Bioorganic & Medicinal Chemistry Letters

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.

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.

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.

Contemporary RNA-binding ligand collections are biased toward aromatic scaffolds, although it remains unclear whether this over-representation reflects an intrinsic requirement for productive RNA recognition or historical discovery bias. Here, using a modular "host-guest" ligand design strategy targeting the env8 cobalamin (Cbl) riboswitch, we established a common molecular framework to directly evaluate whether aromaticity is fundamentally required for RNA binding. We synthesized a focused series of cyclic aliphatic {beta}-axial Cbl derivatives, expanding the ligand library and enabling matched-pair comparisons to isolate the contribution of aromaticity to molecular recognition. Aliphatic ligands supported high-affinity RNA binding and regulatory activity comparable to aromatic analogues, with several derivatives exhibiting equal or greater affinity than their matched aromatic counterparts. Structural analyses revealed that aromatic and aliphatic ligands engage the same cryptic RNA binding site through distinct modes of molecular recognition, including nucleobase {pi}-stacking and alternative van der Waals packing arrangements. Machine learning analyses further demonstrated that the physicochemical features associated with affinity extend beyond aromaticity itself and instead reflect a broader combination of shape, surface, heteroatom, and electronic properties. Together, these findings demonstrate that high-affinity RNA binding can arise from multiple structural and physicochemical solutions, suggesting that aromaticity is not uniquely privileged as a strategy for RNA-targeted ligand design and supporting broader exploration of underrepresented RNA-binding chemotypes.

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.

Glioblastoma (GBM) is the most common primary malignant brain tumor and is typically fatal. GBM therapies are hindered by the impermeability of the blood brain barrier (BBB), the diffuse and infiltrative nature of the tumor, and the high heterogeneity of intratumoral GBM cells. Aptamers are short, synthetic, folded single strands of RNA or DNA or analogs that bind targets with high affinity and specificity. Aptamers are developed via the principles of natural selection, permitting an unbiased approach to therapeutic development. Thus, rather than using rational design to select a target and develop a targeting moiety, cycles of Systematic Evolution of Ligands by Exponential Enrichment (SELEX) are employed in cell culture or in vivo to identify aptamers against unknown targets. Antibody drug conjugates (ADCs) have shown some efficacy for GBM but are limited by their large size and thus depend on leakiness of the BBB. We have recently applied in vivo SELEX to develop anti-GBM aptamers (six-fold smaller in mass than IgG antibodies) and to select aptamer-drug conjugates. Here we report attempts to focus aptamer selection toward internalizing drug-delivery targets and resulting challenges involving loss of tumor specificity in vivo.

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.

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.

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.

Background: Subtype-selectivity predictions are scored against measured selectivity and judged against an assumed noise ceiling. We asked what an 2-adrenergic benchmark rewards and which controls change its interpretation. Research design and methods: On a frozen benchmark of 586 paired 2A/2C compounds we evaluated Glide SP docking, CNN rescoring, ligand-only fingerprint models, receptor descriptors and pose contacts, with dopamine D3/D2 as comparator, applying five controls: a measured ceiling, a cluster-identity null, a nonselective reference, a same-receptor floor and a trivial-descriptor baseline. Results: Five descriptors from SMILES reached Spearman 0.645, 72% of the measured ceiling, against 0.071 for Glide SP and 0.188 for CNN rescoring; receptor properties and pose contacts reduced to size under control, while a non-size signal of 0.258 survived. Measured rather than propagated noise raised that ceiling from 0.704 to 0.897; cluster identity alone reached R2 0.499 on D3/D2 and none on 2; a nonselective reference received +1.43 to +4.79 kcal/mol where zero is expected; and a same-receptor floor reached 1.77-fold against 1.88-fold across subtypes. Conclusions: Such benchmarks reward molecular size first; a method must exceed 0.645 before its score indicates structural reasoning. The controls are inexpensive; conclusions rest on two receptor pairs, a three-pair floor and static structures.

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

Small interfering RNA (siRNA) enables precise post-transcriptional gene silencing for refractory diseases, yet its clinical translation remains limited by the lack of safe and efficient delivery vectors. Inspired by the dissymmetric alkyl chain architecture of natural membrane phospholipids, we designed and synthesized 34 novel ionizable lipids with dissymmetric hydrophobic tails and formulated them into lipid nanoparticles (LNPs). Through systematic physicochemical and biological assessments, we established clear structure-activity relationships and identified two lead LNPs (O14-LNP, H18a-LNP) with superior endosomal escape capacity, enhanced in vivo gene silencing potency, and favorable biosafety relative to the clinical benchmark MC3-LNP. In both streptozotocin-induced and spontaneous db/db type 2 diabetes (T2D) mouse models, lead LNPs delivering ferroptosis-related siRNAs effectively ameliorated glucose and lipid metabolic disorders, restored islet function, and alleviated hepatic steatosis. This study not only lays a theoretical foundation for the rational design of novel ionizable lipids, but also validates the therapeutic potential of siRNA therapy targeting ferroptosis, providing a versatile delivery platform and targeted therapeutic strategy for the treatment of T2D.

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.

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.

