridars pharmacokinetic (PK) properties in the human body. This paper aims to obtain a better understanding of elacridars pharmacokinetic profile and predict the optimal dose and dose interval for its application in the clinic. To begin with, a series of basic PK models were created using fundamental PK equations and the models were fit to elacridar clinical data to obtain elacridar-specific PK parameters. Next, elacridar preclinical PK as well as in vitro inhibition assay were used to determine the therapeutic window. Our final, multiple-dose extravascular PK model reveals that the most convenient and effective dose regimen is 900 mg BID (bis in die; twice per day). With this elacridar PK model, researchers can leverage elacridar human PK to improve clinical outcomes.

Immunogenicity and protective efficacy of a trimeric full-length S protein subunit vaccine for porcine epidemic diarrhea virus

Porcine epidemic diarrhea virus (PEDV) has caused serious economic losses to the pig husbandry worldwide, and the effects of existing commercialized vaccines are suboptimal. Therefore, research to develop an efficacious vaccine for prevention and control of PEDV is essential. In this study, we designed and produced trimerized proteins of full-length PEDV spike (S) protein, S1 subunit, and a tandem of multiple epitopes of S protein using an efficient mammalian expression vector system in HEK 293F cells. The immunogenicity of two commercial adjuvants, M401 and M103, was also evaluated in mice. Enzyme-linked immunosorbent assays demonstrated that all immunized mice generated highly systemic PEDV S-specific IgG and IgA antibodies. Mice in S/M103-immunized group generated the highest neutralizing antibody titer with 1:96. Compared with control group, the subunit vaccines elicited multifunctional CD3+CD4+ and CD3+CD8+ T cells, B220+CD19+ B cells, and CD3-CD49b+ natural killer cells in the spleen. PEDV S/M103 vaccine, which had the best immune effect, was selected for further evaluation in piglets. Immunization with S/M103 vaccine induced high levels of S-specific IgG, IgA, and neutralizing antibodies, and increased the proliferation of peripheral blood mononuclear cells and the expression levels of interferon-γ and interleukin-4 in peripheral blood of piglets. Virus challenge test results showed significantly lower diarrheal index scores and fecal viral loads, and less pathological damage to the intestines in S/M103-immunized piglets than in controls, indicating that S/M103 provides good protection against the virulent virus challenge. Our findings suggest that trimeric PEDV S/M103 has potential as a clinical vaccine candidate.

Design, synthesis and biological evaluation of 6-chloro-quinolin-2-one derivatives as novel FXIa inhibitors

A series of 6-chloro-quinolin-2-one derivatives were designed and synthesized as FXIa inhibitors by exploration of P1, P1 prime and P2 prime groups. Each compound was accessed for inhibitory effect on FXIa and some of them were evaluated in the clotting assay. 14c demonstrated excellent in-vitro potency (FXIa IC50: 15 nM, 2 x aPTT: 6.8 μM) and good in-vivo efficacy (prolonged in-vivo aPTT by more than 1-fold but not PT). Moreover, the pharmacokinetics property of 14c were evaluated following intravenous administration in rats, which indicated that 14c probably will be a clinical candidate for intravenous administration.

A novel antibiotic class targeting the lipopolysaccharide transporter

Carbapenem-resistant Acinetobacter baumannii (CRAB) has emerged as a major global pathogen with limited treatment options1. No new antibiotic chemical class with activity against A. baumannii has reached patients in over 50 years1. Here we report the identification and optimization of tethered macrocyclic peptide (MCP) antibiotics with potent antibacterial activity against CRAB. The mechanism of action of this molecule class involves blocking the transport of bacterial lipopolysaccharide from the inner membrane to its destination on the outer membrane, through inhibition of the LptB2FGC complex. A clinical candidate derived from the MCP class, zosurabalpin (RG6006), effectively treats highly drug-resistant contemporary isolates of CRAB both in vitro and in mouse models of infection, overcoming existing antibiotic resistance mechanisms. This chemical class represents a promising treatment paradigm for patients with invasive infections due to CRAB, for whom current treatment options are inadequate, and additionally identifies LptB2FGC as a tractable target for antimicrobial drug development.

Renalase mediates macrophage-to-fibroblast crosstalk to attenuate pressure overload-induced pathological myocardial fibrosis.

