Abstract Proteolysis-targeting chimera (PROTAC) technology has emerged as a powerful therapeutic strategy in drug discovery. Conventional PROACs are heterobifunctional molecules composed of two distinct ligands that independently bind to a protein of interest (POI) and an E3 ligase. However, their inherently large molecular size often leads to suboptimal drug-like properties. In this study, we report a “2-in-1” PROTAC design strategy for developing more compact HDAC8 degraders by integrating the CRBN-recruiting ligand into the capping group of an HDAC8 warhead, thereby enabling a single structural motif to engage both HDAC8 and CRBN. Compared to our first generation HDAC8 degrader YX862, the new degraders exhibit improved selectivity, reduced molecular weight, improved overall drug-like properties, and, importantly, potent HDAC8 degradation in vivo. These findings highlight the therapeutic potential of this new class of HDAC8 degraders, and demonstrate a conceptual framework for integrating E3 ligase recruitment and target engagement into a more efficient and compact PROTAC design.
Abstract The development of isoform-selective HSP70 inhibitors remains challenging due to the high conservation of this chaperone family. Continuing our efforts to target the peptide-binding pocket within the substrate-binding domain (SBD) of GRP78 (BiP/HSPA5), we identified compound 12 using a Direct-to-Biology (D2B) strategy. Biochemical, biophysical, and cellular characterization showed that 12 directly engages the GRP78 peptide-binding pocket, selectively binds GRP78 over other tested HSP70 isoforms, and potently kills A549 lung cancer cells while showing minimal activity in other tested lung cancer cell lines. Cellular thermal shift assay (CETSA) demonstrated GRP78 engagement in cells, and unfolded protein response (UPR) signaling analysis further showed that 12 modulates GRP78-related stress−response pathways, supporting its functional engagement of GRP78. Collectively, compound 12 represents a well-characterized, isoform-selective, cell active, and cell selective GRP78 inhibitor, providing a valuable chemical probe for dissecting GRP78 biology and a promising starting point for the development of GRP78-targeted therapeutics.
Abstract Error-prone DNA repair pathways promote genomic instability and cancer progression. Notably, Poly(ADP-ribose) polymerase-1 (PARP1)-directed alternative non-homologous end joining plays a pivotal role in cancer, including Multiple Myeloma (MM), conferring vulnerability to PARP1 inhibitors (PARPis). However, despite their clinical efficacy, PARPi-mediated trapping of PARP1 contributes to hematologic toxicities and can be circumvented by resistance mechanisms, highlighting the need for alternative strategies to modulate PARP1 activity. Here, we investigated the non-canonical G-quadruplex (G4) formed within the PARP1 gene promoter and characterized by an adenine bulge as a potential therapeutic target. Computational modeling and biophysical characterization identified a small molecule capable of targeting this distinctive G4 architecture. In MM cells, the compound effectively downregulated PARP1 expression, perturbed downstream DNA repair pathways, and induced DNA damage-associated apoptosis, while sparing healthy blood cells. Overall, our findings reveal a previously unexplored G4-binding mode with potential therapeutic relevance in cancer.
Abstract The EP4 receptor (EP4R) has emerged as a promising target in immuno-oncology due to its role in modulating tumor immunity and inflammation. Here, we describe the discovery and preclinical development of DT-9081, a clinical candidate EP4R-antagonist that has completed a phase I monotherapy trial for the treatment of solid cancers. A scaffold hopping strategy enabled the identification of a novel chemical series with improved activity and pharmacokinetics. Structure–activity relationship (SAR) studies guided the optimization of lead compounds, culminating in the identification of DT-9081, which exhibits potent EP4R antagonism, favorable ADME characteristics, and an excellent preclinical safety profile. Comprehensive in vitro and in vivo pharmacology data further support the utility of DT-9081 in modulating tumor immunity, providing a compelling rationale for its clinical development in the treatment of solid cancers.
Abstract Selective KIT inhibition is an effective strategy for treating mast cell-driven diseases, but highly conserved ATP-binding sites in class III RTKs hinder selective inhibitor design. We employed structure-guided medicinal chemistry to optimize a series of potent, selective KIT inhibitors. An initial triazolopyridine hinge-binding motif enabled potent KIT inhibition with high selectivity and, after central linker optimization, led to a “reverse” arylacetamide series with further improved potency and pharmacokinetic properties. Incorporation of a pyrazole-based linker and modification of the hinge-binding motif led to improved potency in the presence of human serum. Further tuning of the terminal aryl substituent mitigated cytochrome P450 time-dependent inhibition and improved kinome selectivity. Crystallography revealed key elements of KIT inhibition and showed that subtle modulation of linker stereoelectronic effects in the gatekeeper region can drive selectivity. These efforts yielded an optimized lead inhibitor with low-nanomolar cellular potency, high KIT selectivity, and properties favorable for chronic therapy.
