Journal of Medicinal Chemistry

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.

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.

The classical estrogen receptor (ER) is a steroid hormone receptor known for its role in both health and disease. ER, like its counterpart ER{beta}, acts as a receptor for its endogenous ligand 17{beta}-estradiol (E2) and is capable of binding a diverse set of natural and synthetic small molecules. Several screening campaigns have identified various ER ligands, based on the oxabicyclo[3.3.1]-nonene scaffold, that act as ER agonists. One such campaign extensively characterized an oxabicyclic ER agonist, termed AB-1. Herein, we report the synthesis of a series of analogs based on the scaffold of AB-1 (herein termed 1), focusing on the enhancement of their binding to ER and functional potencies in ER mediated assays. We identify a new substituted compound 9 that achieves subnanomolar potencies for ER binding (IC50= 0.45 nM) and ER dependent transcription (EC50= 0.37 nM) and proliferation (EC50= 0.03 nM), with potencies approaching those of E2. The high potency of 9 provides a promising scaffold for the potential development of new ER-targeted therapeutics.

Fatty acid synthase (FASN) is the sole enzyme capable of lipogenesis in cells and is upregulated in many cancers. Herein we report the discovery and development of SBP-5917 (78S), a novel, reversible, small-molecule probe which targets the thioesterase (TE) domain of FASN. SBP-5917 and other compounds in the series possess IC50 < 1 M and < 5 M for inhibiting recombinantly purified FASN-TE and cellular FASN respectively. Co-crystal structures of our inhibitors bound to the FASN-TE domain show that they are positioned in the substrate tunnel of the enzyme. Biochemical and biophysical analyses quantify the strength of compound binding and demonstrate their selectivity for FASN-TE. Preliminary pharmacokinetic studies indicate promising in vivo potency for many of the compounds in the series. SBP-5917 is the first structurally validated, reversible inhibitor of FASN-TE with demonstrated cellular target engagement and can serve as a chemical probe to investigate FASN as a therapeutic target.

Tunicamycins are potent inhibitors of dolichyl-phosphate N-acetylglucosamine phosphotransferase (DPAGT1) but are unsuitable for therapeutic development due to non-selective cytotoxicity, acid-labile glycosidic linkages, and poor physicochemical properties. Although prior structural modifications reduced the promiscuous toxicity of tunicamycins, the intrinsic 11'-{beta}-1''- trehalose-type glycosidic linkage remains chemically unstable, limiting biological durability. Here, we report a rationally engineered scaffold-stabilization strategy in which the acid-labile linkage is replaced with a chemically robust cyclitol framework, enabling the concise synthesis of chemically stable and water-soluble tunicamycin analogues in only 12 synthetic steps. From this platform, TM-Cy-TBPA (4) was identified as a lead DPAGT1 inhibitor that potently suppresses the proliferation of breast cancer cells by inducing G2-phase arrest followed by apoptosis, while exhibiting minimal cytotoxicity toward nontransformed cells. The compound shows improved solubility, and favorable pharmacokinetic exposure. These results establish tunicamycin cyclitol analogues as a structurally distinct class of selective DPAGT1-targeted anticancer agents and demonstrate that stabilization of the glycosidic linkage is an effective strategy for enhancing pharmacological selectivity, improving in vivo performance, and simplifying the synthetic route.