Plant-derived small molecules possess highly diverse physicochemical properties, and the computational design of their protein recognition elements depends not only on the global structural quality of candidate backbones, but also on whether the local binding pocket, ligand-contact pattern, and predefined recognition conformation can be consistently retained after sequence design and structural back-prediction. To explore pocket-design strategies for different types of natural-product small molecules, this study selected capsaicin, (4R)-limonene, and quercetin as model ligands, representing a flexible amphipathic molecule, a compact hydrophobic monoterpene, and a rigid polyphenolic flavonoid scaffold, respectively, and covering the dimensions of pungent sensory flavor, volatile aroma, and flavonoid functional constituents. A ligand- physicochemical-property-guided computational design and multi-stage prioritization framework was established for candidate protein binders. The results showed that candidates with favorable initial global structural scores did not necessarily form reasonable local small-molecule binding pockets, indicating that evaluation of the local ligand environment is essential for candidate prioritization. After screening, 31 partial- pocket candidate backbones for capsaicin, 75 buried hydrophobic-pocket candidate backbones for (4R)-limonene, and 56 pocket-qualified candidate backbones for quercetin were obtained. Further sequence design and structural back-prediction analyses indicated that a subset of candidates could maintain the original pocket geometry and major ligand-contact patterns after sequence realization. Overall, these results suggest that the physicochemical properties of different plant-derived small molecules substantially influence the efficiency of de novo protein pocket formation, with compact hydrophobic ligands being more compatible with buried hydrophobic- pocket strategies, whereas flexible or multipolar ligands require a more refined balance between hydrophobic burial and polar exposure. This study provides a pre- experimental computational prioritization framework for natural-product small- molecule-recognizing proteins and offers candidate resources for subsequent protein expression, in vitro binding validation, active-constituent enrichment, and development of small-molecule biorecognition tools. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/743643v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@8fe6c2org.highwire.dtl.DTLVardef@176cef2org.highwire.dtl.DTLVardef@10c8201org.highwire.dtl.DTLVardef@2b28cf_HPS_FORMAT_FIGEXP M_FIG C_FIG

The initial attachment of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) to host cell sialosides is critical for infection, yet its precise receptor specificity remains poorly understood. Here, we describe a chemoenzymatic methodology to synthesize a comprehensive panel of 6-sulfo sialyl Lewisx (6-sulfo-SLex) containing glycans. Our approach entails the enzymatic assembly of an oligo-lactosamine chain modified at specific positions with N-trifluoroacetyl-glucosamine (GlcNTFA) moieties. Mild base treatment removes the TFA group to yield glucosamine, which effectively blocks enzymatic fucosylation. By leveraging this approach alongside the unique substrate selectivity of GlcNAc-6-O-sulfotransferases 2 (CHST-2), we achieved the selective preparation of fucosylated 6-sulfo-SLex glycans. Microarray screening of these printed glycans revealed that a 6-sulfo-SLex derivative presented on an extended LacNAc chain is the preferred host receptor for MERS-CoV. Conjugation of this lead compound to a polyglycerol-based dendrimer generated a multivalent inhibitor that potently blocks hemagglutination of human red blood cells by the MERS-CoV spike protein N-terminal domain (NTD). Furthermore, computational modeling demonstrated that the fucose moiety does not directly contact the viral spike protein. Instead, it pre-organizes the ligand into a favorable conformation, enabling a critical salt bridge between the glycans sulfate group and the guanidinium side chain of viral residue Arg307.

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.

To combat refractory diseases, such as cancer, multitarget-directed ligands (MTDLs) have become an emerging area of research to exploit synthetic lethality (SL) relationships associated with drug resistance. Herein, we present the in silico design of MTDLs for the polypharmaceutical treatment of endometrial adenocarcinoma (EAC) and our discovery of a novel SL in EAC; PTEN loss of function (LOF) and the inhibition of CDK9. We used high-resolution x-ray crystallographic data to chemically engineer, LCI133, to inhibit CDK9, CDK4/6-and AURKA/B kinases. PTEN LOF in EAC results in augmented deregulated transcription and a massive increase in nascent RNA, a phenotype which encodes a high level of apoptotic sensitivity to LCI133 and CDK9 inhibitors. Treatment with LCI133 results in a rapid decline nose-dive in global nRNA, MYC nRNA levels and TS elongation (TE) in PTEN LOF EAC. PTEN LOF is necessary and sufficient to confer sensitivity of EAC cells to LCI133 and other CDK9 inhibitors.

Many functional RNA motifs adopt structures that deviate from the canonical A-form helix and are emerging targets for RNA-directed therapeutics. The microtubule-associated protein tau (MAPT) A-bulge motif (5'-GCAGU/5'-ACGU) is one such motif. Because its structure is stabilized by a delicate balance of local interactions, its accurate modeling remains a major challenge for molecular dynamics (MD) simulations. The experimentally determined nuclear magnetic resonance (NMR) structure of the MAPT A-bulge motif provides a stringent test of whether RNA force fields can accurately reproduce the experimentally observed conformation. Most current AMBER-family RNA force-field models have incorrectly favored a non-native base-triple state of the MAPT A-bulge motif over the experimentally observed stacked state. Structural comparison of the stacked and base-triple conformations revealed that overly favorable NH-N hydrogen bonds between the bulged adenosine and an adjacent Watson-Crick base pair were the primary source of this imbalance. We developed gHBfix-18Ab, an 18-component hydrogen-bond correction that distinguishes NH and NH2; donors. gHBfix-18Ab was combined with the previously developed OL3CP and NBfix0BPh corrections to generate the composite model gHBfix-18Ab*. This model restored the experimentally observed stacked state as the global minimum in the calculated free-energy profile and improved agreement with NMR-derived distance data for the A-bulge region. Importantly, gHBfix-18Ab* did not produce marked structural destabilization of the cUUCGg tetraloop, a widely used benchmark for RNA force-field validation, suggesting that the refinement preserves the stability of the unrelated RNA motif. These results demonstrate that targeted refinement of hydrogen-bond interactions provides a practical strategy for systematic improvement of RNA force fields toward more accurate modeling of noncanonical RNA motifs.

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.