Abstract Dual inhibition of the histamine H1 and H4 receptors (H1R and H4R) has been shown to provide superior anti-inflammatory efficacy in preclinical models of allergic disease compared to selective inhibition of either receptor alone. Building on this concept, we initiated a fragment-based discovery program and previously identified a quinazoline-containing fragment hit. Here, we describe the lead optimization toward compounds with unique dual H1R/H4R activity. Structure–activity relationship studies yielded potent and balanced ligands with nanomolar affinities for both receptors, as well as pharmacokinetic properties suitable for ocular administration. Among these, quinazoline 35.HCl (GD136) and tetrahydroquinazoline 72.HCl (GD134) demonstrate robust in vivo efficacy in a ragweed-induced mouse model of allergic conjunctivitis, significantly reducing hyperemia and outperforming selective H1R or H4R antagonists. Based on its pharmacological profile, ADME properties, ease of formulation, and in vivo efficacy, GD134 was selected as the clinical development candidate for a first-in-class dual-targeted therapy of allergic conjunctivitis.
The clinical utility of platinum-based chemotherapeutics is limited by severe toxicity and acquired resistance, motivating the development of alternative metallo drugs. Here, we report a series of rhenium complexes, Re[HL1-4]3, synthesized via reduction of Re(VII) precursors followed by coordination with aromatic thiohydrazide ligands. The lead complex, Re[HL2]3, adopts an unusual trigonal-prismatic geometry and a favorable redox profile, correlating with enhanced cytotoxic potency. It demonstrates selective antiproliferative activity against cancer cells with minimal toxicity toward nonmalignant cells in both 2D and 3D culture systems. Mechanistic studies reveal DNA binding, induction of DNA damage, and reactive oxygen species (ROS) generation, triggering apoptosis via PARP and caspase 3 cleavage. Pharmacokinetic analysis indicates moderate stability and plasma retention, supporting sustained therapeutic levels while limiting accumulation-related toxicity. In a syngeneic 4T1 murine breast cancer model, Re[HL2]3 significantly inhibited tumor growth without observable systemic toxicity, supporting its further preclinical evaluation.
Within the tumor microenvironment (TME), regulatory T (Treg) cells promote an immunosuppressive state with limited tumor antigen presentation and antitumor effector T (Teff) cell responses, which can drive resistance to cancer immunotherapies. IKZF2 (Helios) is a transcription factor essential for stabilizing the immunosuppressive Treg cell phenotype in tumors. Herein, we present the discovery of BMS-986449, a selective Cereblon E3 Ligase Modulatory Drug (CELMoD) degrader of IKZF2 and IKZF4 (Eos) that spares the closely related transcription factors IKZF1 (Ikaros) and IKZF3 (Aiolos). BMS-986449 is an orally available degrader that demonstrates single-agent growth inhibition of syngeneic MC38 tumors implanted in humanized Cereblon (CRBN) knock-in mice. Tumor growth inhibition was more robust when BMS-986449 was administered in combination with anti-PD-1. Nonhuman primates administered daily with BMS-986449 show sustained IKZF2 degradation in Treg cells providing confidence in human clinical doses. Collectively, these findings establish BMS-986449 as a promising clinical candidate for cancer immunotherapy.
We report paradoxical findings in properties when secondary sulfonamides are replaced with isosteric secondary sulfoximines. Despite the reduction in polar surface area and the elimination of a hydrogen bond acceptor and a hydrogen bond donor in the RS(O)2NHR to RS(O)(Me) = NR transformation, the resulting sulfoximines generally had increased aqueous solubility and decreased albumin binding, with experimental logP/D values substantially lower than predicted. Despite these characteristics of increased hydrophilicity of the sulfoximine motif, permeability and metabolic stability were similar to those of the corresponding sulfonamide. These studies were enabled by parallel medicinal chemistry via library synthesis facilitated by the use of a palladium precatalyst. We further demonstrate that in comparing molecules over a range of albumin binding, calculating the pseudoaffinity constant (log KHSA) is superior for data interpretation than the more frequently used fraction unbound. The paradoxical findings presented here are indicative of secondary sulfoximines being both more hydrophilic and more lipophilic than their corresponding sulfonamides.
