European Journal of Medicinal Chemistry

Protein Kinase Novel 3 (PKN3) understudied kinase with a diverse array of biological functions that are yet to be fully defined. Here, we report the design and development of a novel advanced functional chemical tool inhibitor for PKN3. A pyridyl imidazole series has been synthesized and evaluated against PKN3 in vitro and in cells. These efforts led to the discovery of 6e (URS03-06), a submicromolar cell active functional inhibitor with a narrow kinome spectrum, to enable the elucidation and interrogation of PKN3 cellular biology.

Histone deacetylases (HDACs) are pivotal epigenetic regulators that modulate diverse cellular pathways by removing acetyl groups from lysine residues on both histone and non-histone proteins. Histone deacetylase 11 (HDAC11), the sole member of class IV HDACs, exhibits both deacetylation and fatty acid deacylation activities. Accumulating evidence implicates HDAC11 as a key epigenetic regulator of fundamental cellular processes, including metabolism, immune responses, and tissue development. Dysregulation of HDAC11 activity has been associated with inflammatory diseases, metabolic disorders, neurodegenerative conditions, and cancer, highlighting its potential as a therapeutic target. Although several HDAC11-specific inhibitors have been identified, none have progressed to clinical development. In this study, we aimed to discover HDAC11-selective inhibitors by integrating in silico and in vitro validation approaches. Homology modelling of the HDAC11 structure was conducted, followed by model validation, structure-based virtual screening, molecular dynamics (MD) simulations, and binding free energy calculations. We identified and validated three lead compounds and their intermediates using biochemical and cell-based assays. Fluorescence-based and HPLC-based enzymatic assays demonstrated potent inhibition of both the deacetylase and deacylase activities of HDAC11, with Inhibitor 6 and Inhibitor 3 exhibiting the strongest effects among the six compounds tested. Further, a decrease in lipid accumulation, reduced stability of the HDAC11 substrate SHMT2, as determined by immunoblot analysis and decreased cell viability, as assessed by MTT assay, confirmed HDAC11 inhibition in cellular models. The study shows that new HDAC11 inhibitors significantly reduce the viability of breast cancer cells and induce apoptosis; inhibitor 6, in particular, showed high potency, similar to the reference compound SIS-17. Flow cytometry showed that treated MDA-MB-231 cells exhibited cell-cycle arrest and increased apoptosis, a finding further confirmed by Annexin V/PI staining. Molecular analysis showed that BAX increased while BCL2 decreased, indicating that apoptotic pathways were activated in novel compound-treated MDA-MB-231 cells. The results suggest that inhibiting HDAC11 is an effective way to induce cancer cell death and provide a basis for further assessment of these compounds as potential treatments for breast cancer. Collectively, this study identifies novel zinc-chelating HDAC11 inhibitors containing a nitro-sp2 group, providing promising candidates for further therapeutic development.

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

IL-4Ra is a key regulatory receptor for type 2 inflammatory responses, signal transduce from IL-4 and IL-13 through binding with IL-13Ra or the gamma c chain to activate the downstream JAK1-STAT6 pathway. IL-4Ra is currently the most successful "golden target" in the field of allergic disease therapeutics. Its representative monoclonal antibody drug, dupilumab, through the dual blockade mechanism of IL-4/IL-13 has pioneered a new era of precision therapy for type 2 inflammation. In our manuscript, we employed large-scale deep learning-based computational design methods to de novo design mini-protein antagonists specific for both human and mouse IL-4Ra. The binding affinity was improved from 22.1 nM to 569 pM through partial diffusion. The design accuracy and binding specificity were verified through X-ray crystallography and biochemical studies. In vitro IL4/IL13 signal blockade assays revealed that de novo designed monomeric mini-protein antagonist exhibited comparable blockade ability to bivalent dupilumab. In vivo pharmacokinetic half-life studies demonstrated that fusion to an HSA-binding domain extended the half-life of the mini-protein antagonist from 2.7 hours to 60.6 hours. The IL-4Ra mini-protein antagonist had excellent expression levels, solubility and thermal stability. The IL4/IL13 signal blockade ability remained unchanged even after being heating to 95 degrees. In conclusion, through large-scale cluster computing and deep learning-based de novo design, we developed well-performed IL-4Ra mini-protein antagonist, and demonstrates certain potential for drug development.

