ChemMedChem

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.

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.

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

Dengue Virus Serotype 2 is a human pathogenic flavivirus that encodes a non-structural protein 3 (DEN2-NS3) containing a helicase domain essential for viral replication. DEN2-NS3 utilizes energy derived from NTP hydrolysis to unwind dsRNA and dsDNA. A galloylated catechin, (-)-epigallocatechin gallate (EGCG), was previously reported to be highly potent against the Zika Virus NS3 helicase, with an IC50 value observed at 295.7 nM. This prompted an investigation to determine if three catechins, namely, (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and EGCG, would act as potent inhibitors of DEN2-NS3. Enzyme-inhibition assays revealed that the helicase catalytic domain, DEN2-NS3(S171-K618), is strongly inhibited by these galloylated catechins. We observed Ki values of 400 {+/-} 86.6 nM for EGCG (mixed-mode inhibition with respect to ATP) and 550 {+/-} 250 nM for ECG (uncompetitive inhibition with respect to ATP). Furthermore, using a computational workflow starting with SiteMap, we provide evidence that a highly druggable pocket exists within the RNA-binding cavity, involving residues ASP290, ARG387, ASP409, MET429, HIS487, ASP541, ARG599, and ASP603. These catechins were each analyzed through 200-ns molecular dynamics (MD) simulations to evaluate the binding stability within the target DEN2-NS3 binding pocket. Computational results revealed that EGCG and ECG maintained high stability, forming shared, highly persistent amino acid contacts (>45% occupancy) with ASP603, ARG599, ASP541, and ARG387. In conclusion, we have demonstrated that EGCG and ECG achieve strong binding and allosteric disruption of the critical RNA-binding channel. We suggest that future structural optimization of these compounds into stable prodrug derivatives could yield promising antiviral therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/733882v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@2db363org.highwire.dtl.DTLVardef@5c2fdaorg.highwire.dtl.DTLVardef@49bf8eorg.highwire.dtl.DTLVardef@1bf31f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

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.

Prostate cancer (PCa) is one of the principal contributors to health burden in the aging male population. PCa develops through dysregulation of androgen receptor (AR) signaling pathways. Despite improvements in diagnostic techniques and interventions, no pharmacological measures with long term efficacy have been established once PCa advances to castration resistant prostate cancer (CRPC). To circumvent this issue, tetra-aryl cyclobutanes (CBs) have been proposed as structurally distinct compounds with a mechanism of action differing from traditional androgen receptor signaling inhibitor (ARSIs). Here, we apply principles of crystal engineering and solid state synthesis to expand the class of CBs through strategic derivatization. The synthesis of the CB occurs quantitatively, producing no side products and eliminating the need for product purification. We demonstrate how head-to-tail stacking interactions of halo-pyrimidine rings can be exploited to stack and align unsymmetrical alkenes to undergo [2+2] photodimerization to generate the CB in the solid state. We examine the structure-function relationships of CBs in vitro by profiling AR mediated transcriptional activity, receptor translocation, and cell viability. Moreover, we explore and identify putative binding interactions within CB/AR complexes and establish an adaptive ligand-binding potential using molecular docking platforms. In total, our data suggests that CBs have unexploited therapeutic potential in CRPC and that green chemistry and crystal engineering principles offer a unique route to generating these drug candidates.

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.

RAB27A and RAB27B are homologous small GTPases that regulate intracellular vesicle trafficking, orchestrating endocytic and exocytic processes that affect cellular communication, immune responses, and dynamics of the cellular microenvironment. Through their interactions with effector proteins, RAB27A/B play roles in tumor metastasis and chronic inflammation. However, pharmacological modulation of their activity faces challenges typical of small GTPases, including a lack of well-defined pockets outside the conserved GTP binding site, and large RAB27-effector protein-protein interaction (PPI) surfaces. Here, we present the discovery and development of the first cell-active, rationally designed covalent inhibitors of the RAB27-effector interaction, targeting a non-conserved cysteine residue flanking the PPI interface. An electrophile-first biochemical screen led to a novel class of acrylamide covalent inhibitors, and X-ray crystallography structure-guided design led to optimized inhibitors and probes that enantioselectively target RAB27A/B-Cys123 in cells. Potency and selectivity were confirmed through biochemical and cellular assays, including chemical proteomics and phenotype recapitulation in melanocytes alongside a matched inactive enantioprobe control. In contrast, a previously reported compound, Nexinhib-20, was found to be toxic and to exert its activity through non-selective reactivity. This work provides the first toolbox of cell-active chemical probes for RAB27 which can be used in future studies to shed light on the function of this protein and its potential as a therapeutic target.