Abstract Chikungunya virus (CHIKV), an arthropod-borne alphavirus, has emerged as a global health threat due to its rapid transmission and the lack of effective antiviral therapies. The cysteine protease activity of the virus-encoded nonstructural protein 2 (nsP2) is critical for CHIKV replication, as it processes viral polyproteins and counteracts host antiviral defenses, establishing it as a highly attractive target for therapeutic intervention. In this study, we present a rapid drug development platform that integrates covalent docking with direct-to-biology (D2B) synthesis and screening to identify nsP2 inhibitors. Candidates prioritized by in silico docking were synthesized and directly tested in FRET enzymatic assays without purification. This approach led to the identification of several nsP2 inhibitors with diverse chemical scaffolds, potent enzymatic inhibition, and antiviral activity. Together, these findings establish a streamlined strategy for covalent inhibitor development and provide promising leads for CHIKV antiviral development.
Heparan sulfate proteoglycans (HSPGs) facilitate interactions between growth factors and their receptors, regulating signaling pathways involved in angiogenesis and cell proliferation. However, the structural heterogeneity and synthetic inaccessibility of native heparan sulfate (HS) have limited systematic exploration of structure-activity relationships (SAR) and the discovery of HS-based inhibitors. Here, we developed 46 aminoglycoside-derived non-native HS mimetics spanning diverse structural and functional group patterns. SAR analysis identified N-acetylation and 6-O-sulfation as key determinants of HS-FGF2 and HS-VEGF165 interaction modulation. Compound 8, synthesized in two steps, competitively disrupted heparin binding to FGF2 (IC50 = 0.23 μM), FGFR1 (IC50 = 0.17 μM), and VEGF165 (IC50 = 0.66 μM), with >140-fold FGF2/FGF4 selectivity and substantially greater potency than native HS pentasaccharide fondaparinux. In NIH3T3 fibroblasts, compound 8 selectively reduced FGF2-induced proliferation in a dose-dependent manner without measurable cytotoxicity, highlighting aminoglycoside-derived HS mimetics as synthetically accessible platforms for modulating growth factor signaling.
Macrocyclization can enhance the selectivity of acyclic compounds toward structurally similar biological targets such as kinases. WEE1 regulates cellular homeostasis and is a promising target in oncology. The clinical candidate AZD1775 (1) failed to progress past Phase II trials because of patient tolerability issues, likely due to off-target inhibition of polo-like kinase 1 (PLK1). Herein, a computer-aided drug design approach was conducted to develop a macrocycle based on the 1-WEE1 X-ray cocrystal structure. Significantly enhanced WEE1 inhibitory selectivity over PLK1 was determined for leading macrocycle 2, which also demonstrated broader kinome-wide selectivity. Patient-derived organoids from colorectal cancer (CRC) peritoneal and liver metastases, treated with 2, demonstrated comparably strong or enhanced anticancer efficacy compared to that of 1. Against patient-matched normal colon vs primary CRC organoids, 2 potently and selectively treated CRC, as well as enhanced DNA damage compared to 1. Finally, the X-ray cocrystal structure of 2 bound to WEE1 validated its computationally predicted bioactive binding mode.
Abstract To study the ″preference″ of bacterial membranes for the secondary structure of antimicrobial peptides (AMPs), we designed a universal palindromic sequence template G(LK)n(KL)nG-NH2 (designated as GnPm, where n = 2–6 and m = 0–3) that can cover various secondary structures of AMPs, with 0 to 3 proline residues uniformly and dispersedly inserted in the middle region to regulate their secondary structures. Results confirmed that both the numbers of LK/KL repeats and proline can regulate secondary structure, thereby controlling the antibacterial activity of the AMPs. The optimized peptide G5P2 exhibits potent antibacterial activity via a typical membrane-disrupting mechanism, is not prone to inducing bacterial drug resistance, can synergize with traditional antibiotics to delay the emergence of antibiotic resistance, and has excellent in vivo antibacterial activity (a 93.52% bacterial clearance) and high safety (LD50 = 89.87 mg/kg), thus providing new chemical strategies and candidate molecules for the clinical management of drug-resistant bacterial infections.