Mucopolysaccharidosis IIIC (Sanfilippo syndrome type C) is a rare lysosomal storage disorder caused by loss-of-function mutations in HGSNAT, which encodes an enzyme involved in heparan sulfate (HS) degradation, leading to impaired HS catabolism, lysosomal accumulation, and progressive neurodegeneration. Because enzyme replacement therapies have limited penetration across the blood-brain barrier, substrate-reduction therapy represents an alternative therapeutic strategy. Here, N-deacetylase/N-sulfotransferase 1 (NDST1), a key enzyme responsible for HS biosynthesis, was investigated as a potential substrate-reduction target. A structure-based computational pipeline was used to identify and evaluate inhibitors targeting the NDST1 sulfotransferase domain. Approximately 4.1 million drug-like compounds and FDA-approved drugs were screened by molecular docking, followed by pharmacokinetic filtering, molecular dynamics simulations, and MM/PBSA binding free energy calculations. In parallel, peptide binders targeting the same site were generated using diffusion-based protein design and evaluated using molecular dynamics and MM/GBSA analysis. Four chemically distinct small-molecule scaffolds and three peptide candidates were identified as stable binders to the NDST1 active site. The lead small-molecule candidate exhibited a predicted binding free energy of -13.36 {+/-} 5.87 kcal mol-1. These provide a focused set of candidates for further investigation and support the feasibility of targeting NDST1 as a substrate-reduction strategy for MPS IIIC.

Synaptic vesicle glycoprotein 2C (SV2C) is a vesicular protein enriched in dopaminergic neurons of the basal ganglia that modulates dopamine storage and release, and its disruption is implicated in Parkinsons disease (PD). Despite strong genetic and pathological links to PD, there are no selective small-molecule probes for SV2C. Here, we describe an AI-enhanced virtual screening (VS) and experimental campaign that identified multiple novel chemotypes with low-micromolar affinity and marked selectivity for SV2C over SV2A and SV2B, starting from a large, general-purpose commercial library. Because no full-length high-resolution SV2C structure was available, we built a homology model using SV2A cryo-EM structures as templates and characterized its conformational landscape by molecular dynamics (MD) and Gaussian accelerated MD (GaMD) simulations in apo form and in complex with known SV2 ligands (plosaracetam, levetiracetam, brivaracetam, and padsevonil). A convolutional neural network-based scoring function (CNN VS), retrospectively validated on a manually curated 39-ligand SV2A benchmark (r = 0.72 vs experimental pIC50), was then applied in a multi-stage funnel to 5.96 million Mcule in-stock compounds, which were sequentially filtered to 3.19 million CNS-relevant molecules before docking and rescoring. From 94 VS-prioritized candidates, 71 compounds were experimentally profiled in an orthogonal primary assay cascade combining a thermal shift assay (TSA) with a [3H]-padsevonil scintillation proximity assay (SPA), followed by Ki determination and isoform selectivity profiling for key hits. This campaign yielded 22 active molecules (31% hit rate) that naturally segregated into two categories: compounds that showed primary site competition, and compounds that did not show primary site competition with [3H]-padsevonil. A subset of competitor compounds also showed thermostabilization activity. Among these, compounds 36 and 56 emerged as particularly attractive leads, with Ki values of 24.6 {micro}M and 3.25 {micro}M at SV2C, respectively, and >10-fold selectivity versus SV2A; compound 56 also maintained[~] 12-fold selectivity relative to SV2B. A complementary subset of SV2C-selective hits behaved as padsevonil-site competitors, providing a lead set that will serve as a template for functional characterization and future drug development for conditions that affect dopaminergic signaling. Docking analysis suggests a common binding mode anchored by conserved tryptophan residues in the SV2 pocket, a prediction independently confirmed by an unpublished SV2A- plosaracetam cryo-EM structure showing 0.76 [A] binding-site C RMSD relative to the SV2C model and complete conservation of the tryptophan cage. Subtle differences in the luminal domain and transmembrane region point to the structural determinants underlying isoform selectivity. Collectively, these results demonstrate that an AI-driven VS pipeline, tightly integrated with medium-throughput biophysical assays, can deliver selective SV2C binders from a general chemical library on a structurally under-characterized membrane target. The identified hits provide multiple starting points for hit-to-lead optimization and tools for probing SV2C biology and its role in PD.