A potential antifibrotic mechanism in pathological myocardial remodeling is the recruitment of beneficial functional subpopulations of macrophages or the transformation of their phenotype. Macrophages are required to activate molecular cascades that regulate fibroblast behavior. Identifying mediators that activate the antifibrotic macrophage phenotype is tantamount to identifying the button that retards pathological remodeling of the myocardium; however, relevant studies are inadequate. Circulating renalase (RNLS) is mainly of renal origin, and cardiac myocytes also secrete it autonomously. Our previous studies revealed that RNLS delivers cell signaling to exert multiple cardiovascular protective effects, including the improvement of myocardial ischemia, and heart failure. Here, we further investigated the potential mechanism by which macrophage phenotypic transformation is targeted by RNLS to mediate stress load-induced myocardial fibrosis. Mice subjected to transverse aortic constriction (TAC) were used as a model of myocardial fibrosis. The co-incubation of macrophages and cardiac fibroblasts was used to study intercellular signaling. The results showed that RNLS co-localized with macrophages and reduced protein expression after cardiac pressure overload. TAC mice exhibited improved cardiac function and alleviated left ventricular fibrosis when exogenous RNLS was administered. Flow sorting showed that RNLS is essential for macrophage polarization towards a restorative phenotype (M2-like), thereby inhibiting myofibroblast activation, as proven by both mouse RAW264.7 and bone marrow-derived macrophage models. Mechanistically, we found that activated protein kinase B is a major pathway by which RNLS promotes M2 polarization in macrophages. RNLS may serve as a prognostic biomarker and a potential clinical candidate for the treatment of myocardial fibrosis.

Structure-guided design of a selective inhibitor of the methyltransferase KMT9 with cellular activity.

Inhibition of epigenetic regulators by small molecules is an attractive strategy for cancer treatment. Recently, we characterised the role of lysine methyltransferase 9 (KMT9) in prostate, lung, and colon cancer. Our observation that the enzymatic activity was required for tumour cell proliferation identified KMT9 as a potential therapeutic target. Here, we report the development of a potent and selective KMT9 inhibitor (compound 4, KMI169) with cellular activity through structure-based drug design. KMI169 functions as a bi-substrate inhibitor targeting the SAM and substrate binding pockets of KMT9 and exhibits high potency, selectivity, and cellular target engagement. KMT9 inhibition selectively downregulates target genes involved incell cycle regulation and impairs proliferation of tumours cells including castration- and enzalutamide-resistant prostate cancer cells. KMI169 represents a valuable tool to probe cellular KMT9 functions and paves the way for the development of clinical candidate inhibitors as therapeutic options to treat malignancies such as therapy-resistant prostate cancer.

Discovery and Characterization of RGH-122, a Potent, Selective, and Orally Bioavailable V1a Receptor Antagonist.

The V1a receptor is a major contributor in mediating the social and emotional effects of arginine-vasopressin (AVP); therefore it represents a promising target in the treatment of several neuropsychiatric conditions. The aim of this research was to design and synthesize novel and selective V1a antagonists with improved in vitro and in vivo profiles. Through optimization and detailed SAR studies, we developed low nanomolar antagonists, and further characterizations led to the discovery of the clinical candidate compound 43 (RGH-122). The CNS activity of the compound was determined in a 3-chamber social preference test of autism in which RGH-122 successfully enhanced social preference with the lowest effective dose of 1.5 mg/kg.

Identification of coumarin derivatives targeting acetylcholinesterase for Alzheimer's disease by field-based 3D-QSAR, pharmacophore model-based virtual screening, molecular docking, MM/GBSA, ADME and MD Simulation study

Alzheimer's disease (AD) leads to gradual memory loss including other compromised cognitive abilities. Acetylcholinesterase (AChE), an important biochemical enzyme from the cholinesterase (ChE) family, is recognized as primary pharmacological target for treating AD. Currently marketed drugs for AD treatment are primarily AChE inhibitors and coumarin derivatives comprising a wide variety of pharmacological activities have proved their efficacy towards AChE inhibition. Ensaculin (KA-672 HCl), a compound that belong to the coumarin family, is a clinical trial candidate for AD treatment. Therefore, a ligand library was prepared with 60 reported coumarin derivatives for field-based 3D-QSAR and pharmacophore modelling. The field-based 3D-QSAR model obtained at partial least square (PLS) factor 7, was the best validated model that predicted activity closer to original activity for each ligand introduced. The contour maps demonstrated spatial distribution of favourable and unfavorable steric, hydrophobic, electrostatic and H-bond donor and acceptor contours around coumarin nucleus. The best pharmacophore model, ADHRR_1 exhibited five essential pharmacophoric features of four different traits for optimum AChE inhibition. Virtual screening through ADHRR_1 accompanied with molecular docking and MM/GBSA identified 10 HITs from a 4,00,000 coumarin derivatives from PubChem database. HITs comprised docking scores ranging from −12.096 kcal/mol to −8.271 kcal/mol and compared with the reference drug Donepezil (-8.271 kcal/mol). ADME properties analysis led into detecting two leads (HIT 1 and HIT 2) among these 10 HITs. Molecular Dynamics Simulation indicated thermodynamic stability of the complex of lead compounds with AChE protein. Finally, thorough survey of the experimental results from 3D-QSAR modelling, pharmacophore modelling and molecular docking interactions led us to develop the lead formula I for future advancements in treating AD through AChE inhibitors.