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.

Cetuximab is a chimeric IgG1 monoclonal antibody that has been a cornerstone therapy for EGFR-driven malignancies for nearly two decades. Its therapeutic activity is governed by competitive displacement of endogenous EGFR ligands, making binding affinity a direct determinant of clinical efficacy. We applied ConvergeAB, a target-aware antibody design platform, in a fully zero-shot configuration to generate a biobetter version of cetuximab. The lead Converge-designed antibody binds EGFR with a mean KD of 315 pM -- approximately 2.1-fold tighter than cetuximab (673 pM) and 4.4-fold tighter than a recently published, computationally designed anti-EGFR antibody from Cradle Bio (1.38 nM). The affinity gain arises from six substitutions that leave the global paratope architecture intact (C RMSD 0.15 [A] vs cetuximab) and instead optimize the binding interface through localized packing and electrostatic adjustments. A panel of biophysical and developability assays -- HIC, DLS, DSF, and PSR ELISA -- shows that the Converge variant matches or exceeds cetuximab on monomericity, monodispersity, polyspecificity, and thermal stability, while remaining within a developable hydrophobicity envelope. Together, these data demonstrate that a single zero-shot ConvergeAB campaign can deliver a biobetter molecule with significantly improved affinity and a clean developability profile, without compromising the parental antibodys drug-like properties.

Structure-based drug discovery is known to apply computational methods in a tiered hierarchy, with each layer narrowing the candidate set and refining the binding picture before committing to the next, more expensive step. We present a four-tiered computational benchmarking study evaluating five engines against a panel of 36 compounds targeting B-secretase 1 (BACE1), a validated Alzheimer's disease target with extensive co-crystal ground truth. This study evaluates Flexible Docking and Boltz2 Cofolding as the primary tier, followed by Ensemble Docking, and then Protein-Ligand MD with MM/PBSA and MM/GBSA post-processing. This is then concluded with Relative Binding Free Energy Perturbation (RevFEP) as the terminal refinement layer. Each method was benchmarked against the experimental binding free energies derived from the co-crystal structures spanning -7.85 to -11.35 kcal/mol. Our findings revealed that Flexible Docking reproduced the co-crystal binding mode for 35 of 36 ligands (97.2% within 2.0 A RMSD) but did not rank potency at this resolution. Boltz2 CoFolding provided an orthogonal structural cross-check with a receptor backbone RMSD of 0.293 A against the experimental co-crystal structure. Ensemble Docking identified the optimal receptor conformation for downstream FEP setup. MD with MM/GBSA decomposition identified van der Waals complementarity as the primary potency driver (Pearson r = +0.855, R2 = 0.732 on a 10-compound subset). RevFEP delivered the highest affinity correlation of any method (Pearson r = +0.662, R2 = 0.438, Spearman p = +0.624, mean absolute error 1.02 kcal/mol across all 36 ligands), resolving potency differences within a narrow 3.5 kcal/mol congeneric window that no other engine could discriminate. We characterize what each engine contributes independently and where RevFEP delivers signals no other engine achieves.

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.

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.

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

Mycobacterium tuberculosis possesses a flexible metabolic system helping it to survive inside the host. The type II NADH dehydrogenase, composed of Ndh and NdhA, essential for bacilli, is a promising drug target. Based on ATP depletion values, two quinoline scaffolds were shortlisted after screening of a library of drug like molecules. Structurally, both 64-9C and 64-9D carry ester moieties at the 5- and 8-positions of the quinoline core, respectively. Ease to re-synthesise 64-9D resulted in synthesis of a focused library of compounds, with MIC values of 0.25-4 g/mL, consistent with ATP depletion. These compounds exhibited bactericidal activity against non-replicating mycobacteria, and showed potent efficacy against multidrug-resistant isolates. Altered, intracellular NADH/NAD+ ratio and reduced respiration was indicative of oxidative phosphorylation inhibition. Inhibition of the purified recombinant NDH protein uncompetitively, SNPs in gene encoding NDH-2 for selected one step mutants and, molecular modelling of 4FQN and 2FQN validated NDH-2 as a target for these compounds. The derivative 2FQN exhibited dose-dependent bactericidal efficacy in mice, underscoring the potential of the series as a promising anti-tuberculosis candidates.