Breast cancer (BC) is the second most common noncutaneous cancer and the second leading cause of cancer-related death in women. BC is classified into three primary subtypes, with triple-negative breast cancer (TNBC) having the poorest prognosis because it lacks specific targetable markers. Preclinical studies on TNBC indicated a common occurrence of diminished tumor-suppressor activity of PTEN, activating the PI3K/AKT/mTOR signaling pathway. Notably, published studies reveal that the WWP1 enzyme plays a pivotal role in driving PTEN degradation via ubiquitination, unveiling a promising therapeutic target for treating TNBC. In the search of new WWP1 inhibitors, we used artificial intelligence (AI)-driven computational strategies for de novo design of peptide-based WWP1 inhibitors and identified a hexapeptide, termed WI23-B, which demonstrated high nanomolar binding affinity to WWP1. In TR-FRET enzymatic assays, WI23-B inhibited WWP1 activity with an IC of approximately 11 {micro}M. In MCF7 and MDA-MB-231 breast cancer cell lines, WI23-B showed promising cytotoxic efficacy, particularly in combination with the PI3K inhibitor BYL719, also when it was loaded into nanocapsules. Collectively, these findings highlight WI23-B as a promising lead peptide with potent WWP1 inhibitory activity and synergistic antiproliferative effects when combined with PI3K inhibitors. While further structural optimization is required to enhance its potency and pharmacological properties, our results provide a strong foundation for the development of next-generation WWP1 inhibitors. Such agents have the potential to reshape therapeutic strategies for BC and TNBC by enabling more effective and less toxic treatment regimens, ultimately reducing the reliance on high-dose chemotherapy and minimizing adverse effects.

Lead optimization, the systematic refinement of therapeutic compounds through iterative structural modification, faces a dual challenge in modern drug discovery: navigating astronomically vast molecular design spaces while balancing conflicting demands on potency, pharmacokinetics, and safety. We present MASCOT (Multi-Agent SearCh for molecular OpTimization), a role-specialized multi-agent framework for molecular optimization. Integrated with a chemically constrained graph-editing search, MASCOT coordinates three specialized agents: a trade-off agent that reprioritizes competing objectives, a strategy agent that adapts how molecular edits are proposed, and a reflection agent that distills lessons from previous decisions. Computational experiments showed that MASCOT achieved the best performance over competing methods on six benchmark settings. On the SARS-CoV-2 main protease task, its mean docking-score improvement was 3.6 times that of the strongest baseline. Applied to the clinically used anesthetic remimazolam (RM), MASCOT prioritized RM-1, which showed a shorter liver microsomal half-life, higher brain exposure, and a larger therapeutic index than RM. Subsequent derivative design yielded RM-7. Extensive animal studies established RM-7 as a rapid-recovery intravenous anesthetic candidate with greater potency, faster functional recovery, a wider safety margin, and preserved flumazenil reversibility. These results demonstrate that multi-agent coordination can link adaptive molecular search to medicinal chemistry and experimental pharmacology.

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

AlphaFold 3 (AF3) and Boltz-2 are state-of-the-art AI-based tools for biomolecular structure prediction, but whether their predictions provide useful guidance for lead optimization, SAR interpretation, and virtual screening remains insufficiently characterized. We benchmarked their performance using newly determined soluble epoxide hydrolase co-crystal structures and matched activity data together with a curated post-training-cutoff dataset spanning kinases, allosteric modulators, covalent systems, PROTACs, molecular glues, fragments, membrane proteins, RNA binders, and activity-cliff pairs. Both models recovered canonical orthosteric enzyme and kinase complexes, including key DFG/C conformational states, whereas allosteric, membrane-protein, and induced-proximity complexes remained challenging. Pharmacophore RMSD was often lower than overall ligand RMSD, indicating preservation of key recognition features despite imperfect whole-ligand alignment. AF3 minPAE correlated with pose accuracy, and very low minPAE values (<0.85 A) were strongly enriched for accurate poses. Model confidence scores were not associated with experimental activity, whereas Boltz-2 predicted affinity captured relative activity trends and distinguished the activity-cliff pair, although its performance varied across ligand series.

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

Disulfide-rich peptides possess exceptional potency and selectivity but are often limited by the instability and synthetic challenges associated with native disulfide bonds. Here, we report the design, synthesis, pharmacological evaluation, and structural characterisation of triazole-based peptidomimetics of the -GI conotoxin, a selective antagonist of the muscle-type nicotinic acetylcholine receptor (nAChR). A series of 1,4- and 1,5-disubstituted triazole analogues were prepared entirely on resin using CuAAC and RuAAC chemistry to replace the native Cys3/13 disulfide bridge. Functional evaluation against human muscle nAChRs revealed that 1,5-triazole analogues retained low-nanomolar potency, with the lead mimetic exhibiting activity comparable to native -GI. Cryo-electron microscopy of the lead compound bound to the muscle-type nAChR provided the first structure of a disulfide-isostere peptidomimetic in complex with a membrane receptor. The structure demonstrates that the 1,5-triazole reproduces the native peptide fold with high fidelity while contributing receptor-facing interactions not available to the native disulfide bridge. Molecular dynamics simulations further revealed conserved hydration networks and similar conformational sampling between the native peptide and lead mimetic. Together, these findings establish triazoles as effective disulfide surrogates and provide a structural framework for the rational design of stabilised conotoxin therapeutics.

