ACS Medicinal Chemistry Letters

Neuroactive steroids modulate GABAA and NMDA receptors allosterically, typically requiring specific structural features for their activity. In this study, we characterize YX84, a novel neuroactive steroid bearing a 3{beta} sulfate and p-trifluoroacetylbenzyl alcohol attached in an ether linkage to a hydroxyl group at steroid carbon 17. This compound and similar analogues exhibit an atypical pharmacological profile, with three distinct actions at GABAA receptors. First, YX84 is a full agonist, with EC50 near 1 {micro}M and comparable efficacy to GABA at GABAA receptors in native hippocampal neurons. It presents as a full agonist relative to GABA at 4/{delta} subunit-containing receptors. Second, YX84 acts as a slow-onset, potent positive allosteric modulator (PAM) of GABAA receptors at concentrations below those that gate a response. Finally, YX84 exhibits rapid desensitizing and/or blocking kinetics; voltage dependence is consistent with a contribution of channel block. Structure- activity relationship analyses reveal that both functional groups are essential for gating activity, while classical requirements such as carbon 3 hydroxyl stereoselectivity and carbon 5 reduction are dispensable. YX84 also modestly inhibits NMDA receptor currents, suggesting weak negative allosteric modulation. Behavioral assays show that intraperitoneal administration of YX84 (30 mg/kg) does not impair sensorimotor function, unlike allopregnanolone. These findings identify YX84 as a structurally distinct neuroactive steroid with dual receptor activity and favorable behavioral tolerability, offering a promising scaffold for therapeutic development targeting excitatory/inhibitory imbalance in neuropsychiatric disorders if pharmacokinetic considerations can be overcome.

TEA domain (TEAD) proteins bind co-activator Yes-associated protein (YAP) to regulate the expression of target genes of the Hippo pathway. The TEAD*YAP protein-protein interaction is not druggable, but TEADs possess a unique and deep palmitate pocket with a highly conserved cysteine located outside the TEAD*YAP protein-protein interaction interface. Here, we screen a fragment library of acrylamide electrophiles and identify a fragment that forms an adduct with the conserved palmitate pocket cysteine and inhibits TEAD4 binding to YAP. Synthesis of a focused set of derivatives and time- and concentration-dependent studies with four TEADs provide reaction rates and binding constants. Co-crystal structures of fragments bound to TEAD2 and TEAD3 reveal reaction at the conserved palmitate pocket cysteine but also at another less conserved cysteine located in the palmitate pocket of TEAD2 closer to the TEAD*YAP interface. These fragments provide a starting point for the development of allosteric acrylamide small-molecule covalent TEAD*YAP inhibitors.

Intrinsically disordered proteins (IDPs) represent major yet challenging therapeutic targets in neurodegenerative disease due to their conformational heterogeneity and aggregation-prone behavior. Tau protein is a prototypical IDP that forms pathological aggregates in Alzheimers disease and related tauopathies. Despite extensive clinical efforts, tau-directed monoclonal antibodies have demonstrated limited efficacy. Concurrently, single-domain antibodies (nanobodies) have been gaining importance because of their small size and membrane penetrating capabilities. New design paradigms are therefore required for nanobodies to enable precise targeting of disease-relevant conformations. Here, we describe a biophysical modelling and AI-guided nanobody discovery targeting the VQIVYK motif of tau, which constitutes the structural core of neurofibrillary tangles in Alzheimers Disease. Biophysical modelling-based target analysis identified low-energy conformational states of VQIVYK. These conformational insights were used to guide AI-driven nanobody design of CDR3 loops. Starting from a nanobody scaffold, we generated 145 candidate nanobodies through systematic backbone sampling and neural network-guided sequence design, followed by multi-dimensional computational prioritization. Two candidates demonstrated robust binding to synthetic full tau protein in ELISA binding assays, achieving binding indices of 148.9% and 140%, relative to reference controls. Notably, one candidate also exhibited strong reactivity in post-mortem Alzheimers disease human brain tissue, with a binding index of 236.1%, exceeding that of the positive control (222.9%). Structural analysis indicates that our nanobodies engineered CDR3 engages VQIVYK through optimized aromatic and hydrophobic interactions. Together, these findings establish a proof-of-concept for biophysics-guided, AI-guided nanobody engineering against IDPs and identifies them as a promising lead for tau-targeted single domain antibody development.