CC10137 is a first-in-class peripherally restricted 2A-adrenergic receptor (2AAR) agonist with broad-spectrum analgesic efficacy and a favorable safety profile. Systematic exploration of the chemical space surrounding first-in-class leads is important for defining series boundaries and guiding continued optimization, but conventional analogue-by-analogue medicinal chemistry samples only a small fraction of the accessible structural space. Here, we used a scaffold-seeded enumeration strategy to expand the chemical space surrounding CC10137 from four SAR-informed seed compounds comprising CC10137 and three closely related structural variants. Application of predefined medicinal chemistry transformation rules in StarDrop generated a virtual library of 16,601,163 unique structures. Morgan fingerprint-based principal component analysis indicated that the library occupied a highly multidimensional structural space involving variation in scaffold substitution, peripheral functional groups, and side-chain composition. A retrospective comparison set of 43 compounds independently designed and experimentally characterized in the earlier CC10137 program represented only approximately 0.00026% of the 16.6-million-member library, yet all 43 were recovered as exact structural matches. Three compounds selected directly from the virtual library retained 2AAR binding affinity and agonist potency below 25 nM. Five representative compounds further showed significant anti-allodynic effects in the in vivo spared nerve injury model, with inhibition rates ranging from 39.3% to 55.7%. These findings support scaffold-seeded computational enumeration as a practical strategy for systematic chemical-space mapping around a first-in-class lead and for identifying additional pharmacologically active structural regions for further optimization.

Modulating autophagy has emerged as a potential strategy for treating age-related diseases. However, commonly used pharmacological approaches to induce autophagy, particularly inhibition of mechanistic target of rapamycin complex 1 (mTORC1), can be associated with adverse effects, including immunosuppression and insulin resistance. This has prompted interest in autophagy modulators that act without directly inhibiting mTORC1. 2,5-Diketopiperazines (DKPs) are bioactive cyclic dipeptide scaffolds with diverse biological activities. However, systematic evaluation of their structure-activity relationships has been hindered by racemization during conventional chemical synthesis, leaving the contribution of stereochemistry to autophagy regulation poorly understood. Here, we used a stereoselective one-pot chemoenzymatic synthesis based on the adenylation domain of tyrocidine synthetase A to generate a DKP library with defined stereochemistry. Phenotypic screening in Caco-2 cells stably expressing the GFP-LC3-RFP autophagic flux probe identified four DKPs that increased autophagic flux: c(DW-DP), c(DW-LP), c(DF-DP), and c(DM-LP). Structure-activity analysis revealed stereochemistry-dependent effects associated with amino acid side-chain properties: D-configured residues were favored among DKPs containing aromatic amino acids or methionine, whereas L-configured residues were favored among those containing branched-chain amino acids. Substitution of the proline residue further altered activity, with glycine substitution tending to increase autophagic flux in some DKP scaffolds. Importantly, the active DKPs did not detectably reduce the phosphorylation of the mTORC1 downstream targets p70 S6K and 4EBP1, indicating that their autophagy-inducing effects do not require detectable suppression of canonical mTORC1 signaling. These findings establish stereochemically defined DKPs as candidate scaffolds for the development of autophagy inducers that act through mechanisms distinct from direct mTORC1 inhibition.

Rv1625c/Cya has emerged as a promising target for the development of treatment-shortening therapies for tuberculosis. Screening of an Enamine compound library identified sBQQ004 as an initial hit, and rapid hit optimization led to compound 1, which was subsequently confirmed as an Rv1625c/Cya agonist. Structure activity relationship studies identified lead compound 25 with potent activity against Mycobacterium tuberculosis H37Rv under cholesterol-dependent growth conditions (MIC = 0.27 uM) and strong intramacrophage activity (EC50 = 0.079 uM). Compounds 1 and 25 showed oral bioavailabilities of 84.5% and 51.9% in mice, respectively. Repeat BID dosing of compound 1 resulted in a dose- and time-dependent decrease in systemic exposure. Despite this pharmacokinetic limitation, the potency and overall profile of this chemotype encouraged continued optimization.

