Bioorganic & Medicinal Chemistry Letters

Metastatic melanoma is an aggressive cutaneous malignancy frequently driven by the oncogenic V600E mutation within the BRAF kinase. While first-generation Type IS BRAF inhibitors, such as dabrafenib, are currently prescribed to target this specific molecular vulnerability, paradoxical MAPK pathway activation, and acquired drug resistance necessitate the continuous development of structurally optimized lead molecules. In this study, chemical intuition, bioisosteric replacement, and computational molecular docking were employed to propose two novel BRAFV600E drug candidates. The proposed therapeutics, engineered to incorporate constrained sp3-hybridized aliphatic rings and a sulfoximine bioisostere, demonstrated thermodynamically superior docking scores within the mutant catalytic cleft compared to dabrafenib. Lastly, a homology analysis determined that Mus musculus is a suitable model organism for future preclinical studies and confirmed crucial structural selectivity against microbial off-target kinases.

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

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 deubiquitinating enzyme, ubiquitin C-terminal hydrolase L3 (UCHL3), has been implicated as a potential therapeutic target for cancer with a role in regulating the DNA damage response pathways. While the target has been studied using genetic methods there is a lack of reliable chemical probes to selectivity target UCHL3. In this study we report hit identification and optimization of a new chemical scaffold that irreversibly inhibits UCHL3. The observed structure-activity relationships are corroborated by ligand-bound crystal structures that confirm covalent adduct formation with the catalytic cysteine of the enzyme. Finally, through gel-shift assays using a ubiquitin activity-based probe we demonstrate on-target engagement with UCHL3 in two cell lines. The work as a whole presents a comprehensive evaluation of the new scaffold that can be utilized to probe UCHL3 in different biological contexts.

Activating KRAS mutations drive millions of cancers diagnosed worldwide,1 yet for decades this oncoprotein was deemed "undruggable", reflecting the challenge of discovering molecules capable of perturbing its complex biological functions, and of translating these discoveries into effective cancer therapeutics.2 Recent advances propelled by innovative screening have identified diverse modalities that bind at or near the switch-II pocket (SII-P) of RAS proteins, including molecular glues,3 macrocyclic peptides,4 fragment-derived small molecules,5 and approved therapies that covalently target KRASG12C.6,7 Unfortunately, resistance to approved therapies has emerged,8,9 highlighting the need for molecules that engage new or underexploited binding sites on RAS oncoproteins with mechanisms complementary to established SII-P inhibitors.10,11 Here we show that mirror-image mRNA display12 enabled the discovery of all-D macrocyclic peptide ligands targeting a cryptic RAS back pocket (CRB-P).13 These ligands engage KRAS(OFF) and KRAS(ON) with equal affinity, exploit a single-residue difference among isoforms to bind KRAS selectively, and successfully inhibit oncogenic signaling in KRAS-mutant cells through a mechanism distinct from SII-P binders. Mirror-image screening directly afforded nanomolar peptide ligands stable toward cellular proteolysis and delivered probes targeting distinct epitopes not accessible by homochiral peptide-display methods. Together, these findings establish the CRB-P as a specifically druggable and mechanistically differentiated site on KRAS with potential for combination with emerging RAS-targeting therapies and substantiate mirror-image mRNA display as a strategy for discovering stable all-D macrocyclic peptides targeting previously inaccessible epitopes on challenging targets.

Ligand optimization is central to drug discovery as hundreds of analogs might be designed and synthesized between an initial hit and a therapeutic candidate. The efficiency of this process is unclear, at least partly because there is no random background for optimization against which to compare. Such a random background might emerge from synthetically accessible but otherwise systematic random small substitutions across starting ligands, measuring likelihood of achieving a substantial improvement in affinity/potency or other property by any single perturbation. Recent literature and ligand-affinity/potency databases suggest that perhaps 10% of analogs with minor modifications improve upon a parents potency substantially (by [≥]10-fold), but this number is clouded by reporting bias, intentional improvement, and inter-group reproducibility. To begin to establish a background expectation for ligand optimization, we comprehensively and systematically modified 18 lead molecules across six targets with single atom changes; 257 compounds were synthesized. Unexpectedly, 11.2% of these random small perturbation analogs improved potency by [≥]10-fold over their parents. Conversely, these more potent analogs typically had worse in vitro pharmacokinetics (e.g. reduced metabolic stability, lower plasma free fraction). While it was possible to find analogs where the potency increase compensated for inferior exposure and half-life, resulting in more potent compounds in vivo, overall a frustrated landscape for ligand optimization is revealed. This study begins to establish a background expectation for ligand potency optimization and offers a simple strategy to do so. It also begins to quantify the challenges confronting the field in moving beyond in vitro potency.

