Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a molecular chaperone implicated in pro-tumorigenic pathways by regulating the folding of substrate proteins (clients) within cancer cells. Recent research has pinpointed a potentially druggable allosteric site within the client binding site (CBS) of TRAP1, suggesting this site might offer a more effective strategy for developing potent and selective TRAP1 inhibitors. However, the absence of reliable assay systems has hindered quantitative evaluation of inhibitors. In this study, we have developed a fluorescent probe, Rho6TPP, designed to target the CBS. Utilizing fluorescence polarization-based high-throughput screening assays, Rho6TPP exhibits excellent signal-to-noise ratio (>20), factor (>0.6), and ' factor (>0.6). Additionally, it facilitates comparative analysis of existing small molecules and discovery of novel binders. MitoTam, a mitochondria-targeted tamoxifen, emerges as a potent CBS-targeting TRAP1 inhibitor. Our findings highlight the potential of Rho6TPP as a crucial tool for advancing the development of CBS-targeting TRAP1 inhibitors.

Akuammicine (), an alkaloid isolated from , is an agonist of the kappa opioid receptor (κOR). To establish structure-activity relationships (SARs) for this structurally unique κOR ligand, a collection of semisynthetic derivatives was synthesized. Evaluating these derivatives for their ability to activate the κOR and mu opioid receptor (μOR) revealed key SAR trends and identified derivatives with enhanced κOR potency. Most notably, substitutions to the C10 position of the aryl ring led to a > 200-fold improvement in κOR potency and nearly complete selectivity for the κOR. A selection of the most potent ligands was shown to possess differing abilities recruitment of β-Arrestin-2 to the κOR, indicating they have distinct signaling properties from each other and existing κOR ligands. The discovery of these κOR agonists underscores the potential of using natural products to identify new classes of potent and selective ligands and provides new tools to probe the κOR.

We describe the discovery and preclinical characterization of a potent and selective lysophosphatidic acid receptor 1 (LPAR1) antagonist with a direct-acting antifibrotic mechanism. was initially identified as a potent non-carboxylic acid LPAR1 antagonist in an LPA-induced myocardin-related transcription factor A (MRTF-A) nuclear translocation assay. Modifications to the aromatic elements in the structure allowed for improvements in metabolic stability and the mitigation of GSH adduct formation, but in vitro to in vivo clearance disconnects were observed with several potent sulfonamides (e.g., ) across preclinical species. Through modification of the sulfonamide, () emerged as a potent LPAR1 antagonist with a suitable in vitro profile and desirable pharmacokinetic properties for oral QD dosing. dose-dependently blocked LPA-induced histamine release and demonstrated efficacy in an interventional model of bleomycin-induced lung fibrosis. However, CNS-related toxicity was observed in dogs, and based on these findings, the clinical development of for IPF was halted.

Insulin-like peptide 5 (INSL5) targets the G protein-coupled receptor, relaxin family peptide receptor 4 (RXFP4), predominantly coexpressed in the colorectum. While INSL5 also binds to the related receptor RXFP3, it does not activate it. The INSL5/RXFP4 axis is a promising target for treating gastrointestinal disorders such as constipation. Despite its therapeutic potential, the clinical application of INSL5 has been hindered by synthesis complexities, necessitating the need for more accessible yet potent mimetics. In this study, we engineered an INSL5 analogue A13:B7-24-GG, featuring a simplified two-chain, two-disulfide scaffold with 32 amino acids, as opposed to the 45 amino acids found in native INSL5 (two-chain, three-disulfide), improving the synthesis yield by 19.5-fold. Additionally, A13:B7-24-GG demonstrates ∼4-fold higher potency (EC = 1.17 nM vs 4.57 nM) and ∼11 times greater selectivity than native INSL5, with significantly reduced RXFP3 binding affinity, positioning it as a promising new therapeutic candidate for the treatment of constipation.

The discovery of d-cycloserine (), a partial agonist of the NMDA receptor that exhibits antidepressant effects without the psychotomimetic effects of ketamine, has fueled interest in new NMDA-targeting antidepressants. Our objective was to identify potent partial agonists mirroring , particularly tailored for the GluN2B subtype of the NMDA receptor. Through a structure-based drug design approach, we discovered compound . This compound acts as a partial agonist of the GluN1/GluN2B complex, exhibiting 24% efficacy, and has an EC value of 78 nM. Subsequent investigations led us to (Lu AF90103), a methyl ester prodrug of capable of penetrating the blood-brain barrier, as confirmed by rat microdialysis studies. In different rat models relevant to neuropsychiatric diseases, administering led to demonstrating both acute effects, observed in a seizure model and EEG, and lasting effects in the stress-sensitive hippocampal pathway and an antidepressant-sensitive model.

