Journal of Medicinal Chemistry

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.

Aldehyde dehydrogenases (ALDH) are enzymes that catalyze the NAD(P)+-dependent oxidation of aldehydes into carboxylic acids, playing roles in detoxification, biosynthesis, and regulatory functions. Dysfunction of ALDH is associated with serious conditions such as alcohol intolerance, cancer, cardiovascular problems, and neurological disorders. In humans, ALDH1A1 and ALDH1A3 isoforms act as retinaldehyde dehydrogenases and are overexpressed in various cancers, where high levels are associated with increased tumor malignancy, cancer stem cell traits, and therapeutic resistance. ALDH1A3 is recognized as a promising target for anticancer therapies, with several inhibitors, mainly reversible, developed to specifically target it or the enzyme family. Since ALDH enzymes can also display esterase activity, we used this property to develop an in vitro assay specifically targeting the esterase function of ALDH1A3. A highly conserved active-site cysteine in ALDH1A3 is located at the bottom of two converging channels, which define the substrate- and cofactor-binding pockets. To target this catalytic cysteine, we screened a library of 3,200 cysteine-focused covalent fragments. This led to the identification of Z3405279217 (Z34), an acrylamide-based covalent compound that inhibits both ALDH1A1 and ALDH1A3 at sub-micromolar levels. Biochemical and biophysical tests confirmed that Z34 acts as a time-dependent, covalent, and irreversible binder to the active-site cysteine. In this work, we determined the Cryo-EM structure of the ALDH1A3-Z34 complex at 2.26 [A] resolution, confirming the covalent attachment to the catalytic cysteine of Z34. Notably, two mutually exclusive covalent binding modes were observed: one occupying the substrate-binding pocket and the other the cofactor-binding region. Z34 displayed unexpected binding modes within the active site and holds promise as a lead compound for future drug development. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/738401v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@982d1forg.highwire.dtl.DTLVardef@ba86f2org.highwire.dtl.DTLVardef@1f19f2borg.highwire.dtl.DTLVardef@8e807_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

GLP-1 receptor agonists achieve weight loss but are associated with clinically significant reductions in lean mass. Activin type II receptors (ActRIIA and ActRIIB) mediate signaling of myostatin and activin A, both of which negatively regulate muscle growth, suggesting that dual blockade of these receptors may preserve or increase lean mass while promoting fat loss. In this study, we developed anti-ActRIIA/B antibodies using AI-driven platforms (AlfaDAX) and selected the lead candidate AB130-165 based on in vitro binding, functional blocking, and developability assessments. Compared with a laboratory-prepared bimagrumab analog, AB130-165 exhibited potent dual inhibition of ActRIIA/B signaling, with a 9.5-fold higher functional blocking activity against activin A-induced SMAD signaling and 1054-fold improvements in binding affinity for ActRIIA (KD = 0.204 pM), 10-fold for ActRIIB (KD = 0.243 pM), respectively. In diet-induced obese mice, combination therapy with AB130-165 and semaglutide resulted in a 33.4% body weight reduction, which was superior to semaglutide monotherapy (-24.3%) and the bimagrumab combination group (-25.5%). Moreover, the combination significantly improved body composition, reducing fat mass percentage by 77.8% (vs. 65.0% in the bimagrumab combination group) and increasing the lean-to-body weight ratio to 67.3% (vs. 62.3%), demonstrating superior fat loss with better preservation of lean mass. Collectively, these findings establish AB130-165 as a differentiated anti-ActRII antibody that enables high-quality weight loss, and its combination with semaglutide shows superior efficacy over bimagrumab-based regimens. With favorable developability and potential for long-acting subcutaneous administration, AB130-165 represents a promising next-generation therapeutic candidate for obesity and muscle-sparing weight management.