The rise of new, rapidly mutating viruses presents increasing challenges for drug developers. Traditional methods, such as high-throughput screening and drug repurposing against mutagenic viral targets, have recently shown their limitations. Our current rational molecular engineering approach offers a sustainable solution by targeting viral ion channels, which generally have low mutation rates. First, extending the amantadine molecule led to the development of new compounds that better match the alternating hydrophobic and hydrophilic patterns of the inner walls of ion channels--a common feature across many viruses. Then, simplifying the structure yielded a cyclohexylamine-based minimalist scaffold that effectively blocks the ion channel and demonstrates improved antiviral activity compared to well-known agents such as amantadine and arterolane. SARS-CoV-2 variants served as test systems in laboratory experiments. The new molecular scaffolds presented here provide a strong foundation for designing potent, broad-spectrum viral ion channel blockers.

We have developed and characterized a potent and cell active tau tubulin kinase 1 and 2 (TTBK1 and TTBK2) inhibitor, 13. Compound 13 demonstrates in-cell, kinome-wide selectivity, and potently inhibits both TTBK1 and TTBK2. As part of our medicinal chemistry campaign, we also identified a structurally similar negative control, compound 5, which lacks in-cell affinity for TTBK1 and TTBK2. Based on their substrates, which include TDP-43, tau, and tubulin, TTBK1 and TTBK2 inhibition has been pursued as a therapeutic approach for Alzheimers disease, frontotemporal lobe dementia, and amyotrophic lateral sclerosis. TTBK2 is also an effector of ciliogenesis, acting in concert with CEP164, CP110, and CEP83 to initiate the biogenesis of primary cilia. The development of selective chemical tools for these kinases facilitates investigation into TTBK1/2-mediated pathways and potential disease-altering ramifications linked to their pharmacological perturbation.

Histone deacetylase 6 (HDAC6) is a class IIb histone deacetylase that regulates diverse cytosolic acetylation through its two catalytic deacetylase domains and a C-terminal zinc finger ubiquitin-binding domain (ZnF-UBD). This ZnF-UBD mediates key protein-protein interactions (PPIs) that couple deacetylation and ubiquitin-dependent degradation. While most HDAC6 inhibitors target the catalytic domains, the ZnF-UBD represents an underexplored target. Here, we validate previously reported small-molecule inhibitors of the HDAC6 ZnF-UBD/ubiquitin interaction and describe novel N-alkyl moieties based on quinazolinone and phthalazinone scaffolds. Starting from known quinazolinone and phthalazinone scaffolds, a literature and modeling-guided scaffold hop revealed potential for an extended phthalazinone series. Results obtained both in fluorescence polarization (FP) and differential scanning fluorimetry (DSF) confirm this hypothesis. Additionally, late-stage diversification yields compounds with improved predicted physicochemical properties. Finally, machine-learning-based co-folding affinity predictions correlate with experimental IC{square}{square} rank order, highlighting their utility in PPI inhibitor design. These studies continue expanding the chemical space of HDAC6 ZnF-UBD inhibitors and build upon existing foundations for future therapeutic and mechanistic exploration of HDAC6- ubiquitin signaling.

Talin-induced integrin activation is a central regulator of cell adhesion and signaling. Intracellular Targeting of this pathway remains to be challenging. Here, we report the structure-guided development of next-generation stapled peptidomimetics derived from the RIAM talin-binding site (TBS). Using biochemical, structural, and cellular analyses, we show that the second-generation S2-TBS enhances talin binding and inhibitory potency but exhibits reduced cell uptake due to an extended linker. Guided by these insights, we designed a minimized third-generation peptide, S3-TBS, which restores structural order, improves thermal stability and cell uptake, and retains strong talin binding. NMR and crystallographic analyses confirm conserved binding to specific interfaces in talin head and talin rod domains. Functionally, optimized peptides potently inhibit talin-mediated integrin interactions and suppress invadopodia-driven matrix degradation in cancer cells. These findings establish key principles for balancing affinity, conformational stability, and molecular size in the design of intracellular stapled peptide inhibitors. HIGHLIGHTSO_LIStructure-guided optimization converts a flexible stapled peptide into a highly ordered, high-affinity talin inhibitor. C_LIO_LIS2-TBS increases binding affinity but introduces conformational heterogeneity due to a longer linker. C_LIO_LIS3-TBS achieves improved stability, reduced size, and a well-defined stapled conformation. C_LIO_LIOptimized peptides inhibit talin-mediated integrin function and suppress cancer cell matrix degradation. C_LI