The blood-brain barrier (BBB) is a highly selective cell layer that restricts the diffusion of diverse chemical entities into the central nervous system (CNS) from systemic circulation. Macromolecular therapeutics including oligonucleotides, peptides, and monoclonal antibodies exhibit only minimal brain distribution after systemic dosing due to exclusion by the BBB. Receptor-mediated transcytosis (RMT) has evolved to transport vital cargo across the BBB through a specialized vesicular transport pathway. Transferrin receptor 1 (TfR1) shuttles transferrin, its natural ligand, across the BBB, as well as TfR1-binding IgG antibodies and conjugates. Here, we describe a novel monovalent TfR1-binding VHH-Fc for the delivery of oligonucleotide cargo, including antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) across the BBB in rodents and non-human primates (NHPs), supporting the translational potential of the VHH-antisense RMT platform for the treatment of neurological disorders. We explore the role of binding affinity, conjugation site, drug-antibody ratio (DAR), and conjugation chemistry, and determine that binding affinity, DAR and conjugation site are major determinants of RMT capacity and brain activity of siRNAs delivered across the BBB. Graphical Abstract / Highlights O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/744307v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@d1d648org.highwire.dtl.DTLVardef@4b22d3org.highwire.dtl.DTLVardef@db8b6borg.highwire.dtl.DTLVardef@19e5ac3_HPS_FORMAT_FIGEXP M_FIG C_FIG - Anti-TfR1 (-TfR1) VHH ligands formatted as heterodimeric, 2-chain monovalent VHH-Fc were engineered for conjugation to siRNA and ASO. - Systematic in vivo evaluation of VHH clones spanning a range of TfR1 binding affinities revealed a relationship between TfR1 binding affinity and the CNS activity of intravenously dosed VHH-Fc-siRNA conjugates. - By optimizing TfR1 binding affinity, conjugation site, and conjugation chemistry, we identified VHH-Fc-siRNA molecules that efficiently cross the BBB via receptor-mediated transcytosis and reduce target mRNA across CNS tissues, including deeper brain regions, after intravenous (IV) or subcutaneous (SC) dosing in mice and non-human primates (NHPs).

The leukocyte-specific {beta}2-integrin receptor family exerts a wide range of functions: {beta}2-integrins are involved in leukocyte trafficking, where they mediate cell adhesion during inflammatory responses via binding to ICAM-1, ICAM-2, or JAM-C. Furthermore, they are essential for the recognition and phagocytosis of pathogens opsonized by complement. Accordingly, the {beta}2-integrin family is known to be involved in autoimmune and inflammatory diseases, such as systemic lupus erythematosus. Owing to their complex biology, involving multiple conformational transitions, different signaling pathways, and a broad spectrum of ligands, the development of {beta}2-integrin-targeted probes and therapeutics has remained challenging. We aimed to develop macrocyclic peptides, derived from phage display screening, which can be used to unravel ligand binding profiles of {beta}2-integrins with an emphasis on the I domain. The selection of suitable lead peptides, and the characterization of their interaction profiles with different I domains, was enabled by an established in-vitro assay platform. Various peptide sequences were enriched during several rounds of phage display against the I-domain of CR3, of which two peptides with particularly low micromolar binding affinity were further characterized. Both peptides showed direct binding to {beta}2-integrin I-domains and, in a competitive assay, dose-dependent inhibition of the I-domains interactions with their main ligands iC3b and ICAM-1, respectively. These ligand-interfering properties were confirmed in bead- and cell-based adhesion assays. The modulators developed here are expected to provide valuable insight into the (patho-)physiology of CR3 and the other members of the {beta}2-integrin family, as the two peptides were able to compete with different ligands. In the future, this may help to identify potential therapeutic approaches for autoimmune, inflammatory, and age-related diseases.