Osteoarthritis (OA) is a chronic and degenerative joint disease affecting more than 500 million patients worldwide with no disease-modifying treatment approved to date. Several publications report on the transforming growth factor β-activated kinase 1 (TAK1) as a potential molecular target for OA, with complementary anti-catabolic and anti-inflammatory effects. We report herein on the development of TAK1 inhibitors with physicochemical properties suitable for intra-articular injection, with the aim to achieve high drug concentration at the affected joint, while avoiding severe toxicity associated with systemic inhibition. More specifically, reducing solubility by increasing crystallinity, while maintaining moderate lipophilicity proved to be a good compromise to ensure high and sustained free drug exposures in the joint. Furthermore, structure-based design allowed for an improvement of selectivity versus interleukin-1 receptor-associated kinases 1 and 4 (IRAK1/4). Finally, TAK-756 was discovered as a potent TAK1 inhibitor with good selectivity versus IRAK1/4 as well as excellent intra-articular pharmacokinetic properties.

The NRF2-KEAP1 interaction is central for cytoprotection against stresses, giving it high clinical significance. Covalent modification of KEAP1 is an efficient approach, but the covalent inhibitors used in the clinic carry undesired side effects originating in their moderate selectivity. Starting with a phenotypic screen, we identified a new covalent inhibitor chemotype that was optimized to deliver a series of potent and highly selective KEAP1 binders. While the developed compounds showed both cellular and in vivo activity, upregulating antioxidant response element-dependent target genes, they showed no genotoxicity in vitro. The lead compound exhibited broad selectivity in activity-based protein profiling and showed no significant interaction with a panel of commonly studied receptors nor with a broad panel of kinases. The nature of its interaction with KEAP1 and the origin of its selectivity were revealed by X-ray crystallography.

Targeting the lysine residue of protein kinases to develop covalent inhibitors is an emerging hotspot. Herein, we have reported an approach to develop lysine-targeted covalent inhibitors of PI3Kδ by in situ interaction upgradation of the H-bonding to covalent bonding. Several warhead groups were introduced and screened in situ, leading to lysine-targeted covalent inhibitors bearing aromatic esters with high bioactivity and PI3Kδ selectivity. Compound bearing phenolic ester was finally optimized to show a long duration of action in SU-DHL-6 cells by multiple assays. Docking simulation and further protein mass spectrometry confirmed that bound to PI3Kδ by covalent-bonding interactions with Lys779. Furthermore, exhibited potently antitumor efficacy without obvious toxicity in the SU-DHL-6 and Pfeiffer xenograft mouse models. This study identified to be a much more effective antitumor agent in vitro and in vivo as a lysine-targeted covalent inhibitor, and it also provided a practical approach for the development of lysine-targeted covalent inhibitors.

Activation of the NLRP3 inflammasome in response to danger signals is a key innate immune mechanism and results in the production of the pro-inflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18) as well as pyroptotic cell death. Aberrant NLRP3 activation has been linked to many acute and chronic conditions ranging from atherosclerosis to Alzheimer's disease and cancer, and based on the clinical success of IL-1-targeting therapies, NLRP3 has emerged as an attractive therapeutic target. Herein we describe our discovery, characterization, and structure-based optimization of a pyridazine-based series of NLRP3 inhibitors initiating from an high-throughput screening campaign. The scaffold, exemplified by lead molecule , has excellent potency and physicochemical and pharmacokinetic properties, including good brain penetration. The establishment of pharmacokinetic/pharmacodynamic relationships in the periphery and central nervous system in mechanistic models facilitates the use of as a tool to further interrogate the biology of NLRP3 in peripheral and neuroinflammatory models.

