Journal of Medicinal Chemistry

Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1), encoded by the gene INPP5D, is a lipid phosphatase that negatively regulates immune receptor signaling in hematopoietic cells and microglia. Here, we describe a pyridyl-pyrazole-piperidine scaffold and the lead compound 3-((2-chlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine (32), which demonstrates SHIP1 target engagement, brain exposure, and evidence of a central pharmacodynamic response in vivo. Structure-activity relationship studies, guided by biochemical and cellular assays using multiple human and murine protein constructs and cells, identified SHIP1-active ligands. A thermal shift assay using full-length SHIP1 was used to assess compounds for cellular target engagement, while studies in IL-4 conditioned THP-1 cells was used to demonstrate changes in downstream AKT signaling. Targeted lipidomics revealed changes in the overall phosphoinositide pool consistent with SHIP1 target engagement and reduction of phospho-AKT levels. In a protein-lipid overlay assay, compound 32 induced changes in the relative association of SHIP1 with multiple phosphatidylinositols on a membrane surface. In high-content cellular imaging assays, compound 32 enhanced the uptake of myelin/membrane debris and fibrillar amyloid by primary murine microglia, phenocopying a genetic model with reduced SHIP1 expression. Finally, oral administration of compound 32 resulted in brain exposure sufficient to alter gene expression and reduce IL-1{beta} levels as pharmacodynamic markers of microglial activation and neuroinflammation in an amyloidosis mouse model of Alzheimers disease. Collectively, these results define a scaffold with SHIP1 target engagement, CNS exposure, and in vivo activity, providing a foundation for the optimization of brain-penetrant SHIP1 ligands suitable for further mechanistic studies and therapeutic development for the treatment of Alzheimers disease.

The pivotal role of receptor-interacting protein kinase 1 (RIPK1) as a scaffold protein in mediating tumor resistance to immune checkpoint inhibitors (ICBs) underscores the significance of pharmacological RIPK1 degradation as a therapeutic strategy to enhance antitumor immunity. In this study, we present the design, synthesis, and evaluation of a novel series of RIPK1 degraders, derived from the optimization of the previously identified compound LD4172. Through systematic refinement of the linker, exit vector of the RIPK1 warhead, and the VHL ligand portion, we identified compound LD5097 (24b), which exhibited potent RIPK1 degradation activity across various cancer cell lines, with DC50 values of single digit nanomolar range and inducing more than 95% maximum degradation. Remarkably, LD5097 (24b) induced rapid and complete degradation of RIPK1 within 2 hours of treatment and enhanced TNF-mediated apoptosis in Jurkat cells. Furthermore, proteomic profiling unveiled the high selectivity of LD5097 (24b) in degrading RIPK1. LD5097 (24b) exhibited excellent metabolic stability and pharmacokinetic properties, characterized by low clearance, an extended half-life, and high plasma drug concentrations. Notably, a single administration of LD5097 (24b) effectively reduced RIPK1 protein levels in Jurkat xenograft tumor tissues in mice at both 6- and 24-hour post-administration. These findings underscore LD5097 (24b) as a promising RIPK1 degrader candidate, offering potent activity, favorable pharmaco-kinetic profiles, and notable pharmacodynamic effects, thereby holding significant promise in cancer immunology therapies.

Receptor-interacting protein kinase 1 (RIPK1) is a critical regulator of programmed cell death and is implicated in various pathological conditions, particularly in mediating tumor resistance to immune checkpoint inhibitors (ICBs). In this study, we have pioneered the development of a novel cereblon (CRBN)-recruiting RIPK1 degrader, LD5095, through systematic optimization of linker and CRBN ligand portion. LD5095 demonstrates potent and selective RIPK1 degradation across cell lines, with rapid kinetics and sustained degradation over 72h post-washout. Functionally, RIPK1 degradation by LD5095 significantly sensitized Jurkat cells to TNF-induced apoptosis. Furthermore, LD5095 exhibited favorable pharmacokinetics, including metabolic stability and an extended half-life. Strikingly, in vivo, a single dose of LD5095 achieved durable RIPK1 degradation in xenograft tumors over 6 days. These findings underscore the potential of LD5095 as a chemical probe for studying RIPK1 biology and a promising candidate for cancer treatment.