Excessive osteoclast activity contributes to pathological bone loss in osteoporosis, rheumatoid arthritis, and osteolytic malignancies. The effects of small catechol derivatives on receptor activator of nuclear factor-{kappa}B ligand (RANKL)-induced osteoclastogenesis remain poorly understood. This study investigated the effects of 4-methylcatechol (4-MC) on RANKL-induced NF-{kappa}B activation and osteoclast differentiation. 4-MC reduced RANKL-induced NF-{kappa}B luciferase activity in HEK-293T/RANK cells. 4-MC also suppressed RANKL-induced TRAP activity in RAW264.7 cells in a concentration-dependent manner and reduced the number of TRAP-positive multinucleated osteoclasts, without affecting cell viability. Molecular docking predicted non-covalent binding of 4-MC within the ATP-binding hinge region of IKK{beta} (PDB: 4KIK), forming a close polar contact with Glu97, predicted hydrogen bonds with Cys99, and a hydrophobic contact with Ile165, within the pocket occupied by the co-crystallized inhibitor K252a. Covalent docking predicted that the oxidized quinone form of 4-MC engages Cys179 in the IKK{beta} activation loop. Quantum chemical calculations confirmed a markedly higher electrophilicity index for the oxidized quinone than for the parent catechol, supporting this mechanism. In silico ADMET profiling indicated favorable drug-likeness and safety. These findings identify IKK{beta} as a plausible molecular target of 4-MC through both non-covalent and covalent mechanisms. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/741661v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@35a0d3org.highwire.dtl.DTLVardef@d19458org.highwire.dtl.DTLVardef@1623fadorg.highwire.dtl.DTLVardef@1429e8b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Isocitrate dehydrogenase 1 (IDH1) catalyses the interconversion of isocitrate and -ketoglutarate and is overexpressed in several cancers, supporting tumour survival and treatment resistance. Inhibitors targeting the oncogenic mutant forms of IDH1 also inhibit the wild-type (WT) protein, but lose substantial potency due to greater competition with native substrates (isocitrate and Mg2+). We hypothesised that a covalent inhibitor may be more effective at overcoming the higher substrate affinity of wild-type IDH1. Here, a covalent fragment-based approach was used to develop IDH1-C269 selective inhibitors which prevent the key regulatory segment from forming an -helix required for catalytic competency. Following the identification of C269-selective fragments, we used X-ray crystallography to characterise IDH1-fragment bound complexes and guide structure-based design efforts. Inspired by the unexpected detection of bound isocitrate molecule in an X-ray crystal structure, fragment expansion yielded a series of compounds exploiting both an adjacent site and a unique water-mediated hydrogen bonding network. This series achieved strong potency (reaching IC50 = 47.5 nM, a 155-fold improvement in potency relative to the original fragment), retained activity in the presence of competing Mg2+, showed selectivity against IDH2 and reduced NADPH concentrations in a relevant PDAC cell model. Together, these findings present a mechanistic rationale for the covalent targeting of wild-type IDH1 and provide structurally validated inhibitors for further development.

Cereblon (CRBN) is widely used in targeted protein degradation, but its ligand space has remained dominated by a narrow set of cyclic imide chemotypes. Here, we show that the accessible CRBN ligand space extends substantially beyond this canonical space. A high-throughput screen of > 40,000 compounds, followed by orthogonal biophysical validation, X-ray crystallography and SAR analyses, identified several chemically distinct ligand classes. These include linear acetyl-based motifs, a phthalide-derived scaffold, steroidal compounds, and a range of bicyclic ligands. They engage CRBN through distinct recognition modes, several of which deviate from the canonical hydrogen-bonding pattern. Steroidal scaffolds were particularly notable: cortisone binds the human CRBN thalidomide-binding domain with an affinity comparable to thalidomide, with its A-ring occupying the tri-tryptophan pocket in a glutarimide-like orientation despite lacking the canonical imide NH donor. SAR within this series showed substantial tolerance for chemical modification and scaffold simplification, raising the possibility that endogenous steroidal metabolites may contribute to the physiological ligand landscape of CRBN. Bicyclic lactams additionally provided synthetically accessible scaffolds with tunable affinity and promising sites for linker attachment. Across the identified ligand classes, none of the tested representatives induced detectable degradation of canonical CRBN neosubstrates, and several showed largely clean proteomic profiles. Together, these findings broaden the chemical, mechanistic and potential physiological landscape of CRBN recognition and provide diverse starting points for alternative, potentially neosubstrate-sparing CRBN recruiters.