Small-molecule drug discovery relies on identifying compounds that modulate specific protein targets, a process often hindered by cellular complexity. Through phenotypic screening of a kinase-focused diazaquinazoline library, we serendipitously identified CEM198 as the first high-affinity ligand of tubulin-tyrosine ligase (TTL). Functional assays combining live-cell TTL inhibition, microtubule polymerization, cell cycle analysis, and proteomics revealed that CEM198 acts through a dual mechanism: directly binding to TTL and altering /{beta}-tubulin conformation. This interaction restricts -tubulin tyrosination and disrupts tubulin polymerization, leading to microtubule destabilization. The differential effects observed between SH-SY5Y and HEK293T cells indicate that effective TTL inhibition depends on both direct binding and structural modulation of the tubulin heterodimer. These findings introduce CEM198 as a chemical probe for investigating the tubulin tyrosination-detyrosination and demonstrate the potential of chemoproteomics to uncover novel modulators of microtubule dynamics.

BackgroundCannabis derivatives have been reported to possess antinociceptive properties. However, oral delivery is limited by poor bioavailability, stability, and reliability of effects. Previously, we reported an analgesic effect of the aqueous complex {Delta}9-tetrahydrocannabinol/2-hydroxypropyl-{beta}-cyclodextrin (THC/HP{beta}CD) after intracerebroventricular administration in male rats. MethodsHere, we investigated the analgesic effects of the THC/HP{beta}CD complex after oral administration (0.3 and 3 mg/kg) by the tail flick test after both acute and chronic administration (15 days) in female rats. Locomotor activity and anxiety-like behavior were also evaluated at the same experimental conditions. Moreover, dopamine and glutamate content in the periaqueductal gray (PAG), a key area for the antinociceptive action of THC, were also measured by HPLC. ResultsAfter acute administration, the antinociceptive effect of the complex was seen at 3 but not 0.3 mg/kg THC, with a maximum effect observed at 30 min (MPE 60%). Similar results were obtained after 15 days of treatment, although partially reduced (max MPE 20%). Reductions in locomotor activity with the dose of 3 mg/kg and a slight biphasic effect of the two doses on anxiety-like behavior were also observed. Finally, neurochemical analyses revealed that the dose of 3 mg/kg significantly increased dopamine and glutamate content in the PAG, an effect no longer present after 15 days of treatment. ConclusionsOur results highlight the antinociceptive efficacy of the THC/HP{beta}CD complex also after oral administration, notably higher than that previously seen with other carriers, although with some degree of tolerance after chronic administration. From a translational point of view, these results are relevant for the development of THC-based oral formulations with analgesic properties for the treatment of pain in humans. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/742765v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@fff791org.highwire.dtl.DTLVardef@d672f4org.highwire.dtl.DTLVardef@1150b3forg.highwire.dtl.DTLVardef@956403_HPS_FORMAT_FIGEXP M_FIG C_FIG

We previously identified a series of heterobenzamides (HBAs) with potent growth inhibitory activity against Mycobacterium tuberculosis in axenic culture. We also provided evidence that these target QcrB, a component of the terminal cytochrome oxidase in the electron transport chain. We expanded our studies to look at the full microbiological profile: key molecules from the series were tested for activity under different conditions and against additional strains. HBA analogs were active against intracellular bacteria where they exhibited bacteriostatic activity. A strain of M. tuberculosis with a mutation in QcrB (T313I) was resistant to HBAs in both axenic culture and inside macrophages. HBAs retained potency against lineages and mono-resistant strains of M. tuberculosis. HBAs had a narrow spectrum of activity, since they were not active against the ESKAPEE pathogens. Combination of the key HBA with bedaquiline was synergistic, as expected for a QcrB inhibitor, but there was no strong synergy with other drugs. Exposure of M. tuberculosis to the key HBA led to ATP depletion and boosted the oxygen consumption rate. This effect was specific to M. tuberculosis, since human THP-1 macrophage-like cells were unaffected by exposure to the HBA. HBA did not induce the production of reactive oxygen species or affect membrane potential but did affect pH homeostasis. Taken together, these data provide further evidence to support the identification of QcrB as the target and indicate that they are suitable for further drug development.