Chitinase-3-like protein 1 (CHI3L1) is a key driver of glioblastoma (GBM) progression and an emerging therapeutic target. Building on the CHI3L1 inhibitor 11g, we optimized the scaffold through medicinal chemistry to assess structure-property relationships and improve pharmacokinetics. Using microscale thermophoresis (MST) and computational studies, we validated 10p, which exhibits a CHI31 binding affinity (Kd) of 13.22 {micro}M. Notably, 10p overcomes previous developability hurdles by achieving a kinetic solubility of 758 {micro}M, a five-fold improvement over 11g. It further demonstrates high metabolic stability across species and no hERG inhibition. In 3D GBM spheroid models, 10p significantly reduced tumor viability, mass, and migration, exceeding the efficacy of prior analogues. Collectively, these findings establish 10p as a potent CHI3L1 inhibitor with a superior pharmacokinetic profile and robust functional activity, marking it as a promising candidate for further GBM drug development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/702243v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@dff733org.highwire.dtl.DTLVardef@1de4e56org.highwire.dtl.DTLVardef@1e910dcorg.highwire.dtl.DTLVardef@51e9d4_HPS_FORMAT_FIGEXP M_FIG C_FIG

GPR183 is an oxysterol-sensing GPCR predominantly expressed in lymphoid organs and tissues. Activation of the receptor by oxysterol 7,25-OHC leads to Gi protein-mediated signaling as well as {beta}-arrestin2 recruitment. GPR183/oxysterol signaling modulates localization of lymphoid cells, consequently the receptor is associated with several inflammation-associated diseases and is an interesting potential drug target. Previously, we reported the discovery of moderately potent G protein-biased partial agonists for GPR183 from a virtual screening based on the scaffold of the antagonist NIBR189. Herein, we present the detailed structure-activity investigations and optimizations, which led to the identification of full agonists for GPR183 with complete bias for Gi protein signaling and low nanomolar potency, including 63 (TUG-2604) with potency and efficacy similar to 7,25-OHC. Notably, 63 was unable to induce migration of human dendritic cells but inhibited migration induced by 7,25-OHC. This compound will be valuable for further explorations of the signaling-specific function and drug target potential of GPR183.

Triggering receptor expressed on myeloid cells-2 (TREM2) is a key immune receptor in the central nervous system that regulates microglial phagocytosis, survival, and neuroinflammatory responses. TRME2 variants have been established as genetic risk factors for Alzheimers disease (AD). However, the therapeutic development of TREM2 modulators has been limited to antibody-based approaches that face limitations in blood-brain barrier penetration and manufacturing scalability. Furthermore, there are no FDA approved TREM2 therapeutics available to date marking an unmet therapeutic gap. Herein, we report the identification of the first TREM2 small molecule submicromolar binders as a result of optimizing compound 4a to yield S9 with TREM2 binding affinity of 0.95 {micro}M. S9 demonstrated robust TREM2 agonism in cellular assays where it induced proximal Syk phosphorylation, activated downstream NFAT transcriptional signaling, enhanced APOE internalization and microglial phagocytic capacity. Pharmacokinetic profiling of the optimized hits revealed S9 to exhibit improved drug-likeness compared to 4a with 7-fold enhanced aqueous solubility, superior metabolic stability, reduced intrinsic clearance and a 9-fold improved hERG safety margin. Functional validation in human iPSC-derived microglia confirmed that S9 suppresses amyloid-beta (A{beta})-induced IL-1{beta} secretion through a TREM2-dependent mechanism. In human neuron-microglia co-culture models exposed to amyloid stress, S9 treatment preserved synaptic integrity as measured by PSD95 expression that indicates promising neuroprotective activity. Together, these findings establish S9 as a first-TREM2 submicromolar small molecule TREM2 agonist which is orally bioavailable with favorable pharmacokinetic properties and promising therapeutic potential for the treatment of Alzheimers disease.

The emergence of mosquito-borne alphaviruses that cause chronic arthritis or encephalitis underscores the urgent need for broad-spectrum antiviral therapeutics. The viral nsP2 cysteine protease, which is essential for alphavirus replication, is a promising antiviral target. Vinyl sulfone-based inhibitors, such as RA-2034, potently inhibit nsP2 protease but suffer from glutathione reactivity and species-dependent systemic clearance catalyzed by glutathione S-transferase. To address these liabilities, we explored alternative electrophilic warheads and identified reverse amide inhibitors bearing N-alkyl sulfamate warheads with improved biochemical and antiviral profiles. N-methyl sulfamate acetamide 5 emerged as a lead compound with potency against both New and Old World alphaviruses, low GSH reactivity, and high proteome-wide selectivity. Despite its promising antialphaviral activity, 5 exhibited rapid clearance due to hepatic glucuronidation. Structure-activity studies revealed modifications that improve metabolic stability while retaining antiviral activity. These findings introduce sulfamate acetamides as a new class of covalent nsP2 protease inhibitors and advance the discovery of direct acting pan-alphavirus drugs.