Human Neutrophil Elastase (HNE) plays a vital role in several inflammatory diseases, however its role in the tumour microenvironment and the potential in cancer treatment is still unrevealed. Considering the potential of {beta}-lactams as HNE inhibitors, the present work describes the development of a synthetic strategy to obtain two different types (Type I and Type II) of quenched activity-based probes (qABPs), using a {beta}-lactam ring as a warhead and BODIPY-FL as a fluorophore. The two types differ in mechanism and relative position between the fluorophore and the quencher moiety. The qABPs synthesized presented IC50 values against HNE lower than 0.5 {micro}M, and high selectivity compared with homologous serine hydrolases. Type II qABPs showed a more efficient turn-on mechanism, and selectively targeted HNE in different cell lysates. The qABP 22 was internalized in U937 cells and in human neutrophils and successfully targeted HNE in both.

Microorganisms in the human gut influence the efficacy and metabolism of host-targeted small molecule therapeutics, including the frontline Parkinsons disease drug levodopa (L-dopa). Previous work has identified a mechanism-based inhibitor of gut bacterial decarboxylases that degrade L-dopa, -fluoromethyltyrosine (AFMT). However, early experiments with AFMT in rodent models suggested undesirable in vivo metabolism by host tyrosine hydroxylase, producing a metabolite likely to worsen Parkinsons phenotypes and prevent application as an L-dopa co-treatment. Here, we demonstrate oxidation of AFMT in vitro by recombinant human tyrosine hydroxylase. We then develop AFMT analogs that retain activity against bacterial decarboxylases but have reduced susceptibility to host hydroxylation. Suitable arenes for inhibitor design were identified using assays with commercially available noncanonical amino acids, which revealed aryl difluorination as a promising modification. Difluoroaryl AFMT derivatives are less prone to degradation by tyrosine hydroxylase in vitro yet still inhibit L-dopa metabolism by bacterial decarboxylases. This work exemplifies how substrate reactivity can streamline design of mechanism-based enzyme inhibitors, as well as how constraints posed by the host can be incorporated during development of microbiome-targeted therapeutics. The compounds reported here are promising starting points for future studies in animal models and further exploration of gut bacterial effects on L-dopa treatment efficacy.

Targeting protein-protein interactions (PPIs) with small molecules is historically challenging due to shallow, solvent-exposed interfaces that lack classical binding pockets. Furthermore, employing traditional structure-based virtual screening (SBVS) across ultra-large chemical spaces to find novel chemotypes imposes prohibitive computational bottlenecks. Here, we report the first prospective, real-world application of the PyRMD2Dock platform, an AI-enforced SBVS workflow that integrates machine learning and standard docking available within the PyRMD Studio suite. To target the structurally demanding immune receptor CD28, a chemically diverse subset of 2.4 million molecules from the Enamine REAL Diversity Space was docked into a cleft adjacent to the canonical ligand interface. These data were used to train 672 classification models, and the optimized model rapidly screened the remaining [~]46 million compounds. Following interaction filtering and clustering, 232 highly prioritized ligands were identified. Experimental validation of 150 purchased candidates yielded a remarkable hit rate, identifying multiple direct CD28 binders. Lead compounds 100 and 104 exhibited submicromolar affinity (Kd = 343.8 nM and 407.1 nM, respectively), potent CD28-CD80 disruption, and functional blockade in cellular reporter assays. Furthermore, these compounds successfully reduced cytokine secretion in primary human tumor-PBMC and epithelial tissue co-culture models. This study validates PyRMD2Dock as a highly scalable, effective protocol for mining massive chemical libraries to discover small-molecule modulators of challenging immune receptor interfaces.