Calcineurin (Cn) is a protein phosphatase that initiates T-cell activation by dephosphorylating the transcription factor NFAT, driving its nuclear translocation and the transcription of immune-related genes. While clinical immunosuppressants like Cyclosporine A (CsA) potently inhibit Cn, they completely block its catalytic site, leading to non-specific inhibition and severe off-target toxicity. Selectively targeting the specific protein-protein interaction (PPI) between Cn and NFAT presents a safer therapeutic strategy. We previously identified the C16orf74 (C16) peptide as a high-affinity Cn-NFAT PPI inhibitor; however, its utility in cellular systems is restricted by poor membrane permeability. In this study, we evaluated cell-penetrating peptide (CPP) conjugates of C16 with an N-terminus transactivator of transcription (TAT) and polyarginine (R11) to enable efficient intracellular delivery. Structural modeling, fluorescence polarization displacement, and pull-down assays confirmed that the CPP-C16 conjugates retain the ability to compete with an NFAT-derived peptide and bind Cn. Fluorescence microscopy demonstrated efficient intracellular entry of TAT-C16 and R11-C16 in mammalian cells, and effective inhibition of NFAT nuclear translocation and attenuation of downstream NFAT-dependent transcriptional activity of the IL-2 gene in human T cells at concentrations of 10 {micro}M or lower. Crucially, unlike CsA, the CPP-C16 peptides exhibited minimal cytotoxicity even at high concentrations of up to 50 {micro}M, establishing a potential safe therapeutic window. These findings establish CPP-C16 conjugates as effective, cell-permeable, and non-toxic inhibitors of the Cn-NFAT signaling axis, providing the basis for the development of PPI-directed immunosuppressants.

Premarineosin A undergoes rapid, chemoselective C12 halogenation under mild conditions, providing brominated, chlorinated, fluorinated, and iodinated analogs. These derivatives retained potent antiplasmodial activity against both chloroquine-sensitive and -resistant Plasmodium falciparum strains and displayed smaller reductions in potency against the resistant strain than the parent compound.

Homeodomain-interacting protein kinase 4 (HIPK4) remains an understudied member of the dark kinome. While genetic knockout studies suggest roles for HIPK4 in spermiogenesis and cutaneous squamous cell carcinoma, whether these cellular functions can be recapitulated by pharmacological inhibition remains to be determined. However, such investigations have been hampered by a lack of high-quality chemical tools. To address this, we employed a rational design strategy utilizing macrocyclization of a bosutinib-based scaffold. Systematic optimization led to the discovery of AZ137 (28e), a potent and selective HIPK4 inhibitor (IC50 = 11 nM; cellular EC50 = 76 nM). AZ137 exhibits exceptional selectivity across three comprehensive orthogonal panels, high solubility, and no detectable cytotoxicity. Its cellular activity was confirmed in cell-based assays of HIPK4-dependent F-actin remodeling. Together with a negative control compound, this probe set provides a foundational framework for the validating HIPK4 as a therapeutic target and a high-quality resource to elucidate its roles in normal physiology and disease. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/720179v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@12438borg.highwire.dtl.DTLVardef@11083beorg.highwire.dtl.DTLVardef@1395fb4org.highwire.dtl.DTLVardef@1ba3db8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cancer immunotherapy has transformed cancer treatment; however, durable responses remain limited by suppressive myeloid populations within the tumor microenvironment. Leukocyte immunoglobulin-like receptor B4 (LILRB4/ILT3) is an emerging myeloid immune checkpoint implicated in immune evasion and resistance to immunotherapy, yet small molecule targeting of ILT3 remains largely unexplored. Here, we report the discovery of small molecule ILT3 modulators identified using a Dianthus-based temperature-related intensity change (TRIC) screening platform. Screening of an 8,961-member Enamine Library identified multiple direct ILT3 binders, with lead compound ICB-7 demonstrating high-affinity binding to recombinant human ILT3 by microscale thermophoresis and robust cellular target engagement in CETSA assays. Molecular docking and molecular dynamics simulations revealed a stable hydrophobic binding pocket within the D2 domain of ILT3. Functionally, ICB-7 disrupted the ILT3-SCG2 interaction and inhibited downstream SHP1, SHP2, and STAT3 signaling. In patient-derived colorectal cancer and acute myeloid leukemia co-culture models, ICB-7 enhanced cytotoxic T-cell activity, and reduced tumor-cell viability. The compound also demonstrated favorable pharmacokinetic and safety properties together with significant anti-tumor efficacy in the CT26 syngeneic colorectal carcinoma model. Collectively, these findings establish ILT3 as a tractable target for small-molecule immunomodulation and support pharmacological targeting of suppressive myeloid checkpoints as a promising cancer immunotherapy strategy.