Targeting viral macrodomains (Mac) has emerged as a promising strategy for antiviral drug development, especially after the outbreak of COVID-19 that claimed millions of lives worldwide. Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Mac1 inhibitors have been reported in the past few years. In the present work, we converted GS-441524 (IC50 of [~]10 M for SARS-CoV-2 Mac1) to KP-S54 (18c), a potent inhibitor of both SARS-CoV-2 Mac1 (IC50: 44 nM) and Middle East respiratory syndrome coronavirus (MERS-CoV) Mac1 (IC50: 91 nM) through an iterative direct-to-biology approach. This approach leverages efficient amide-coupling reaction and the mix-and-read fluorescence polarization (FP) assays where reaction mixtures could be screened directly without purification. Cocrystal structure of a selected derivative (12p) binding to SARS-CoV-2 Mac1 revealed the binding mode, which will guide future drug development against viral macrodomains.

The p21-activated kinases (PAKs) are a group of serine-threonine kinases central to multiple signaling pathways that govern cell survival and proliferation. Aberrant activity of PAK1, the most well characterized member of the PAK family, drives progression of several malignancies and brain disorders, including Alzheimers disease and neurodevelopmental disorders. Despite growing interest in PAK1 as a drug target for these diseases, there is no assay to evaluate the intracellular target engagement of PAK1 inhibitors. To address this need, we developed first-in-class NanoBRET assays for wild-type PAK1 and a neurodevelopmental disorder-causing gain-of-function PAK1 mutant. Furthermore, we executed our novel PAK1 NanoBRET assay to evaluate target engagement of PAK1 inhibitors in primary hippocampal neurons. To the best of our knowledge, this is the first demonstration of a NanoBRET cellular target engagement assay in primary neurons, thereby increasing the relevance of our work by confirming PAK1 inhibitor binding to the aberrant form of the protein in primary neurons.

The use of covalent warheads targeting the catalytic cysteine has been a cornerstone in coronavirus main protease (Mpro) inhibitor development, where various electrophilic motifs have been used including aldehydes, nitriles, ketoamides, and hydroxymethyl ketones (HMKs). Recent efforts have been mostly centered around nitrile warheads, given the success of compounds like Nirmatrelvir and Ensitrelvir in the clinic. However, finding and advancing alternative chemotypes with differentiating chemical and pharmacological profiles is essential for future pandemic preparedness. Among such alternatives, HMKs hold special interest because they balance reduced intrinsic electrophilicity with an excellent selectivity profile. Nevertheless, early HMK-based compounds, such as the clinical-stage Mpro inhibitor PF-00835231, suffered from poor oral bioavailability and therefore required intravenous administration, with or without prodrug derivatization of the hydroxyl group. Here, we describe our efforts in advancing the HMK field via the discovery of mCMX110, a lead that has superior potency, increased unbound exposure in vivo, and favorable oral bioavailability in preclinical studies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/725542v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@abe1c9org.highwire.dtl.DTLVardef@746a08org.highwire.dtl.DTLVardef@dd5861org.highwire.dtl.DTLVardef@1d572c7_HPS_FORMAT_FIGEXP M_FIG C_FIG

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/707154v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1e81c3borg.highwire.dtl.DTLVardef@1958c6borg.highwire.dtl.DTLVardef@1360015org.highwire.dtl.DTLVardef@3f9388_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG Protein-protein interactions (PPIs) mediated by extracellular ligands remain challenging targets for small molecule intervention due to their large and dynamic interfaces. The interaction between SLIT2 and its receptor ROBO1 plays a critical role in cell migration and tumor progression, yet remains largely unexplored. Here, we report the discovery and optimization of small molecule inhibitors of the SLIT2/ROBO1 interaction enabled by DNA-encoded library (DEL) screening. Affinity selection against SLIT2 identified four structurally diverse hit compounds, which were subsequently validated using orthogonal biophysical assays. Among these, one hit exhibited measurable SLIT2 binding and functional inhibition of the SLIT2/ROBO1 interaction in a time-resolved FRET assay. Guided by physicochemical considerations, a solubility-optimized analog was designed, resulting in a [~]50-fold improvement in binding affinity and an [~]9-fold enhancement in functional potency. Molecular dynamics simulations and induced-fit docking revealed a stable binding mode within the SLIT2 LRR2 domain and suggested that a benzothiophene substituent was dispensable for target engagement. Fragment-based experimental validation confirmed this prediction, leading to the identification of a minimal azaindole-based pharmacophore that retained nanomolar binding affinity. Collectively, this study demonstrates how DEL-enabled hit discovery combined with rational optimization and fragment deconstruction can yield potent small molecule modulators of a challenging extracellular PPI, providing a foundation for further development of SLIT2/ROBO1 pathway inhibitors.