Lung cancer remains the leading cause of cancer-related deaths worldwide, with cisplatin as the primary chemotherapy despite its limitations. Organotin(IV) dithiocarbamates have emerged as promising anticancer agents due to their potent cytotoxicity and stability. This study reports the successful synthesis of four novel organotin(IV) dithiocarbamates: dimethyltin(IV) N-methyl-N-benzyldithiocarbamate (DioSn-1), diphenyltin(IV) N-methyl-N-benzyldithiocarbamate (DioSn-2), triphenyltin(IV) N-methyl-N-benzyldithiocarbamate (TriSn-3), and triphenyltin(IV) N-ethyl-N-benzyldithiocarbamate (TriSn-4). Their cytotoxicity against A549 lung carcinoma cells was evaluated via MTT assay, while Annexin V-FITC/PI staining determined the mode of cell death. DioSn-2, TriSn-3, and TriSn-4 exhibited potent cytotoxicity (IC: 0.52-1.86 M), whereas DioSn-1 was inactive (IC > 50 M). Apoptotic features such as cell shrinkage and membrane blebbing were observed, with apoptosis rates ranging from 58% to 91%. DioSn-2 was the most selective (SI = 6.45) and induced early DNA damage within 30 minutes, followed by mitochondrial depolarization and excessive ROS generation. Caspase-9 activation exceeded caspase-8, confirming intrinsic apoptosis. NAC treatment reduced apoptosis by 52%, highlighting oxidative stress as a key cytotoxic mechanism. These findings suggest DioSn-2 as a promising alternative to cisplatin for lung cancer therapy.

The glucagon-like peptide-1 receptor (GLP-1R) plays a central role in metabolic regulation and is a major therapeutic target for obesity and diabetes. Peptide agonists, like semaglutide, targeting the GLP-1R remain among the most effective regulators of glucose metabolism and appetite. Nonetheless, recent reports about weight regain have limited the effectiveness of GLP1R peptide agonists, sustaining the interest in expanding the chemical diversity of GLP-1R ligands through drug discovery strategies. However, the structural complexity and conformational plasticity of class B1 GPCRs make conventional single-method virtual screening approaches prone to bias and limited chemotype recovery. Using an integrated ligand- and structure-based virtual screening pipeline, explicitly combining complementary ligand-based descriptors, multi-fingerprint similarity, electrostatic similarity, pharmacophore modeling, and multi-conformation docking under a consensus-driven selection strategy, we were able to identify three chemically distinct classes of GLP-1R agonist candidates: GQB47810, a non-peptidic molecule; neuromedin C, a peptide, and 2,5-Pen-enkephalin (DPDPE), a small peptide. From all of them, DPDPE showed the greatest effectiveness, reaching values similar to those of GLP-1, although with lower potency. Further in vitro characterization confirmed that pen-enkephalin behaved as a full agonist and exhibited dual GLP-1R/GIPR agonistic activity. These findings establish a consensus-driven and transferable computational framework for chemotype-diverse agonist discovery at conformationally flexible GPCR targets, and revealed a pentapeptide with GLP-1-like efficacy as a promising lead for next-generation small peptide therapeutics.