CAPON (NOS1AP) is an adaptor protein involved in neuronal nitric oxide synthase (nNOS) signaling and has been implicated in Alzheimers disease (AD), excitotoxicity, and tau-associated neurodegeneration. Here, we report the identification of cyclic peptide ligands targeting CAPON using phage display screening of a disulfide-constrained peptide library. Phage enrichment, ELISA validation, microscale thermophoresis (MST), and biolayer interferometry (BLI) identified CAP1 as the lead peptide, exhibiting low micromolar binding affinity toward CAPON. Computational studies further supported stable CAPON-CAP1 interactions through complementary hydrophobic and electrostatic contacts. Functionally, CAP1 attenuated A{beta}42-induced neuronal toxicity, suppressed NMDA-driven nitric oxide production, and reduced pathological tau phosphorylation in neuronal models under AD-relevant stress conditions. In addition, CAP1 demonstrated favorable preliminary pharmacokinetic properties, including good aqueous solubility, plasma stability, and measurable membrane permeability. Collectively, these findings establish the first cyclic peptide ligands targeting CAPON and identify CAP1 as a promising scaffold for modulation of CAPON-dependent neurodegenerative signaling.

We report here a method for the chemical synthesis of Fmocprotected -hydroxyglyine (-OH-Gly) dipeptides. Our method features operational simplicity and is compatible with protecting groups for peptide synthesis. Utility is then demonstrated through substitution at the -OH-Gly position to yield myriad non-natural amino acid-containing dipeptide fragments.

Positive allosteric modulators (PAMs) of the M4 muscarinic acetylcholine receptor (mAChR) represent a promising therapeutic strategy for treating cognitive deficits and neuropsychiatric disorders. While first-generation M4 mAChR PAMs, like LY2033298, demonstrated proof-of-concept, second-generation compounds, such as MK-97, exhibit substantially improved potency and reduced species variability. Here we report the cryo-EM structure of the M4 mAChR bound to the endogenous agonist, acetylcholine, and MK-97 at 2.7 [A] resolution, revealing the molecular basis for improved M4 mAChR PAM activity. MK-97 adopts a distinctive boomerang-shaped conformation within the extracellular-facing allosteric binding site, with a central pyridine vertex, a lower cyclopentylmethylpyrazole arm extending toward the floor of the orthosteric site, and an upper isoindolinone arm projecting toward extracellular loop 2 (ECL2). This extended binding mode establishes a distributed interaction network across transmembrane helices TM2, TM3, TM5, TM6, and TM7, with key contacts including a hydrogen bond with Y922.64 and a {pi}-{pi} stacking interaction with W4357.35. Integration of structural data, molecular dynamics simulations, and mutagenesis validation reveals that the high affinity of MK-97 derives from optimized engagement across all three binding regions rather than dependence on any single critical contact. Insights from comprehensive structure-activity relationship (SAR) studies provide a molecular framework for the rational design of next-generation M4 mAChR PAMs with improved pharmacological properties. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/723386v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1ab9c78org.highwire.dtl.DTLVardef@1adb532org.highwire.dtl.DTLVardef@152f9f7org.highwire.dtl.DTLVardef@990768_HPS_FORMAT_FIGEXP M_FIG C_FIG