Pyrrolizidine alkaloids (PA) are well-known and widespread natural products from plants, which have also been identified in several different bacteria. In the latter case, the core structure is constructed by a non-ribosomal peptide synthetase (NRPS), which then undergoes oxidative ring contraction catalyzed by a Baeyer-Villiger monooxygenase. By deploying various NRPS engineering strategies, we have successfully generated five novel peptides carrying the unusual PA moiety at their C-terminus. Nonetheless, efforts to obtain a larger library of PAs were unsuccessful. Combined computational modelling and docking experiments suggest that this failure stems from the strict specificity of the thioesterase (TE) domain at the end of the NRPS, which discriminates against peptides carrying more than two amino acids. Our work thus suggests protein design strategies by which this intrinsic limitation to NRPS engineering may be overcome in future.

The cysteine to serine mutation at residue 481 of Bruton's tyrosine kinase (BTK) is the most common mechanism of clinical resistance against ibrutinib for the treatment of mantle cell lymphoma and chronic lymphocytic leukemia. We report small molecule ligands containing chiral {beta}-lactone electrophiles to address this challenge. The asymmetric warhead enabled stereoselective covalent modification of wild-type and ibrutinib-resistant mutant BTK(C481S) through distinct sites of reactivity. Building on these findings, we developed kinase-directed {beta}-lactone probes and demonstrated that individual enantiomers preferentially engage distinct subsets of the kinome. These studies establish {beta}-lactones as stereochemically encodable covalent warheads whose stereochemistry can serve as a selectivity filter in covalent drug discovery.

Polo-like kinase 1 (PLK1), a key regulatory protein controlling entry into and passage through mitosis, has been targeted through its kinase domain (KD) with mixed clinical success. Inhibition through the Polo-box domain (PBD) is a viable alternative through targeting the sub-cellular localization and kinase activity of PLK1. Novel non-peptidic compounds, termed abbapolins, were discovered through the REPLACE strategy and have been lead optimized through structure-based strategies and screening analogs in the NCI-60 tumor cell panel. Proteomic analysis revealed a correlation between abbapolin activity and PLK1 protein levels in the cell lines part of the NCI-60. Prostate cell lines were identified as among the most sensitive and led to further detailed studies of their activity in prostate cancer models. Compounds were evaluated for their pharmacokinetic properties, and in vivo efficacy, and results showed significant antitumor xenograft activity with no observable gross toxicity. Treated tumors were analyzed for loss of PLK1, which was previously shown to be induced by abbapolin binding. Results obtained showed a significant degradation of PLK1 in abbapolin-treated vs untreated tumors, thereby confirming on-target action in vivo and revealing PLK1 levels as a potential pharmacodynamic marker. Lead compounds were shown to sensitize PC tumors resistant to androgen deprivation therapy paving the way for future combination studies in vivo. These data provide an alternative pathway for effective PLK1 therapeutics that avoid the reported problems of molecules targeting the KD, in vivo proof-of-concept for the REPLACE strategy and validation for targeting the PBD as an anti-tumor drug development strategy.

Small molecule inhibitors of cell death have wide-ranging potential applications, both as tool compounds in the laboratory and as clinical modulators of pathologic cell death. Previous screening efforts have identified candidate compounds targeting the pro-apoptotic, pore-forming BCL-2 family proteins BAX and BAK, but the complex interactions of these proteins at the mitochondrial outer membrane (with other proteins and the membrane itself) present challenges for compound screening. Although no inhibitors of BAX or BAK have advanced to clinical testing to date, candidate inhibitors have thus far been identified via screening of membrane-containing systems such as liposomes and isolated mitochondria. To address some of the challenges of chemical screening for apoptosis inhibitors, we conducted a small molecule screen utilizing BH3 profiling, a method that quantifies mitochondrial outer membrane permeabilization (MOMP) upon treatment with pro-apoptotic peptides derived from BCL-2 family proteins. Of over 40,000 compounds screened, we identified a series of compounds that prevent MOMP in response to pro-apoptotic peptides. The most potent of these, CDL36, binds to BAX and prevents MOMP at early timepoints. In longer term viability assays, the cytoprotective effect of CDL36 is most potent against death induced by doxorubicin, a widely used chemotherapeutic agent that causes dose-limiting cardiovascular toxicity. Our results elucidate the mechanism of action of new and existing cell death inhibitors, providing a foundation for further development of these inhibitors and potential insights into the mechanisms mediating doxorubicin toxicity in patients.