Cationic polymers, which mimic the structure of antimicrobial peptides (AMPs), are increasingly recognized as promising antimicrobial materials. Here, we report the synthesis and evaluation of a new class of cationic lipid-terminated oligomers (CLOs), comprised of 2C18-hydrophobic lipid tails, and short oligomeric cationic chains synthesised via Cu(0)-mediated reversible-deactivation radical polymerization (RDRP). Two 2-vinyl-4,4-dimethyl-5-oxazolone (VDM) oligomers with degrees of polymerization (DP) of 20 or 50 were synthesized using the lipid functional initiator (R)-3-((2-bromo-2-methylpropanoyl) oxy)propane-1,2-diyl dioctadecanoate (2C18-Br). Post-polymerization modification of the pendant oxazolone moieties was carried out using reactive amines, including N-Boc-ethylenediamine (BEDA) and N,N-dimethylethylenediamine (DMEN). Subsequent deprotection of the BEDA groups and quaternization of DMEN groups enabled the synthesis of six functional CLOs exhibiting distinct cationic functionalities. Antimicrobial assays against a panel of WHO bacterial and fungal priority pathogens (methicillin-resistant Staphylococcus aureus [MRSA], Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Candida albicans, and Cryptococcus neoformans) revealed that these CLOs exhibited potent and selective structure-dependent antibacterial activity, particularly against MRSA, with minimum inhibitory concentrations (MICs) in the clinically relevant range, below 4 {micro}g mL-1, comparable to antibiotics vancomycin and colistin. Among these, BEDA-functionalized CLOs demonstrated the strongest antimicrobial profile, which was significantly increased by increasing DP, as evidenced by a reduction in MIC values from 64 {micro}g mL-1 (for DP20) to [≤] 4 {micro}g mL-1 (for DP50) against A. baumannii. Biocompatibility assays against red blood cells and HEK293 cells indicated negligible toxicity, with haemolytic (HC50) and cytotoxic (CC50) values exceeding 512 {micro}g mL-1 across all CLOs. All CLOs displayed minimal activity against C. albicans (MIC [≥] 512 {micro}g mL-1). In contrast, activity against C. neoformans was influenced by both cationic functionality and DP, with DMEN-based CLOs exhibited superior antifungal activity at higher DP relative to their BEDA-based counterparts. Most CLOs displayed high selectivity (SI) toward MRSA (SI >128), while 2C18-O(BEDA)50 exhibited the broadest spectrum, showing potent antimicrobial activity and high selectivity against E. coli (MIC [≤] 4 {micro}g mL-1, SI [≥] 128), A. baumannii (MIC [≤] 4 {micro}g mL-1, SI [≥] 128), and MRSA (MIC [≤] 4 {micro}g mL-1, SI [≥] 128), along with moderate activity against P. aeruginosa (MIC = 32 {micro}g mL-1, SI > 16). Taken together, these findings elucidate the combined influence of end-group lipidation, cationic functionality, and polymer length in modulating antimicrobial activity, thereby establishing 2C18-terminated CLOs as a rationally tunable and biocompatible platform for antimicrobial material development.

Prasugrel is a prodrug, widely used in antiplatelet strategy for secondary prevention after acute coronary syndrome. The metabolism of prasugrel leads to the formation of the Prasugrel Active Metabolite (PAM), an irreversible P2Y12 receptor antagonist. Its mode of binding has not yet been fully established, although it is known that it binds covalently to P2Y12 by forming a disulfide bridge with cysteines and its sulfur moiety. PAM is a molecule with two chiral centers, resulting in four stereoisomers which appear to be stereoselective upon binding. A combination of different molecular modeling methods, such as molecular dynamics, ensemble docking, and Density Functional Theory (DFT), were used to rationalize these differences in antagonism observed in vitro and to elucidate the mode of binding of PAM to P2Y12. PAM is found to bind to the closed P2Y12 conformation in a preferential way. Although the four stereoisomers have comparable affinity, the location of the RS stereoisomer makes the formation of a disulfide bond with cysteines more favorable, particularly with cysteine 175. Compared to the RR stereoisomer, the RS stereoisomer interacts less deeply with the P2Y12 receptor, interacting in particular with the second and third extracellular loops, explaining the competition observed with cangrelor and an intermediate metabolite of prasugrel. Furthermore, DFT calculations have shown that the formation of a disulfide bridge is energetically more favorable with the RS stereoisomer than with the RR stereoisomer. The physical interactions and chemical reaction between the RS stereoisomer and the P2Y12 receptor are key factors in explaining the stereoselective binding of PAM to P2Y12.

Sirtuins (SIRTs), which remove protein lysine acyl modifications, play crucial roles in diverse cellular processes, including metabolism, gene transcription, DNA damage repair, cell survival, and stress response. Several sirtuins are considered non-oncogene addiction of cancer cells and promising targets for anticancer drug development. High-throughput screening (HTS) methods for sirtuins are critical for the development of potent and isoform-selective sirtuin inhibitors, which are needed to validate the therapeutic potential. Herein, we designed and synthesized a fluorescent polarization (FP) tracer, KP-SC-1. Using this high-affinity tracer, we developed a robust, high-throughput FP competition assay for screening SIRT1-3 inhibitors. The assay was validated by testing known SIRT1-3 inhibitors. The assay can detect NAD+-independent SIRT1-3 inhibitors, as well as NAD+-dependent inhibitors, such as Ex-527 and TM. Finally, our assay showed satisfactory stability and outstanding performance in a pilot library screening. Compared to previous assays, the FP assay uses much less SIRT1-3 enzymes, a feature important for high-throughput library screening. We believe that the FP assay developed here will accelerate the discovery and development of SIRT1-3 inhibitors.