Efficient extrahepatic delivery of siRNAs remains a major limitation for broadening their therapeutic potential. Using a modular, orthogonal click chemistry platform, we generated 28 siRNA conjugates varying in ligand class, valency, and spatial arrangement. Following systemic administration, fatty acid conjugates - particularly palmitic acid (C16) - outperformed sterol- and phospholipid-based designs in promoting extrahepatic gene silencing, with preferential activity observed in heart and skeletal muscle. Increasing ligand valency through 3',5'-bis-conjugation generally enhanced activity compared to 5-mono conjugation. Nevertheless, bis-C22 conjugates showed increased hepatic activity, suggesting a shift in tissue distribution linked to hydrophobicity. Architectural parameters further modulated outcomes: Branched 5' C16 conjugates, bearing two lipids on one terminus, were markedly less active than their bis counterparts and required short PEG spacers to restore activity. Notably, bis-lipid conjugation strategies that enhanced extrahepatic activity for an siRNA did not translate to an ASO gapmer, underscoring modality-specific constraints. Together, these findings delineate structure-activity relationships and establish bis-fatty-acid conjugation as a robust design principle for achieving extrahepatic RNAi. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/726808v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@287a47org.highwire.dtl.DTLVardef@17407eborg.highwire.dtl.DTLVardef@b40435org.highwire.dtl.DTLVardef@804352_HPS_FORMAT_FIGEXP M_FIG C_FIG

In a recent study of a mice model it was suggested that after myocardial infarction Serum Amyloid A (SAA) aggregates are formed that contribute to the long-term complications of the infarct, and that a similar mechanism may exist for humans. Motivated by this hypothesis we have designed four peptide candidates that may interfere with formation of SAA3 fibrils, and using all-atom molecular dynamics have evaluated their ability to destabilize SAA fibrils. As the lifetime of peptide drugs can be increased by replacing L-amino acids with their mirror D-amino acids, we have built the peptides from D-amino acids. We identify two of these peptides, DRI-R5S and DRI-H6A, as promising drug candidates.

"Mirror life", self-replicating organisms composed of non-natural-chirality biomacromolecules, presents a future threat with potentially global consequences. Consequently, there is strong agreement among experts that it should not be created. However, there is some disagreement over how effective existing medical countermeasures might prove against mirror bacteria in the event that they were created. Here, we leverage computational chemistry methods including docking and molecular dynamics to determine the likely binding efficacy of existing antibiotics against natural and mirror bacterial protein targets. We find that most existing antibiotics fail to bind to mirror bacterial protein targets, unlike their natural-chirality targets. This suggests altered binding of current medical countermeasures, which may impact the antimicrobial activity against mirror bacteria were the latter were created.

c-Myc is a transcription factor that drives tumorigenesis in many cancers. It is notoriously difficult to directly target c-Myc, mainly due to its lack of well-defined druggable pockets. O-linked {beta}-N-acetylglucosamine modification (O-GlcNAcylation) is a post-translational modification (PTM) playing an important role in regulating c-Myc functions in cancer. However, previous studies have primarily relied on global perturbations to investigate c-Myc O-GlcNAcylation, making it difficult to determine its direct functional consequences due to concurrent cellular effects. Here, we report a bifunctional O-GlcNAcylation TArgeting Chimera (OGTAC) molecule, which can induce the proximity of c-Myc and O-GlcNAc transferase (OGT) in living cells, thereby enhancing the O-GlcNAcylation of c-Myc. The c-Myc-targeting OGTAC exhibits anti-proliferation effect against cancer cells. Mapping of c-Myc occupancy on genome indicates that OGTAC rewires c-Myc transcriptional activity and reprograms expression of the downstream oncogene MALAT1, in an O-GlcNAcylation-dependent manner. Overall, OGTAC presents a novel chemically induced proximity (CIP)-based tool to target and rewire c-Myc activity in cancer. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/722559v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@d1c640org.highwire.dtl.DTLVardef@2eb70corg.highwire.dtl.DTLVardef@f38970org.highwire.dtl.DTLVardef@c421c8_HPS_FORMAT_FIGEXP M_FIG C_FIG