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

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by a dense desmoplastic tumor microenvironment (TME) that limits drug penetration and promotes immune evasion. Effective treatment, therefore, requires simultaneous modulation of multiple signaling pathways. Here, we describe a directed polypharmacological strategy to identify dual modulators of c-MET and Smoothened (SMO), aiming to disrupt the protective stroma through SMO inhibition while directly suppressing tumor cell survival via c-MET targeting. An AI-guided virtual screening workflow combining the machine-learning platform PyRMD, trained on known c-MET and SMO ligands, with structure-based molecular docking was applied to a library of over 9 million compounds. This approach led to the identification of compound 21, an aminopyrimidine-benzamide-phenoxyquinoline derivative, as a dual c-MET/SMO inhibitor. Biochemical and cellular studies demonstrated that compound 21 selectively binds the SMO orthosteric site (pKi = 5.60), inhibits agonist-induced GLI (Glioma-associated oncogene) signaling (pIC50 = 5.50), and potently suppresses c-MET kinase activity (pIC50 = 6.94). Western blot analyses further revealed that compound 21 promotes ubiquitin-proteasome-mediated degradation of c-MET, eliminating receptor availability and limiting compensatory resistance signaling. In 3D heterotypic models comprising MIAPaCa2 pancreatic cancer cells and CAF154-hTERT fibroblasts, dual inhibition of SMO-mediated stromal support and c-MET-driven tumor progression resulted in greater cytotoxicity than the combination of the selective inhibitors Sonidegib and PHA-665752. Overall, compound 21 overcomes stromal-mediated resistance, enhances tumor cell death, and validates dual SMO/c-MET targeting as a promising single-agent therapeutic strategy for PDAC. One Sentence SummaryAn AI-identified dual SMO/c-MET inhibitor overcomes stromal resistance and degrades c-MET to suppress pancreatic cancer.

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.

EIF2alpha kinase heme-regulated inhibitor (HRI) is a novel target for haematological malignancies with modulators reported to trigger cell death via the HRI-eIF2alpha-ATF4 pathway. We report a protocol for producing the minimal kinase domain of full-length human HRI, termed HRIKD-delta-KI, where the unstructured 140 amino acid (aa) kinase insert (KI) within HRI kinase domain (HRIKD) is replaced with a 2aa glycine/serine (GS) linker. X-ray crystal structures were determined of apo-HRIKD-delta-KI and of its complex with ATP at 2.1 & 2.5 Angstrom resolution respectively. Both structures display a canonical bi-lobal kinase fold. However, they remain in a non-productive state with a displaced C-helix, disassembled R-spine, and a disordered activation segment hindering the substrate site. Biophysical assays (fluorescence based thermal shift & Synchrotron Radiation Circular Dichroism) demonstrate HRIKD-delta-KI retains its functional ligand-binding conformation. All together, these findings define structural and ligand-binding features of HRI to support ongoing drug discovery efforts in blood cancer.