The blood-brain barrier (BBB) is crucial for neural homeostasis, tightly regulating molecular exchange between the circulation and brain. However, this selective protection also greatly limits drug delivery to the central nervous system, posing a major challenge for treating neurological disorders. Pharmacological strategies that transiently and safely increase BBB permeability could therefore transform brain drug delivery, yet systematic discovery of such modulators remains hampered by the limitations of current in vitro and in vivo approaches. Here we present FishNAP, a non-invasive, high-throughput zebrafish platform for real-time assessment of BBB permeability in vivo. FishNAP captures developmental changes in barrier function and detects dysfunction in genetic mutants. Using this platform, we screened 2,320 FDA-approved small molecules for compounds capable of opening an intact BBB and identified 11 that reproducibly increased permeability. Seven of these molecules allowed entry of a 1 kDa tracer into brain tissue, and five also permitted passage of a larger 10 kDa Dextran. Barrier integrity recovered within 24 hours for all seven compounds, indicating reversible modulation. Finally, testing three representative molecules (Calcitriol, Lovastatin, and Sunitinib) in adult mice revealed increased BBB permeability and reduced Claudin-5 expression, demonstrating conserved mechanisms of BBB-regulation across vertebrates. FishNAP thus enables systematic discovery of BBB modulators with direct translational potential for brain drug delivery.

Neurosteroids are endogenous neuromodulators and emerging therapeutics, but understanding but understanding how these compounds modulate receptor signaling within defined neuronal populations and networks has been limited by an inability to deliver these molecules with receptor-level and cell-type specificity. Here, we developed a neurosteroid DART (Drug Acutely Restricted by Tethering) that combines the GABAA receptor subunit-selectivity of a neuroactive steroid (NAS) with the cell-type specificity of the DART platform. Screening of seventeen NAS analogs identified seven scaffolds suitable for further engineering, and structure-activity analysis revealed that DART linker attachment at the C11 position preserved NAS activity on GABAA receptors, whereas C2 and C17 attachment failed to exhibit activity. Functional profiling of C11-linked NAS-DARTs slowed IPSC decay kinetics and showed variable off-target modulation of NMDA and AMPA EPSCs. The most selective compound, YX85.1DART.2, potentiated GABA-evoked currents in neurons expressing pharmacogenetically isolated 4/{delta}-containing GABAA receptors but not in {gamma}2-expressing neurons. A previously validated BZP.1DART.2 produced complementary selectivity on the two receptor populations. Together, these findings establish new tools for interrogating subunit-specific NAS actions on inhibitory signaling in defined neuronal populations.

Despite decades of research, current understanding of the spectrum of targets bound by kinase inhibitors remains incomplete. This complicates mechanism of action studies, drug repurposing, and understanding of adverse responses. Here, we describe kinome-wide profiling of an optimal kinase library (OKL) comprising 192 small molecules selected based on stage of clinical development, chemical diversity, and target coverage. Our results show that polypharmacology is widespread among kinase inhibitors independent of regulatory approval. The generally understood ("assigned") targets of approved molecules are not necessarily the most potently inhibited and off targets include multiple understudied kinases. Moreover, median selectivity has not increased over time. We illustrate the use of synoptic OKL-kinome profiles in identifying potential toxicity targets, repurposing anti-inflammatory drugs for neurodegenerative and infectious diseases, and performing chemical genetic studies. Our studies illustrate how much remains to be discovered about the chemistry and biology of one of the largest classes of human therapeutics.