Epigenetics regulate cell-cycle kinetics, differentiation, apoptosis, and migration. Nuclear receptor-binding SET Domain (NSD) histone methyltransferases represent a family of oncoproteins with aberrant expression in cancer. Emerging reports suggest that NSD1 could be an attractive target as its expression is correlated with poor prognosis and tumorigenesis. Previously, we reported the target validation and structure-based virtual screening against NSD1, leading to the selection of several hit molecules with relatively high docking and MMGBSA delta G Bind scores. One of the best-fit molecules identified was 5-O-sulfamoyl adenosine (5-SA) and was compared with the S-Adenosyl-l-Cysteine (SAC), a structural analog of S-Adenosyl-l-Methionine (SAM) for its inhibitory activity against NSD1. IC50 values for 5-SA and SAC against NSD1 were 53.819 {micro}M and 115.003 {micro}M respectively. 5-SA significantly reduced the viability of DU145 and HepG2 cells with IC50 values calculated as 198{micro}M and 168.3 {micro}M respectively. It also reduced the RNA and protein expression levels of NSD1 and subsequently prevented dimethylation of lysine 36 on histone H3 (H3K36me2). Furthermore, 5-SA impeded proliferation, and migration, altered the cell cycle phase, and induced cell apoptosis. Interestingly, 5-SA potentiated the anticancer activity of 5-Fluorouracil (5-FU) against cancer cells. The xenograft model of prostate cancer also showed that 5-SA significantly reduced the tumor growth kinetics. However, the combination of 5-SA and 5-FU synergistically reduced tumor growth and improved survival of animals. To the best of our knowledge, we report for the first time that 5-SA mediated inhibition of NSD1 enhanced the tumor sensitivity to 5-FU and thereby, improved the tumor growth and progression. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/731397v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@e2f0bdorg.highwire.dtl.DTLVardef@12b2ca6org.highwire.dtl.DTLVardef@1807613org.highwire.dtl.DTLVardef@c7f8f0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Small molecule targeting of suppressive myeloid immune checkpoints remains a major challenge in cancer immunotherapy, particularly for non-enzymatic receptors lacking conventional druggable active sites. Leukocyte immunoglobulin-like receptor B4 (LILRB4/ILT3) is an immunosuppressive myeloid checkpoint implicated in tumor immune evasion, T-cell dysfunction, and resistance to immunotherapy across both solid and hematologic malignancies. Here, we report the discovery and characterization of GL-4512, a direct small molecule modulator of LILRB4 identified through a Dianthus-based temperature-related intensity change (TRIC) screening platform. Orthogonal biophysical studies, including microscale thermophoresis, surface plasmon resonance, and cellular thermal shift assays, confirmed direct target engagement with nanomolar affinity. Extensive microsecond molecular dynamics simulations combined with site-directed mutagenesis identified a previously unrecognized ligandable pocket within the flexible extracellular domain of LILRB4. Functionally, GL-4512 disrupted the immunosuppressive LILRB4-SCG2 signaling axis and suppressed downstream SHP1/SHP2 and STAT3 signaling. In patient-derived colorectal cancer and acute myeloid leukemia co-culture systems, pharmacological inhibition of LILRB4 restored anti-tumor immune activity, enhanced IFN-{gamma} and IL-2 production, increased cytotoxic T-cell activation, and reduced tumor-cell viability. GL-4512 additionally demonstrated favorable pharmacokinetic and safety properties supporting oral in vivo administration. In immunocompetent CT26 syngeneic colorectal tumors, once-daily oral treatment significantly suppressed tumor growth and enhanced intratumoral immune activation. Collectively, these findings establish LILRB4 as a tractable target for direct small molecule immunomodulation and support therapeutic targeting of suppressive myeloid immune checkpoints for cancer using non-biologic modalities.

Antibody and protein-drug conjugates (XDCs) have emerged as promising cancer therapeutics, yet their clinical utility remains constrained by dose-limiting toxicities and narrow therapeutic windows. These safety challenges stem primarily from two factors: premature payload release during systemic circulation, and poor physicochemical properties inherent to the hydrophobic cytotoxic drugs they carry. Prior strategies attempted to address these limitations by appending water-soluble tags to reduce overall conjugate hydrophobicity, but achieved only modest improvements. As a result, the hydrophobic nature of cytotoxic payloads has remained a persistent obstacle in XDC development. Here, we report a fundamentally different chemical strategy that reframes this liability as a design opportunity. Rather than masking drug hydrophobicity, we exploit it as the driving force for self-assembly of facially amphiphilic protein-drug conjugates with programmable drug moieties (PDCs). In this architecture, the hydrophobic cytotoxic drug and the hydrophilic protein serve as the core and shell, respectively, spontaneously assembling into monodisperse, well-defined spherical protein nanotherapeutics of controlled size. This design principle transforms a longstanding physicochemical challenge into a functional engineering tool, enabling precise nanostructure formation without sacrificing potency. In vitro studies confirm that the resulting nanotherapeutics effectively kill cancer cells, establishing a strong foundation for further therapeutic development.