Background & Aims: Activation of apoptosis signal-regulating kinase 1 (ASK1), a ubiquitous redox-sensitive kinase, results in inflammation, apoptosis, and fibrosis, key common pathways in human liver disease. SRT-015 is a novel, small molecule inhibitor of ASK1. This study evaluated the in vitro efficacy of SRT-015, compared it to other ASK1 inhibitors, and determined the in vivo efficacy of SRT-015 across multiple acute and chronic liver disease models. Methods: In vitro studies determined the kinase potency and selectivity of SRT-015, and cellular studies were used to demonstrate direct mechanisms of action. The cardiac hERG channel inhibition was assessed and PK determined in rodents and nonhuman primates. In vivo studies evaluated SRT-015 efficacy in rodent models of drug-induced hepatotoxicity (acetaminophen (APAP) overdose), alcohol-associated liver disease (ALD), metabolic-disease associated steatohepatitis (MASH) and cholestatic disease (bile duct ligation, BDL). Results: SRT-015, was demonstrated a selective ASK1 kinase, and SRT-015 treatment directly inhibited fibrosis, apoptosis and inflammation in activated human fibroblasts, hepatocytes and PBMCs, respectively without safety signals or hERG inhibition. Other ASK1 inhibitors had safety concerns or limited functional activity. Liver and kidney selective PK were observed for SRT-015 in all species evaluated. In vivo, SRT-015 treatment was efficacious in the acute mouse APAP overdose and ALD model significantly (P<0.05) decreasing serum ALT. Using a therapeutic diet-induced obesity (DIO)-MASH model with biopsy-verified fibrosis, SRT-015 treatment significantly (P<0.05) inhibited DIO-induced liver enzymes, hepatomegaly, fibrosis, inflammation, and apoptosis independent of body weight loss whereas treatment with selonsertib was ineffective. In a rat cholestatic model, SRT-015 treatment significantly (P<0.05) decreased fibrosis and stellate cell activation. Conclusions: These findings support SRT-015 as a potential therapeutic for human liver diseases of any etiology.

IntroductionIn this study, we developed a modular in vitro platform that integrates advanced polymer-drug conjugation chemistry with stepwise cytotoxicity screening in both 2D (monolayer) and 3D (spheroids) glioblastoma (GBM) models. Buparlisib was selected as the model therapeutic due to its well-characterised mechanism of action, high blood-brain barrier permeability, and relevance to PI3K-targeted therapy. MethodsTwo mechanistically distinct conjugation strategies were explored using N-(2-hydroxypropyl)methacrylamide-based copolymers. The first strategy was based on a redox-sensitive disulphide linkage designed for intracellular glutathione-triggered release, whereas the second used an azide-bearing derivative compatible with strain-promoted azide-alkyne cycloaddition. Drug release was assessed by high-performance liquid chromatography. Biological activity was systematically evaluated in U87MG, U118MG, and T98G cells under 2D conditions using a resazurin-based metabolic activity assay. Subsequently, the more promising disulphide-based formulations were assessed in 3D spheroids by metabolic activity measurements and live-cell monitoring of spheroid growth dynamics. ResultsFree Buparlisib showed the strongest inhibitory effect, while its modification and polymer conjugation reduced the apparent activity. Nevertheless, the disulphide-based derivative and polymer conjugate retained concentration-dependent activity, whereas the azide-based polymer conjugate showed minimal effects. Moreover, treatment responses differed between cell lines and between 2D and 3D models. DiscussionOverall, linker chemistry, cell-line-specific behaviour, and model dimensionality strongly influenced the biological performance of the polymeric Buparlisib formulations. The redox-sensitive polymer conjugate therefore represents the more promising strategy for further development.

The inhibition of A{beta}42 ({beta}-amyloid) fibril formation is a key therapeutic strategy in Alzheimer's disease research. Paddlewheel diruthenium complexes have shown promising activity against A{beta}42 aggregation and preformed fibril disaggregation, yet their molecular mode of action remains poorly understood. In this work, we perform atomistic simulations to explore how charge modulation influences the interactions of three analogous paddlewheel diruthenium complexes, the parent neutral complex [Ru2Cl(D-p-FPhF)(O2CCH3)3], and its anionic [Ru2Cl2(D-p-FPhF)(O2CCH3)3]- and cationic [Ru2(D-p-FPhF)(O2CCH3)3]+ counterparts (D-p-FPhF- is the N,N' -bis(4-fluorophenyl)formamidinato ligand) with A{beta}42. Our results indicate that electrostatic tuning governs binding affinity and the extent of interaction across the A{beta}42 fibril surface. As the complexes' charge changes from -1 to +1, the interaction pattern shifts from localized contacts to widespread, multi-site engagement encompassing key charged, aromatic, and hydrophobic regions of A{beta}42. This enhanced binding correlates with longer-lived, thermodynamically stable interactions at the fibril interface, which effectively lower the free energy penalty for fibril disassembly. Overall, our findings propose a mechanism in which charge-dependent activation through ligand exchange enhances fibril recognition and promotes disruptive binding modes, demonstrating the potential of charge-tunable diruthenium complexes as therapeutic modulators of A{beta}42 fibril stability.