The ATP-gated P2X7 receptor (P2X7R) activates inflammatory signaling pathways in the central nervous system. In particular, P2X7Rs drive chronic glia-mediated neuroinflammation, which is increasingly recognized as a key contributor to Alzheimers disease, a neurodegenerative disorder that lacks effective disease-modifying therapies. Here we identify a potent and selective negative allosteric modulator of P2X7Rs with therapeutic potential. We synthesize a series of small molecules based on a polycyclic scaffold and confirm blood-brain barrier penetration by testing a radiolabeled analogue using positron emission tomography imaging. Through a structure-guided medicinal chemistry campaign centered on our scaffold, we identify four promising P2X7R antagonists. Of these, UB-ALT-P2 exhibits the most favorable safety profile, high oral bioavailability and robust brain penetration. High-resolution cryo-EM structures of UB-ALT-P2 bound to human, mouse, and rat P2X7Rs reveal a conserved antagonist binding mode with steric features that favor prolonged binding to human receptors. In the 5xFAD mouse model of AD, oral UB-ALT-P2 blunts weight loss, improves short- and long-term memory, reduces amyloid-{beta} plaque burden, lowers hyperphosphorylated tau, and diminishes oxidative and inflammatory markers. These results establish UB-ALT-P2 as a potent and safe P2X7R antagonist that can mitigate core AD pathologies, providing a compelling foundation for further development.

Angiogenesis, the process by which new blood vessels form from existing vasculature, is fundamental to tissue repair and regeneration but also underlies pathological conditions such as cancer progression. Targeting angiogenesis has thus become a promising approach for developing novel cancer therapeutics. While various phytochemicals have demonstrated anti-angiogenic effects, the role of 2-5(H)-Furanone, a naturally occurring lactone found in various plants and marine sources with diverse biological activities, remains insufficiently explored. In this study, we systematically evaluate the anti-angiogenic potential of 2-5(H)-Furanone using Human Umbilical Vein Endothelial Cells (HUVECs) as an in vitro model and zebrafish embryos as an in vivo model. Experimental findings demonstrated that treatment of HUVECs with increasing concentrations of 2-5(H)-Furanone led to significant, dose-dependent reductions in proliferation, invasion, migration, and tube formation. Analyses of gene expression revealed marked downregulation of key pro-angiogenic mediators, VEGF, and HIF-1. Complementing these in vitro results, in vivo studies in zebrafish embryos showed robust, dose-dependent inhibition of intersegmental vessel (ISV) formation, accompanied by suppression of critical angiogenesis-related genes. Molecular docking further supported these observations by indicating stable binding of 2-5(H)-Furanone to major angiogenic targets, including VEGFR2, MMP2, HIF-1, and PIK3CA. Collectively, our data demonstrate that 2-5(H)-Furanone potently inhibits angiogenesis, as evidenced in both HUVEC and zebrafish models, through functional and molecular mechanisms. These findings support the further development of 2-5(H)-Furanone as a promising anti-angiogenic therapy candidate.

Mutant KRAS is a potent oncogene, serving as a tumor driver in many solid human cancers. Current small-molecule inhibitors target the highly conserved G-domain, but to gain further mechanistic insight into the roles of different isoforms, we investigated the strategy of sterically shielding the unstructured hypervariable regions (HVRs). KRAS HVRs undergo a series of post-translational modifications that enable intracellular trafficking and membrane attachment. Previous attempts to drug KRAS by preventing its post-translational modification, based on inhibition of the involved prenylation enzymes have been largely unsuccessful. In this study, we explored the property of Designed Armadillo Repeat Proteins (dArmRPs) to specifically bind unstructured regions. We assembled a dArmRP to recognize the unstructured KRAS4B-HVR and developed it into a high-affinity binder by directed evolution. The resulting dArmRP recognizes the 14 C-terminal residues of unprocessed KRAS4B, thereby blocking the farnesyltransferase-binding epitope. This steric shielding disrupts KRAS4B post-translational modification and thereby significantly reduces its plasma membrane localization, while demonstrating complete selectivity over KRAS4A, NRAS, and HRAS. This work establishes the shielding of intrinsically disordered regions as a precise biochemical strategy to control protein function and provides an isoform-specific tool to dissect KRAS biology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/712636v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@791ac4org.highwire.dtl.DTLVardef@cc4c91org.highwire.dtl.DTLVardef@b6c920org.highwire.dtl.DTLVardef@4e8a9c_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical representation of how the unstructured KRAS4B-HVR is occupied by a dArmRP, making it inaccessible for the FTase.