Deubiquitinase-targeting chimeras (DUBTACs) have emerged as a promising strategy for targeted protein stabilization, but their broader application remains limited by the scarcity of ligandable deubiquitinase recruiters. Here, we report a previously unexplored four-membered-ring OTUB1 recruiter chemotype. Through systematic structure-activity relationship studies, we identified compound 21 (MS2159) as a potent and selective covalent OTUB1 ligand. Biochemical and intact protein mass spectrometric analyses demonstrated that MS2159 selectively engages the non-catalytic C23 residue of OTUB1, shows minimal reactivity toward other tested proteins, and preserves OTUB1 deubiquitinase activity. Conjugation of MS2159 with the CFTR ligand lumacaftor yielded compound 25 (MS2134), which effectively stabilized {Delta}F508-CFTR. Collectively, these findings establish a new OTUB1 recruiter scaffold, expand the ligandable chemical space of OTUB1, and provide additional opportunities for developing next-generation DUBTACs.

The unique cell surface composition of tumor cells forms the molecular basis for many targeting and cell-based therapies. Here, we describe the development of novel peptide-based targeting agents for programmed death ligand 1 (PD-L1). Molecular imaging by peptide agents, coupled with therapeutic intervention using the same modality, represents a critical advancement in cancer management. Whole-body PET imaging of PD-L1 expression offers a superior alternative to traditional immuno-histochemistry, making PD-L1 radiodiagnostic imaging a highly sought-after modality. PD-L1 targeting modalities developed for clinical imaging to date can be divided into antibodies, protein domains, and small macrocyclic peptides with fewer than 20 amino acids. The latter modalities can address many challenges seen in antibody-based targeting vectors. All potent PD-L1 targeting peptide modalities reported to date rely extensively on non-canonical amino acids (ncAAs). Here, we report a comprehensive structure-activity relationship (SAR) analysis of a family of macrocycles discovered from an Sx2Cx8Cx2 phage-display library composed entirely of natural amino acids (x represents 19 natural amino acids excluding Cys). Using >10,000 variants in ''focused'' phage-display libraries, we optimized these macrocycles to achieve single-digit-nanomolar potency in protein- and cell-based assays. En route to this optimization, the activity of 216 synthetic macrocycles towards PD-L1 was measured in five distinct assays; two leads have been evaluated by imaging in tumor xenografts in mice, and the X-ray structure of one advanced lead in complex with PD-L1 has been determined at 2.78 [A] resolution. This publication demonstrates the development potential of PD-L1-targeting macrocycles that do not require extensive incorporation of ncAAs and the democratization of discovery by mapping the optimization path to single-digit-nanomolar assets for targeted radiopharmaceuticals via canonical phage-display technology.

The folate receptor (FR) is overexpressed in a range of human tumours including ovarian cancer cells. We propose that the overexpression of the FR on the surface of ovarian tumour cells could be exploited for the selective delivery of a DNA damage response inhibitor (DDRi) in the form of an intact folate drug conjugate (FDC). This approach would improve the therapeutic index of the parent DDRi facilitating combination studies of the DDRi-based FDC with DNA damaging chemotherapy. FR-mediated cellular uptake of the proposed folate drug conjugates is requisite for FDC selective delivery into tumours. In this study, we synthesised a series of olaparib-based folate conjugates that maintained the biochemical PARP1 inhibition associated with olaparib and showed binding affinity for the folate receptor. Significantly, we identified compounds 10b and 11 that selectively enter FR overexpressing tumour cells via folate receptor-mediated endocytosis in their intact form and engage with their target as demonstrated by the potent inhibition of PARylation (KB cells, PARylation IC50 = 5.7 and 3.9 nM; respectively).