Human epidermal growth factor receptor 2 (HER2) is an oncogenic receptor tyrosine kinase in breast cancer and other malignancies. A subset of HER2-positive tumours expresses 611-CTF-p95HER2, a tumour-specific, hyperactive truncated isoform associated with metastasis and treatment resistance that lacks most of the extracellular domain targeted by conventional HER2-directed antibodies. We previously developed NAZ-mAb (formerly known as Oslo-2), a monoclonal antibody against 611-CTF-p95HER2. Here, we describe a computational antibody-engineering workflow for designing variants of NAZ-mAb. Starting from the sequence alone, we modeled the NAZ-mAb-611-CTF-p95HER2 complex, generated a combinatorial mutational landscape using FoldX 5.0, and prioritized candidate variants using predicted interaction energy and developability criteria. Two variants representing distinct design strategies were selected for validation: an aromatic double mutant, NAZ-mAb v1 (L:S31W/L:H107W), and a conservative single mutant, NAZ-mAb v2 (L:S31M). Both variants were successfully expressed as recombinant IgGs; NAZ-mAb v2 achieved a five-fold higher recombinant expression yield than parental NAZ-mAb, while both variants retained antigen binding with a higher apparent signal than the parental antibody in indirect ELISA. However, Biacore two-state kinetic analysis revealed weaker affinities than the parental antibody (KD NAZ-mAb v1: 32.6 nM, NAZ-mAb v2: 9.45 nM vs. parental NAZ-mAb: 5.33 nM). These findings show that the computational workflow can generate experimentally tractable, antigen-engaging NAZ-mAb variants, while also highlighting the limitations of fixed-backbone interaction-energy ranking as a predictor of binding affinity and yield. This study provides a practical framework for computationally driven, developability-aware antibody optimization in the absence of experimental structural data.

The platelet P2Y1 receptor (P2Y1R) is necessary for inflammation, signalling via Rho-GTPase pathways to elicit functions that are distinct from aggregation (PLC-dependent canonical signalling pathway). Whether these distinct platelet inflammatory functions can be selectively suppressed to preserve hemostasis through the rational design of P2Y1R antagonists has not been explored. In silico molecular docking analysis examined biased nucleotide interactions within the P2Y1R binding pocket. The identified possible key amino acid residues guided rational design to synthesize compounds for pathway selective inhibition, evolving from nucleotide to non-nucleotide structures. The nucleotide analogue KMR-82-13 was predicted to engage distinct regions of the binding pocket and selectively inhibited platelet chemotaxis while preserving aggregation. These findings informed the design of a non-nucleotide compound KSN-159-27, aiming to retain key KMR-82-13-like interactions while improving drug-like properties. Docking and molecular dynamics simulation supported a stable but dynamic binding mode for KSN-159-27 within the P2Y1R pocket, consistent with pathway-selective inhibition. KSN-159-27 displayed characteristics of a pathway selective inverse agonist at P2Y1R towards G12/13-mediated pathways, but not those associated by Gq activation in P2Y1R-transfected HEK293T cells. KSN-159-27 showed functionally selective inhibition for platelet P2Y1R-mediated functions. In vivo, KSN-159-27 suppressed inflammatory cell recruitment, whilst preserving bleeding time and ADP-induced thromboembolic responses, in contrast to the neutral P2Y1R antagonist MRS2500. This first demonstration for the rational design of a pathway selective inverse agonist at platelet P2Y1Rs has significant implications for novel therapeutic strategies developed to safely target platelet activation during inflammation, in contrast to current anti-platelet drugs used in the prevention of thrombosis. Key PointsO_LIBiased inverse platelet P2Y1R agonists selectively supress inflammation whilst preserving hemostasis and the ability of platelets to aggregate. C_LIO_LIBiased inverse agonism selectively inhibited P2Y1R G12/13 (Rho-GTPAse functions) but not Gq activities (PLC functions). C_LI