Abstract Although injectable anemoside B4 (AB4) has therapeutic potential for atopic dermatitis (AD), its clinical use is restricted by safety and pharmacokinetic issues. Therefore, we designed 42 AB4 derivatives, established a preliminary SAR for anti-inflammatory and antiallergic activities, and selected three promising compounds for in vivo evaluation in an AD mouse model, leading to the identification of B4-39 as the lead compound. In a DNCB-induced AD mouse model, topical B4-39 (6.6 mg/kg) was more effective than dexamethasone and free from its side effects, while matching the efficacy of crisaborole at a much lower dose and providing better skin barrier repair. Mechanistically, B4-39 targets pyruvate carboxylase (PC)─a novel therapeutic target in AD─modulating the TCA cycle to suppress dendritic cell activation and concurrently inhibiting NF-κB and NLRP3 inflammasome signaling. Given its enhanced efficacy, steroid-sparing safety, novel PC-targeted action, and favorable topical delivery, B4-39 is a highly promising candidate for AD treatment.
Antibody-oligonucleotide conjugates (AOCs) enable targeted delivery of small interfering RNAs (siRNAs) by coupling them to antibodies for receptor-mediated uptake, enhancing tissue specificity and therapeutic potential. A key determinant of AOC performance is the drug-to-antibody ratio (DAR), which influences pharmacokinetics, biodistribution, and knockdown efficiency. Prior studies with transferrin receptor 1 (TfR1)-targeted AOCs revealed that DAR ≥2 constructs exhibit rapid plasma clearance, preferential hepatic uptake, and reduced muscle delivery compared with DAR1. We hypothesized that increased negative charge from multiple phosphodiester and phosphorothioate linkages drives nonspecific plasma protein binding and scavenger receptor-mediated clearance of DAR2 AOCs. To test this, we investigated siRNA modifications designed to reduce charge and phosphorothioate content, including phosphorothioate removal and incorporation of neutral backbone chemistries such as phosphoryl guanidine (PG), methoxypropylphosphonate, and triester linkages. PG-modified DAR2 AOCs improved pharmacokinetics and tissue distribution in mice while maintaining activity. These findings lay the groundwork for optimizing high-DAR AOCs.
Clinically approved therapies have established that combining β-lactam antibiotics with β-lactamase inhibitors is an effective strategy to counter antibiotic resistance; however, infections caused by metallo-β-lactamase (MBL) producing bacteria remain a critical unmet medical need. Building on our previous efforts targeting clinically relevant MBLs, including New Delhi metallo-β-lactamase (NDM), Verona integron-encoded metallo-β-lactamase (VIM), and imipenemase (IMP), we report the design and synthesis of 40 novel dihydro-chromeno-pyrrole (dCP) derivatives using a scaffold-hopping approach. Several compounds displayed potent MBL inhibition and lead compounds 74 and 91 effectively restored the activity of mmeropenem and imipenem against carbapenem-resistant Gram-negative pathogens. Optimization efforts focused on improving ADME properties while maintaining potency, and lead compounds were further evaluated for their pharmacokinetic profiles. Docking studies revealed favorable binding interactions of inhibitors with MBL proteins, supporting and justifying the observed inhibitory activity. Overall, this study provides a robust framework for lead optimization and identifies promising candidates for preclinical development.
Direct-to-biology (D2B) is a powerful strategy that accelerates early drug discovery. It enables compounds to be synthesized in miniaturized formats and evaluated directly as crude reaction mixtures. This bypasses the need for purification during the initial design-make-test cycle. Advances in robust synthetic methodologies, automation, reaction miniaturization, and biological screening have transformed D2B from a proof-of-concept approach into a versatile medicinal chemistry platform. This platform is applicable to fragment optimization, covalent ligands, macrocycles, proteolysis-targeting chimeras (PROTACs), molecular glues, and cellular phenotypic screening. This perspective focuses on the synthetic transformations, assay technologies, and platform implementations that drive modern D2B workflows. It emphasizes reaction robustness, assay compatibility, and practical implementation. Analysis of the current literature revealed that D2B is more governed by reaction reliability than synthetic diversity. Amide coupling and click chemistry dominate reported workflows, while more complex transformations remain underexplored. We discuss the complementary strengths and limitations of biochemical, biophysical, and cellular readouts, identify current bottlenecks in reaction scope and data management, and highlight emerging opportunities arising from reaction miniaturization, machine learning, automated experimentation, and advanced synthetic methodologies. Rather than replacing conventional medicinal chemistry, D2B fundamentally shifts experimental effort from purification toward early biological validation and is poised to become an integral component of future medicinal chemistry workflows.