The de novo design of proteins that bind chemically complex small molecules has broad chemical and biological implications, but strategies typically rely on a small set of protein scaffolds and require extensive experimental screening. Here, we computationally designed proteins around a minimal aromatic {pi}-stacking motif to bind the anthracycline anticancer drug doxorubicin. Experimental characterization of twelve proteins revealed a {micro}M doxorubicin binder; two additional design cycles improved scaffold stability and binding affinity to yield an 85-residue protein that binds doxorubicin with a dissociation constant of 85 nM. An X-ray crystal structure of the protein-drug complex confirmed the accuracy of the designed {pi}-{pi} stacking interactions. The designed protein could act to protect cultured cells from doxorubicin-induced cytotoxicity. Unlike previous ligand-binding protein designs based on repeat proteins or naturally occurring folds, the designed protein adopts a previously unobserved 5-helix globular fold, indicating that a broader space of folded, functional proteins exists even for compact tertiary structures smaller than 100 residues. These results demonstrate that motif-guided generative protein design can discover compact de novo protein folds capable of high-affinity recognition of chemically complex small molecules.

The cooperative integration of photoredox catalysis and metalloenzyme catalysis has emerged as a powerful strategy for enabling stereoselective radical transformations beyond the capabilities of either catalytic mode alone. Herein, we report a photometallobiocatalytic enantioselective intermolecular C-C cross-coupling of pyridotriazoles and secondary alkyltrifluoroborate salts through cooperative catalysis between an organic photosensitizer and an engineered protoglobin. By combining visible-light-mediated radical generation with enzymatic activation of pyridotriazoles to form reactive Fe carbenoid intermediates, this transformation enabled highly enantioselective radical C-C bond formation through a proposed outer-sphere coupling mechanism. Through biocatalyst mining and directed evolution, engineered Aeropyrum pernix protoglobin catalysts were developed that catalyzed this radical C-C coupling with excellent efficiency and stereocontrol. The photobiocatalytic platform exhibited a broad substrate scope with respect to both secondary alkyltrifluoroborate salts and pyridotriazoles, affording a range of valuable N-heterocyclic products in excellent yields and enantioselectivities. Mechanistic studies supported the involvement of radical intermediates and revealed spontaneous binding between the photocatalyst eosin B and the engineered metalloenzyme. By leveraging cooperative photometallobiocatalysis, this work established an underexplored strategy for asymmetric intermolecular radical cross-coupling via an outer-sphere mechanism, further expanding the catalytic repertoire of transition-metal carbenoid chemistry. Entry for the Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/744224v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@132b69corg.highwire.dtl.DTLVardef@72eea5org.highwire.dtl.DTLVardef@1919e26org.highwire.dtl.DTLVardef@125fba6_HPS_FORMAT_FIGEXP M_FIG An enantioselective photometallobiocatalytic cross-coupling of pyridotriazoles and secondary alkyltrifluoroborate salts is developed. Cooperative catalysis using eosin B and an engineered protoglobin combines visible-light-mediated radical generation with enzymatic metal carbenoid activation, affording valuable N-heterocyclic products in excellent yield and enantioselectivity through an outer-sphere radical coupling pathway. C_FIG

G protein-coupled receptor (GPCR) allosteric modulators (AMs) offer significant therapeutic advantages over orthosteric drugs, yet structure-based virtual screening lacks validated protocols accounting for the conformational complexity of GPCR allosteric sites. We benchmark docking protocols using PDB experimental structures and structural ensembles derived from Gaussian accelerated Molecular Dynamics (GaMD) simulations across four Class A GPCRs (including the muscarinic M2 and M4 receptors, the {beta}2-adrenergic receptor, and the C-C chemokine receptor type 2) with four programs (Glide HTVS, AutoDock Vina, DOCK3.8, and Boltz-2) against experimentally validated modulator libraries and property-matched decoys. GaMD ensemble docking improved early AM enrichment across all four targets under at least one program. Glide ensemble docking was the only protocol to consistently improve early AM recovery across all four targets, ranking known actives almost exclusively within the top 0.5% of compounds at CCR2 and improving M2R active recovery nearly 9-fold relative to the PDB structure. GaMD free-energy landscape topology governed ensemble re-ranking strategy selection: population-skewed landscapes favored top binding energy ranking (BEmin) while flat, multi-populated landscapes favored average binding energy ranking (BEavg), and at targets with dominant low-energy states, a single GaMD cluster matched or exceeded full ensemble or PDB performance. Taking the union of top percentile hits identified by both ensemble re-ranking methods, BEmin / BEavg, maximizes chemical diversity at the earliest percentiles. Program-specific scaffold recovery biases further motivated a consensus BEmin / BEavg approach to maximize hit diversity. The Boltz-2 deep-learning program showed minimal sensitivity to GaMD templates and underperformed conventional docking, suggesting its affinity predictions complement rather than replace physics- and empirical-based docking approaches for GPCR AM screening.