Background: Medulloblastoma (MB) is the most common malignant brain tumour in children, and aggressive subgroups are frequently driven by the oncoproteins MYC or MYCN. Direct therapeutic targeting of MYC/MYCN has been challenging because of their intrinsically disordered protein structures. The aim of this study was to determine whether novel SE486-11 analogues (UNSW-SCs) can therapeutically target MYC/MYCN-driven MB. Methods: The anticancer activity of UNSW-SCs was assessed in MB cell lines with differential MYC/MYCN expression. Target engagement was evaluated using surface plasmon resonance and drug affinity responsive target stability assays. Blood-brain barrier penetration, MYC/MYCN protein degradation, cell cycle effects, apoptosis, DNA damage, and synergy with histone deacetylase (HDAC) inhibitors were examined. Therapeutic efficacy was evaluated in murine models of MYC- and MYCN-driven human MB. Results: UNSW-SCs showed potent anticancer activity, with preferential selectivity toward MB cells expressing high MYC/MYCN levels and IC50 values ranging from 0.22 to 1.18 M. The lead molecule, UNSW-SC-22, directly bound MYC, crossed the blood-brain barrier, and achieved a brain-to-plasma ratio of 1.44 at peak concentrations. UNSW-SC-22 induced MYC/MYCN-dependent cytotoxicity associated with enhanced proteasomal degradation, cell cycle arrest, apoptosis, and DNA damage. Combined treatment with HDAC inhibitors further reduced MYC/MYCN protein levels, increased DNA damage, and enhanced apoptosis. In vivo, UNSW-SC-22, either alone or with entinostat, significantly suppressed intracranial tumour growth and prolonged survival. Conclusions: UNSW-SC-22 is a brain-penetrant MYC/MYCN-targeting molecule with potent preclinical activity in MYC/MYCN-driven MB, supporting its development as a monotherapy or combination strategy with HDAC inhibition.

Kaitocephalin (KCP) is a fungal neuroactive natural product bearing a peptide-like yet nonpeptidic amino acid-derived scaffold in which amino acid-like units are connected by C-C bonds rather than peptide bonds. The enzymatic construction of this unusual scaffold has remained unresolved. Here, we identify KpbH as a PLP-dependent enzyme that couples pyrroline-5-carboxylate, generated from L-ornithine, with L-aspartate to form (2S,5R)-5-((S)-2-amino-2-carboxyethyl)pyrrolidine-2-carboxylic acid (ACPCA), which corresponds to the nonpeptidic Ala-Pro substructure of KCP. D2O-labeling experiments showed enzyme-controlled, solvent-derived deuterium incorporation at C7 of ACPCA, supporting a decarboxylative Mannich-type mechanism. Feeding of a deuterium-enriched ACPCA-containing reaction mixture to the KCP-producing fungus Eupenicillium shearii resulted in deuterium incorporation into KCP, linking ACPCA to KCP biosynthesis. These results identify KpbH as the first native PLP-dependent enzyme that catalyzes an L-aspartate-dependent decarboxylative Mannich-type C-C bond-forming reaction and reveal a biosynthetic strategy for constructing a noncanonical amino acid-like C-C bond scaffold. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/733665v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@a27fb7org.highwire.dtl.DTLVardef@6eea95org.highwire.dtl.DTLVardef@1eae086org.highwire.dtl.DTLVardef@13a92e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cell penetrating thyclotides (CPTs) are synthetic molecules that promote highly efficient cellular uptake and endosomal escape of bioactive peptides. While peptides are valuable as medicinal agents, their translation to therapies is often limited by their inability to cross cell membranes. CPTs have a unique combination of chiral tetrahydrofurans and polar sidechains within a molecular scaffold that can be optimized to efficiently deliver peptide cargo into cells. The cellular uptake and endosomal escape of two peptides with anticancer biological activities but low bioavailabilities were remarkably improved after conjugation to a CPT. Using CPTs to overcome barriers to cellular uptake represents a new direction for the intracellular delivery of bioactive molecules, and will accelerate drug development for new medical therapies.