Harnessing benzotriazole as a sustainable ligand in metal-catalyzed coupling reactions.

Coupling reactions play a crucial role in drug development enabling the rapid expansion of structure-activity relationships (SARs) during drug discovery programs to identify a clinical candidate and simplify subsequent drug development processes. In particular, their relevance in clinical medicine and drug discovery has increased significantly in the last two decades. To facilitate these metal-catalyzed coupling reactions, suitably designed ligands are necessary and from the industrial point of view, sustainable and cost-effective ligands are of current need. Benzotriazole, a non-toxic, thermally stable, and inexpensive bidentate ligand, exhibits strong electron donating and electron accepting properties, along with excellent solubility in various organic solvents. It has been extensively explored as a synthetic auxiliary in the past; in recent years, benzotriazole and its derivatives have been used as ligands in metal-catalyzed coupling reactions. The facile construction of carbon-carbon and carbon-heteroatom bonds in the presence of versatile benzotriazole ligands makes it an indispensable ligand for catalytic transformations. The present feature article mainly emphasizes the advances in the utilization of benzotriazole as a ligand in a diverse range of C-C, C-N, C-O, and C-S coupling reactions.

Optimization of Selectivity and Pharmacokinetic Properties of Salt-Inducible Kinase Inhibitors that Led to the Discovery of Pan-SIK Inhibitor GLPG3312.

Salt-inducible kinases (SIKs) SIK1, SIK2, and SIK3 are serine/threonine kinases and form a subfamily of the protein kinase AMP-activated protein kinase (AMPK) family. Inhibition of SIKs in stimulated innate immune cells and mouse models has been associated with a dual mechanism of action consisting of a reduction of pro-inflammatory cytokines and an increase of immunoregulatory cytokine production, suggesting a therapeutic potential for inflammatory diseases. Following a high-throughput screening campaign, subsequent hit to lead optimization through synthesis, structure-activity relationship, kinome selectivity, and pharmacokinetic investigations led to the discovery of clinical candidate GLPG3312 (compound 28), a potent and selective pan-SIK inhibitor (IC50: 2.0 nM for SIK1, 0.7 nM for SIK2, and 0.6 nM for SIK3). Characterization of the first human SIK3 crystal structure provided an understanding of the binding mode and kinome selectivity of the chemical series. GLPG3312 demonstrated both anti-inflammatory and immunoregulatory activities in vitro in human primary myeloid cells and in vivo in mouse models.

Targeting Dysregulated Neuroinflammation in CNS Disorders

A critical mechanism driving neurodegenerative disease progression is dysregulated brain inflammation. Neuroinflammatory repair processes are fundamental to CNS homeostasis, but inflammation that is inefficient, excessive, or prolonged can contribute to neurodegeneration. A specific aspect of neuroinflammation, stressor-induced proinflammatory cytokine overproduction from abnormally activated glia, has been linked to subsequent neurological damage and cognitive deficits in both acute and chronic CNS disorders. This cytokine/synaptic dysfunction axis (Fig. 1) has emerged as an attractive druggable target for neurodegenerative diseases. However, despite advances in our understanding of these molecular neuroinflammatory mechanisms, approved therapeutics that target this pathological process are lacking. To address this need, we used an integrated, recursive drug discovery platform (Fig. 2) to develop brain-penetrant, small molecule drug candidates that normalize cytokine levels and exhibit efficacy at low doses in multiple animal models of CNS disorders. The molecule design stage began with a pyridazine chemotype that was diversified using established chemistries to create a library of compounds. Compounds were screened for concentration dependence and selectivity using either a single molecular target approach or the historically successful functional/pathway-based approach. A pharmacology assessment stage was incorporated to assess compounds for drug-like properties, such as chemical and metabolic stability, CNS penetrance, and potential for safety. Prioritized compounds were then tested in relevant animal models, and the most promising drug candidates taken into formal IND-enabling preclinical testing. The platform yielded three clinical drug candidates, MW150, MW151, and MW189, that are all in human phase 1b/2a clinical trials.