Pseudomonas aeruginosa is an adaptable organism, frequently found in chronic infections, and for which antimicrobial resistance is a growing concern. Therefore, there is an urgent need for alternative therapeutic strategies. Cationic antimicrobial peptides (AMPs) offer potent bactericidal activity but suffer from limited selectivity and potential host toxicity. To enhance species-specific targeting, we designed two prodrug variants of the AMP D-Bac8CLeu2,5 - EEEE-D-Bac8CLeu2,5 and ELEG-D-Bac8CLeu2,5 -- engineered for activation by the P. aeruginosa extracellular aminopeptidase PaAP. While both prodrug motifs effectively neutralized the positive charge of D-Bac8CLeu2,5 and prevented DNA-peptide complex formation, EEEE-D-Bac8CLeu2,5 showed negligible antimicrobial activity due to slow and incomplete activation. In contrast, ELEG-D-Bac8CLeu2,5 underwent rapid PaAP-mediated activation, restoring bactericidal activity in planktonic cultures and biofilms. PaAP contributed significantly to complete prodrug activation, particularly within biofilms, where the accumulation of partially activated intermediates correlated with biphasic killing kinetics. The prodrug showed reduced activity against other ESKAPEE pathogens, demonstrating selective activation by P. aeruginosa. Experiments selecting resistant bacteria revealed distinct mutations in lipopolysaccharide biosynthesis pathways for D-Bac8CLeu2,5 and the prodrug, with limited cross-resistance. These findings establish aminopeptidase-activated AMP prodrugs as a promising approach for species-selective antimicrobial therapy and highlight the feasibility of exploiting bacterial enzymes for controlled antimicrobial peptide activation. Table of contents graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/715093v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@4a5505org.highwire.dtl.DTLVardef@13e578org.highwire.dtl.DTLVardef@3e3080org.highwire.dtl.DTLVardef@e24266_HPS_FORMAT_FIGEXP M_FIG C_FIG

Tuberculosis, often considered the worlds deadliest infectious disease, is associated with over one million deaths annually. The emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) makes anti-tuberculosis drug development a critical priority. Griselimycin (GM) is a cyclic peptide that targets the essential DNA sliding clamp of Mtb. While GM is a promising Mtb antibiotic, its poorly understood structure-activity relationship has stalled derivatization. To investigate the contribution of each amino acid towards its activity, we assessed the antibiotic activity of an alanine scan library in M. tuberculosis and M. smegmatis. Residues essential for activity and tolerable to modification were identified, and the impact of backbone N-methylation at each position was determined. Edits to cyclization chemistry, unnatural amino acid incorporation, and replacing the acetylated N-terminus with a free amine were also investigated. Lastly, incorporation of an N-terminal fluorophore enabled visualization of GM accumulation inside of mycobacteria both in and outside of macrophage cells, where Mtb natively resides. These findings present the first comprehensive structure-activity investigation into GM and can be used to rationally design future analogues.

Cannabinoid receptors are therapeutically promising GPCRs that are also interesting test systems for structure-based methods, which have targeted them previously. Here we used the CB2 receptor as a template to explore several topical questions in library docking. Whereas an earlier campaign against the CB1 receptor led to potent but relatively non-selective ligands, here we found that targeting interactions with polar, orthosteric site residues led to subtype-selective ligands. Docking hit rate and especially hit affinity improved in moving from a 7 million to a 2.6 billion molecule library. Similar to earlier studies, docking against active and inactive states of the receptor did not reliably bias toward the discovery of agonists or inverse agonists. Cryo-EM structures of two of the new agonists, each in a different chemotype, superposed well on the docking predictions. Correspondingly, structure-based optimization led to 10- to 140-fold improvements within three different series, also consistent with well-behaved ligand families. Hit rates with a fully enumerated 2.6 billion molecule library resembled those of an implied 11 billion molecule library from a building-block method, consistent with the latters ability to explore this space, though higher affinities were discovered from the fully enumerated set. Overall, eight diverse families of ligands, with potencies <100 nM and mostly unrelated to previously known ligands were found. Implications for future studies are considered.