Screening of the open-access CRESTdb small-molecule library identified the imidazothiazole carboxamide sCQG200 (1) as an initial hit, with activity against H37Rv and Erdman Mycobacterium tuberculosis strains in cholesterol-containing medium (MIC90= 6.88 uM and 4.48 uM, respectively) and against intracellular Mtb (EC50= 3.80 uM). sCQG200 retained activity against drug-resistant Mtb strains, including clinical MDR/XDR isolates. SAR optimization led to analogs 21 and 24 with substantially improved antimycobacterial potency. Mouse pharmacokinetic studies following oral and intravenous administration showed 62.1% and 29.5% oral bioavailability for 21 and 24, respectively. This structurally differentiated chemotype provides a promising starting point for further optimization as antitubercular agent.

The cytosolic histone deacetylase 6 (HDAC6) plays a key role not only in cancer but also in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) peripheral neuropathies. Pharmacological inhibition as well as genetic silencing of HDAC6 is able to rescue several defects. Therefore, developing pharmacological compounds targeting this enzyme is of crucial interest. Here, we report the design, synthesis, and characterization of Methyl Fluorescent Hydroxamic Acid (MeFluHyA), a novel fluorescent HDAC6-selective probe designed for cell imaging. By integrating a Cy5 fluorophore into a phenyl hydroxamic acid scaffold, MeFluHyA shows binding to the catalytically active CD2 domain of HDAC6 without significantly inhibiting its deacetylating function in the sub-micromolar range. Biochemical enzymatic activity assays confirmed its selectivity over other HDAC isoforms. Fluorescent imaging studies in HeLa cells demonstrated strong colocalization with HDAC6-eGFP and a commercial HDAC6 antibody. Competitive binding assays revealed that MeFluHyA effectively identifies known HDAC6 inhibitors, with reduced probe-binding serving as a readout for successful target engagement. MeFluHyA is a cell-permeable, fast and easy to use probe that can be added organism-independent, and its red-shifted fluorophore makes it compatible with multiplex, live and fixed imaging. Overall, we provide a novel tool for screening platforms aimed at identifying novel HDAC6-targeting inhibitors.

Rho guanosine triphosphatases (GTPases) are molecular-switches implicated in neurodegenerative diseases, yet targeting them through competitive inhibition remains a challenge due to their high affinity for guanine nucleotides. Guanine nucleotide exchange factors (GEFs) catalyse GDP-to-GTP nucleotide exchange to activate GTPases, providing an alternative opportunity for GTPase modulation. Herein, we describe a complex-targeted strategy to inhibit nucleotide exchange with covalent molecular glues that engage the Kalirin-Rac1 GEF-GTPase complex at the nucleotide binding site are sequester the GEF Kalirin. Fragment hits were identified through XChem and in silico screening, and a fragment merging approach resulted in the generation of covalent inhibitors RS-009 and MC-278. Multiple analyses demonstrate our compounds inhibit nucleotide exchange both through competition with the nucleotides and by stabilising a ternary inhibitor-Rac1-Kalirin complex, thereby trapping Kalirin in a non-productive state and reducing GEF turnover. Biochemical selectivity screening and cellular activity-based protein profiling (ABPP) show that selectivity can be achieved across distinct GEF-GTPase complexes, which may result in improved spatiotemporal control over targeting the GTPase alone. This work provides evidence for targeting GTPase signalling via stabilization of the GEF-GTPase complex in a unique covalent molecular glue mechanism and provides the basis of a chemical probe or therapeutic.