Arginase is a promising immuno-oncology target that can restore the innate immune response. However, it's highly polar active site often requires potent inhibitors to mimic amino acids, leading to poor passive permeability and low oral exposure. Using structure-based drug design, we discovered a novel proline-based arginase inhibitor () that was potent but had low oral bioavailability in rat. This issue was addressed by incorporating amino acids to target PepT1/2 active transport, followed by in vivo hydrolysis post absorption. The hydrolysis rate was highly tunable, and the valine prodrug () showed the best balance of stability and exposure of the potent payload. Dosing of in mouse xenograft models significantly increased arginine in the tumor microenvironment, resulting in tumor growth inhibition as a monotherapy and in combination with an anti-PD-L1 antibody. Compound (AZD0011) displays good pharmacokinetics and was selected as a clinical drug candidate for cancer.

caseinolytic protease P (ClpP) is essential for maintaining mitochondrial proteome homeostasis, and its activation is increasingly recognized as a promising cancer therapy strategy. Herein, based on structure-guided drug design, we discovered a series of potent ClpP activators by introducing a methyl group to the imipridone scaffold of the ClpP activator in Phase III clinical trials. Through structural optimization of the lead compound, the most optimal compound, , exhibited exceptionally potent ClpP agonistic activity (EC = 23.5 nM, 107.1-fold stronger than ) and inhibited the proliferation of HL60 cells (IC = 4.6 nM, 169.2-fold stronger than ). possesses good pharmacokinetic properties and effectively prolongs the lifespan in the MOLM13 and HL60 xenograft models in mice through oral administration. is the most potent and orally efficacious ClpP activator reported to date.

Chronic hepatitis B (CHB) represents a significant unmet medical need with few options beyond lifelong treatment with nucleoside analogues, which rarely leads to a functional cure. Novel agents that reduce levels of HBV DNA, RNA and other viral antigens could lead to better treatment outcomes. The capsid assembly modulator (CAM) class of compounds represents an important modality for chronic suppression and to improve functional cure rates, either alone or in combination. is a potent CAM, which in this work was optimized for potency, safety, and other drug-like properties leading to . was further advanced through clinical development as the highly soluble prodrug . is currently being explored in multiple clinical trials in HBV-infected subjects where unprecedented reductions in HBV DNA, RNA and other viral antigens have been observed, making a promising candidate to become a cornerstone for future chronic suppressive and combination treatment regimens for CHB.

Bromodomain-containing protein 4 (BRD4) is the most promising target for the treatment of triple-negative breast cancer (TNBC). However, its inherent resistant and acquired drug resistance limits its potential clinical application. Recently it has been shown that cyclin-dependent kinases 4/6 (CDK4/6) inhibitors can reincrease the sensitivity of TNBC cells to BRD4 inhibitors by combination therapy, so we designed a series of dual target CDK6/BRD4 inhibitors. Among the newly synthesized compounds, exhibited potent inhibitory activity against CDK6 and BRD4. It also displayed potent antiproliferative activity against TNBC cells. In vivo experiments showed that has potent antitumor activity in the MDA-MB-231 xenograft mouse model, without observable side effects. demonstrates profound synergistic antitumor effects with ferroptosis inducer in TNBC cells. Therefore, is a novel dual inhibitor of CDK6/BRD4 for the treatment of TNBC either as a single agent or in combination with RSL3.

Beyond the rapid achievements of therapeutic heterobifunctional molecules, some recent efforts have focused on constructing heterotrifunctional molecules, aiming at developing more potent and selective therapeutic agents or emerging additional functions to heterobifunctional molecules. However, the synthesis of these complex molecules requires a specific design and lengthy steps. We have developed a two-step strategy for the modular construction of heterotrifunctional molecules, enabled by the sustainable and convenient iodosulfonylation of allenes followed by S2'-selective amination. This strategy successfully incorporates a broad range of biologically active molecules, labeling them with a fluorescent group. The applications of the obtained compounds in selective protein labeling, subcellular imaging, and targeted inhibition of tumor cells make this strategy highly appealing.

The physiological and pharmacological benefits of hydrogen sulfide (HS) are well established, and various HS and persulfide donors have been developed. However, few studies have examined the pharmacokinetics of sulfur donors, as most activity and metabolism tests are conducted , limiting insights into their clinical applications. This study utilized butylphthalide (NBP), an approved drug for ischemic stroke, by integrating HS and persulfide moieties directly into NBP's carbonyl groups. We systematically compared drug metabolism and and evaluated donor efficacy in ischemia-reperfusion models. Results revealed notable / metabolic differences, with thioacid-containing donors showing promising therapeutic effects in cerebral ischemia, reducing infarct size, oxidative stress, and neuronal apoptosis.