Human Neutrophil Elastase (HNE) plays a vital role in several inflammatory diseases, however its role in the tumour microenvironment and the potential in cancer treatment is still unrevealed. Considering the potential of {beta}-lactams as HNE inhibitors, the present work describes the development of a synthetic strategy to obtain two different types (Type I and Type II) of quenched activity-based probes (qABPs), using a {beta}-lactam ring as a warhead and BODIPY-FL as a fluorophore. The two types differ in mechanism and relative position between the fluorophore and the quencher moiety. The qABPs synthesized presented IC50 values against HNE lower than 0.5 {micro}M, and high selectivity compared with homologous serine hydrolases. Type II qABPs showed a more efficient turn-on mechanism, and selectively targeted HNE in different cell lysates. The qABP 22 was internalized in U937 cells and in human neutrophils and successfully targeted HNE in both.

Previously we identified a non-nucleotide tricyclic agonist BDW568 that activates human STING (stimulator of interferon genes) gene variant containing A230 in a human monocyte cell line (THP-1). STINGA230 alleles, including HAQ and AQ, are less common STING variants in human population. To further characterize the mechanism of BDW568, we obtained the crystal structure of the C-terminal domain of STINGA230 complexed with BDW-OH (active metabolite of BDW568) at 1.95 [A] resolution and found the planar tricyclic structure in BDW-OH dimerizes in the STING binding pocket and mimics the two nucleobases of the endogenous STING ligand 2,3-cGAMP. This binding mode also resembles a known synthetic ligand of human STING, MSA-2, but not another tricyclic mouse STING agonist DMXAA. Structure-activity-relationship (SAR) studies revealed that all three heterocycles in BDW568 and the S-acetate side chain are critical for retaining the compounds activity. BDW568 could robustly activate the STING pathway in human primary peripheral blood mononuclear cells (PBMCs) with STINGA230 genotype from healthy individuals. We also observed BDW568 could robustly activate type I interferon signaling in purified human primary macrophages that were transduced with lentivirus expressing STINGA230, suggesting its potential use to selectively activate genetically engineered macrophages in macrophage-based approaches, such as chimeric antigen receptor (CAR)-macrophage immunotherapies.

Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide (Pom) bind to cereblon (CRBN) and trigger proteasomal degradation of neo-substrates IKZF1/3 leading to multiple myeloma (MM) cell apoptosis. Pomalidomide (Pom) also binds albeit weakly to p53-related protein kinase (PRPK, aka TP53RK), an understudied kinase reported to be associated with poor prognosis in MM patients. Here, we developed a series of IMiDs based on Pom and conducted a structure-activity relationship (SAR) study to identify a potent and selective PRPK binder. Structural analysis showed that IMiDs bind PRPK in a fundamentally different way from CRBN, and suggested specific derivatization to improve affinity. We employed a structure-guided strategy to develop compound TXM-02-118, which exhibited nanomolar affinityfor PRPK in binding assays, and showed high selectivity for PRPK over CRBN. Overall, the work represents an initial effort to develop tool compounds for studying PRPK. Moreover, it illustrates how a single class of molecules can use different recognition elements to bind diverse targets using fundamentally different binding poses. This has broad implications for chemical probe and lead compound selectivity profiling, and argues for more wide-spread use of global proteomics or similar methodologies.

The development of a small library of 4-anilinoquinolines led to the identification of 7-iodo-N-(3,4,5-trimethoxyphenyl)quinolin-4-amine 16 as a potent inhibitor of Protein Kinase Novel 3 (PKN3) with an IC50 of 1.3 M in cells. Compound 16 presents a useful potential tool compound to study the biology of PKN3 including links to pancreatic and prostate cancer, along with T-cell acute lymphoblastic leukemia. These compounds may be useful tools to explore the therapeutic potential of PKN3 inhibition in prevention of a broad range of infectious and systemic diseases.