Selective peptide agonists for complement C5a receptor 1 (C5aR1) are valuable tools for dissecting receptor-specific inflammatory signalling, but their optimisation is complicated by overlap with closely related anaphylatoxin receptors and by pathway-dependent pharmacology. Here, we applied a structure-guided computational workflow to prioritise mutations within two C5a-derived peptide agonist scaffolds. FoldX-guided modelling identified position 5 as a candidate optimisation site, with hydrophobic substitutions predicted to improve C5aR1 engagement without corresponding gains at C3aR. Predicted BM1 and BM221 analogues were synthesised by solid-phase peptide synthesis and evaluated across C3aR, C5aR1 and C5aR2 using ERK1/2 phosphorylation and {beta}-arrestin recruitment assays. Position-5 substitutions enhanced C5aR1 functional preference in ERK assays, although additional replacement of Leu6 with Ala reduced target potency and revealed pathway-dependent receptor discrimination. BM1 P5M provided the clearest overall improvement across ERK and {beta}-arrestin readouts. In the BM221 series, A5Nle improved C5aR1 preference over C3aR, whereas A5Nle Abu6Ala produced the most favourable serum stability profile. These findings support position 5 as a transferable optimisation site and demonstrate that C5aR1 potency and receptor selectivity must be balanced during next-generation agonist design.

Alkaline ceramidase 2 (ACER2) catalyzes the hydrolysis of ceramides into sphingosine and free fatty acids, playing crucial roles in regulating various biological processes, including but not limited to cell proliferation, survival, and the DNA damage response. However, a pharmacological tool that regulates its catalytic and signaling functions remains to be developed. In this study, N-[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]dodecane-1-sulfonamide (KPB82) was identified as a potent and selective ACER2 inhibitor. In vitro enzymatic assays showed that KPB82 inhibited ACER2 with an IC50 of 190 nM, while displaying minimal activity against acid and neutral ceramidases (IC50> 100 {micro}M) and no significant inhibition of alkaline ceramidase 1 or alkaline ceramidase 3. Enzymatic kinetic studies revealed that KPB82 is a competitive inhibitor of ACER2. The three-dimensional structure of ACER2 was modeled in the AlphaFold3 server, revealing key Zn2+-coordinating histidine residues and a substrate-binding pocket. Molecular docking studies demonstrated that KPB82 binds ACER2 within this pocket, forming hydrogen bonds with the catalytically important amino residues, Asp93 and His215, and interacting with several hydrophobic residues. Lactate dehydrogenase release assays revealed that KPB82 significantly reduced cytotoxicity in HeLa cells overexpressing ACER2, as well as in HCT116 cells, RT4 cells, and human umbilical vein endothelial cells treated with the DNA-damaging agent doxorubicin. Lipidomic analysis showed that KPB82 inhibited sphingosine production in response to ACER2 overexpression or doxorubicin treatment. Collectively, these findings demonstrate that KPB82 is a potent and selective ACER2 inhibitor capable of suppressing ACER2-dependent biological processes and highlight its potential as a tool for studying ceramide metabolism and ACER2-mediated signaling pathways.

Allosteric modulation of G protein-coupled receptors (GPCR) is a rapidly advancing area of drug discovery with considerable therapeutic potential. In this study, we used various bioluminescence resonance energy transfer (BRET) assay configurations to investigate the differential activity of LY3154207, a dopamine D1 receptor (D1R)- positive allosteric modulator (PAM), in potentiating D1R-mediated signaling pathways. Our data showed that LY3154207 demonstrated clear bias toward {beta}-arrestin recruitment over G-protein engagement. Among the G-protein subtypes, LY3154207 preferentially potentiated Gs signaling induced by DA. Interestingly, when tested for its effect on the Gs-selective D1R agonist DHX and the Golf-selective agonist TAV, LY3154207 further enhanced the selectivity of these compounds, suggesting a probe-dependent effect. We also studied the structural mechanism underlying this subtype selectivity and found that W123 and R130 in the intracellular loop 2 (ICL2) of D1R were crucial for the activity of LY3154207. Furthermore, the N domain of the G-protein appeared to make minor contributions to this G-protein subtype selectivity of LY3154207. Lastly, our data demonstrated that LY3154207 induced cAMP production in the absence of the orthosteric ligand, indicating that this compound is an ago-PAM.