Poly-ADP-ribose-polymerase 1/2 (PARP1/2) inhibitors have been approved for cancers with homologous recombination deficiency (HRD). However, their narrow therapeutic indexes largely due to hematologic toxicities have limited their clinical usefulness. Developing selective PARP1 inhibitors has emerged as an attractive strategy to achieve equivalent antitumor activity while alleviating the hematological toxicity caused by PARP2 inhibition. Herein, we report the discovery of pyrazolo[1,5,4-de]quinoxalin-2(3)-one as a novel selective PARP1 inhibitor. formed tighter PARP1-DNA trapping than AZD9574, leading to better potency in inhibiting cancer cell proliferation. achieved tumor regression in the BRCA1-mutated MDA-MB-436 xenograft model and showed synergistic efficacy in combination with carboplatin in the SUM149PT xenograft model. In the rat hematological toxicity study, exhibited minimal impact on hematological parameters at 25 mg/kg, while AZD5305 at 1 mg/kg caused 56.5% reduction of reticulocyte. Taken together, we discovered compound with a therapeutic index superior to that of PARP1 inhibitors AZD5305 and AZD9574 in the preclinical setting.

Inhibition of branched-chain ketoacid dehydrogenase kinase (BDK or BCKDK), a negative regulator of branched-chain amino acid (BCAA) metabolism, is hypothesized to treat cardio-metabolic diseases. From a starting point with potential idiosyncratic toxicity risk, modification to a benzothiophene core and discovery of a cryptic pocket allowed for improved potency with 3-aryl substitution to arrive at PF-07328948, which was largely devoid of protein covalent binding liability. This BDK inhibitor was shown also to be a BDK degrader in cells and in vivo rodent studies. Plasma biomarkers, including BCAAs and branched-chain ketoacids (BCKAs), were lowered in vivo with enhanced pharmacodynamic effect upon chronic dosing due to BDK degradation. This molecule improves metabolic and heart failure end points in rodent models. PF-07328948 is the first known selective BDK inhibitor candidate to be examined in clinical studies, with Phase 1 single ascending dose data showing good tolerability and a pharmacokinetic profile commensurate with once-daily dosing.

We describe the identification of selective SMARCA2, VHL-based heterobifunctional degraders. Structurally novel indolo[1,2-]quinazolin-5(7)-one SMARCA bromodomain binders were optimized and then converted to SMARCA2 degraders by linking them to well-defined VHL ligands. Our exploration led to the discovery of potent and selective degraders of SMARCA2 over the SMARCA4 paralog, leading to potent and selective growth inhibition of SMARCA4 mutant versus wild type cell lines. We further highlight the optimization of the pharmacokinetic profile of a subset of compounds leading to potent and selective degradation of SMARCA2 in the xenograft model. These compounds provide valuable tools with desirable properties for continued exploration of the biology defining the susceptibility of SMARCA4 mutant cancers to selective loss of SMARCA2.

The pH-dependent solubility of the weakly basic TYK2 inhibitor posed a risk to its advancement, given that drugs with such profiles have exhibited drug-drug interaction (DDI) with stomach acid-reducing agents in humans. In a rat model of pH dependence, preadministration of famotidine caused a 2.4-fold lower exposure of when compared to control rats, implying that pH-dependent oral absorption can reduce the active drug's exposure and translate to subtherapeutic treatment. As part of risk mitigation, a prodrug strategy was explored by synthesizing solubility-enhancing prodrugs, resulting in the identification of lead prodrug with acceptable stability and solubility profiles. In rats, the prodrug eliminated the significant difference in AUC and between pentagastrin and famotidine arms, thereby effectively mitigating the impaired drug absorption at the elevated pH relevant for absorption and DDI with famotidine. The prodrug also facilitated dose-proportional systemic exposure of following dose escalation in rats and monkeys.

Rhodium(III) complexes have gained attention for their anticancer potential. In this study, we investigated a rhodium(III) bipyridylsulfonamide complex () and its ligand () for their effects on breast cancer (SKBr3) and noncancerous mammary cells (HB2). Both compounds significantly reduced oxidative phosphorylation (OXPHOS) and mitochondrial function in SKBr3 cells while sparing HB2 cells. Compound also increased glycolysis in both lines, suggesting a metabolic shift. Mitochondrial size and shape were altered, particularly in SKBr3 cells. Additionally, both compounds reduced cancer cell migration by disrupting actin cytoskeleton organization and the Rac1/VASP signaling pathway. These findings suggest that the rhodium(III) bipyridylsulfonamide complex selectively impairs mitochondrial dynamics and cell migration in cancer cells while sparing healthy cells, providing insight into its mechanism of action and toward its use as targeted anticancer therapy. This study lays the groundwork for future in vivo studies and further optimization of these metal-based therapeutics for clinical applications.