Blockade of the TGF{beta} signalling pathway has emerged from preclinical studies as a potential treatment to enhance the efficacy of immune checkpoint inhibition in advanced colorectal cancer (CRC) and several other types of cancer. However, clinical translation of first-generation inhibitors has known little success. Here, we report the synthesis and characterization of HYL001, a potent inhibitor of TGF{beta} receptor 1 (ALK5), that is approximately 9 times more efficacious than the structurally related compound galunisertib, while maintaining a favourable safety profile. HYL001 in combination with immune checkpoint blockade (anti-PD1) eradicates liver metastases generated in mice by microsatellite stable, aggressive colorectal cancer tumours at doses where galunisertib is ineffective. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/593510v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1909963org.highwire.dtl.DTLVardef@4644d2org.highwire.dtl.DTLVardef@1506d80org.highwire.dtl.DTLVardef@14504b7_HPS_FORMAT_FIGEXP M_FIG C_FIG

USP5 is a deubiquitinase that has been implicated in a range of diseases, including cancer, but no USP5-targeting chemical probe has been reported to date. Here, we present the progression of a chemical series that occupies the C-terminal ubiquitin-binding site of a poorly characterized zinc-finger ubiquitin binding domain (ZnF-UBD) of USP5 and allosterically inhibits the catalytic activity of the enzyme. Systematic exploration of the structure-activity relationship, complemented with crystallographic characterization of the ZnF-UBD bound to multiple ligands, led to the identification of 64, which binds to the USP5 ZnF-UBD with a KD of 2.8 {micro}M. 64 is selective over the structurally similar ZnF-UBD domain of HDAC6 and inhibits USP5 catalytic activity in vitro with an IC50 of 26 {micro}M. This study provides a chemical and structural framework for the discovery of a chemical probe to delineate USP5 function in cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/444542v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@3fc7bcorg.highwire.dtl.DTLVardef@15233e0org.highwire.dtl.DTLVardef@1cbf34corg.highwire.dtl.DTLVardef@d237d0_HPS_FORMAT_FIGEXP M_FIG Table of Contents Graphic C_FIG

Compared to most ATP-site kinase inhibitors, small molecules that target an allosteric pocket have the potential for improved selectivity due to the often observed lower structural similarity at these distal sites. Despite their promise, relatively few examples of structurally confirmed, high-affinity allosteric kinase inhibitors exist. Cyclin-dependent kinase 2 (CDK2) is a target for many therapeutic indications, including non-hormonal contraception.1 However, an inhibitor against this kinase with exquisite selectivity has not reached the market because of the structural similarity between CDKs.1-2 In this paper, we describe the development and mechanism of action of new type III inhibitors that bind CDK2 with nanomolar affinity, making them the highest affinity, structurally confirmed allosteric CDK inhibitors reported. Notably, these anthranilic acid inhibitors exhibit a strong negative cooperative relationship with cyclin binding, which remains an underexplored mechanism for CDK2 inhibition. Furthermore, the binding profile of these compounds in both biophysical and cellular assays demonstrate the promise of this series for further development into a therapeutic selective for CDK2 over highly similar kinases like CDK1. The potential of these inhibitors as efficacious contraceptive agents is seen by incubation with mouse testicular explants, where they recapitulate Cdk2-/- and Spdya-/- phenotypes.

Protein Kinase Novel 3 (PKN3) understudied kinase with a diverse array of biological functions that are yet to be fully defined. Here, we report the design and development of a novel advanced functional chemical tool inhibitor for PKN3. A pyridyl imidazole series has been synthesized and evaluated against PKN3 in vitro and in cells. These efforts led to the discovery of 6e (URS03-06), a submicromolar cell active functional inhibitor with a narrow kinome spectrum, to enable the elucidation and interrogation of PKN3 cellular biology.

Brutons Tyrosine Kinase (BTK) is a validated target for haematological malignancies, with numerous FDA approved inhibitors on the market. Current therapies target the highly conserved ATP binding site and hence limit the therapeutic index given the sites highly conserved nature across the kinome. We explore a novel approach for BTK inhibition, by targeting the PH domain-mediated membrane recruitment and activation of BTK. We have identified a fragment which covalently labels a lysine in the inositol phosphate (PIP3) binding site. Fragment growth and an extensive structure-binding relationship study uncovered 27 crystal structures and a best-in-class analog, 24. Evaluation of pKa values of the targeted lysine in BTK and other PH domains suggests this as a more general approach to PH domain inhibition.