Ananya Naick

Methods Mol Biol. 2026;3034:145-170. doi: 10.1007/978-1-0716-5268-8_7.

ABSTRACT

In comparison to single-molecule localization microscopy (SMLM) of proteins located on the cell surface, SMLM of proteins within the nucleus requires fluorescent tags that are generally brighter, longer-lasting and (in the case of fluorescent ligands) membrane-permeable. Although fluorescent ligands typically have superior optical qualities compared to those of fluorescent protein (FP) tags, making them ideal candidates for nuclear SMLM, unlike FPs, they are unable to be expressed in cells as a fusion protein with the protein of interest. Self-labelling tags, on the other hand (e.g., HaloTag, SNAP-tag, and CLIP-tag), combine the ability to express a protein tag (e.g., HaloTag protein) as a fusion protein, with the enhanced optical qualities of externally added fluorescent dyes (e.g., dye-conjugated HaloTag ligand which covalently binds to the HaloTag protein). Excitation of only a subset of fluorescently labelled molecules is essential for achieving single-molecule resolution, and therefore SMLM of nuclear proteins requires a microscope setup capable of penetrating the nucleus with a thin beam that is powerful enough to produce an appropriate signal-to-noise ratio. Highly Inclined and Laminated Optical sheet (HILO) microscopy uses an oblique angle (just below that of Total Internal Reflection Fluorescence (TIRF)) to excite fluorescently labelled molecules within a thin, highly inclined plane, the signal of which can be amplified using a high-power TIRF filter.This chapter describes the following protocols: (i) seeding of cells into glass-bottom imaging dishes, (ii) transfection of cells with a HaloTag-tagged nuclear protein of interest, (iii) incubation of transfected cells with a fluorescent nuclear marker and a fluorescent HaloTag ligand, and (iv) single-molecule imaging of the fluorescently labelled HaloTag-tagged protein of interest in the nucleus of live cells using HILO microscopy with a high-powered TIRF filter.

PMID:42091813 | DOI:10.1007/978-1-0716-5268-8_7

Angew Chem Int Ed Engl. 2026 May 14:e8698780. doi: 10.1002/anie.8698780. Online ahead of print.

ABSTRACT

We report the design, synthesis, and characterization of a novel class of all-peptide macrocycles, Cyclo-Polyprolines (CP). Exploiting the precision of Fmoc-based solid-phase peptide synthesis (SPPS) and head-to-tail macrocyclization, this platform grants unparalleled control over the macrocycle's primary sequence and secondary structure, offering a viable route toward exo-/endo-functionalization and addressing a bottleneck of traditional synthetic host macrocycles. The resulting CP scaffold is highly amphiphilic, exhibiting excellent solubility in both organic and aqueous media. Structural analysis via NMR spectroscopy and single-crystal x-ray diffraction reveals a distinct chameleonic character: the macrocycle shifts from an all-junctions-cis conformation in organic solvents to a predominantly all-junctions-trans isomer in water. We demonstrate that this transition is driven by a cooperative hydration effect, wherein water molecules stabilize the expanded framework through precise two-point hydrogen bonding. Demonstrating responsive host-guest capabilities, CP undergoes induced-fit isomerization to bind ligands, successfully forming, among other species, an all-peptide pseudo-rotaxane. This methodology establishes a robust platform for creating functionalized, proline-based hosts with significant potential in medicinal chemistry, drug delivery, and organocatalysis, thereby bridging the gap between supramolecular systems and enzyme mimetics.

PMID:42131948 | DOI:10.1002/anie.8698780

Chembiochem. 2026 May 27;27(10):e70378. doi: 10.1002/cbic.70378.

ABSTRACT

Lasso peptides are structurally unique natural products endowed with high thermal and proteolytic stability, making them attractive as scaffolds for drug discovery. Recently, a new class of lasso peptides containing a second macrocycle, formed between a lysine sidechain and the C-terminus, was discovered, resulting in an even more compact architecture. Here, we report the first NMR structure of the class V lasso peptide, gelatinamin A. Using heterologous expression of the gelatinamin biosynthetic gene cluster (BGC) in Bacillus subtilis, we delineated the biosynthetic pathway through targeted gene deletions. We expressed and characterized the predicted transpeptidase, GelP, that catalyzes the formation of an isopeptide bond between Lys2 and the C-terminus and mediates the reversible conversion of gelatinamin B to gelatinamin A. In addition, we characterize GelT, an N-acetyltransferase that inactivates lasso peptide antimicrobial activity by acetylating a key lysine residue. Furthermore, we demonstrate that gelatinamin is highly potent against several important pathogens and that the activity is strongly bicarbonate-dependent. Finally, we propose a complete biosynthetic pathway for gelatinamin. The structural insight of gelatinamin A and the functional characterization of GelP provide the foundation for future discovery of class V lasso peptides and for engineering transpeptidases to modify other lasso peptide scaffolds.

PMID:42138325 | PMC:PMC13178200 | DOI:10.1002/cbic.70378

ACS Infect Dis. 2026 May 14. doi: 10.1021/acsinfecdis.6c00123. Online ahead of print.

ABSTRACT

Globally, Mycobacterium tuberculosis remains a significant burden. Although effective treatment regimens exist, drug resistance has continued to emerge. This clinical resistance, combined with side effects and protracted treatment times from the current front-line therapies, means that there is a need to identify novel agents to combat this disease. Here, we report on a new chemical series, identified by whole-cell phenotypic growth inhibition screening, that demonstrates significant activity across multiple media. Mode of action studies indicate that this series targets the same biological pathway as ethambutol (EMB), a drug used in the current front-line treatment of tuberculosis. Screening selected analogues against clinical isolates, resistant to EMB, demonstrated differential sensitivity both across the molecules and against the different specific resistant mutations. The data obtained suggest that this series has potential to be developed into a viable alternative to EMB.

PMID:42135210 | DOI:10.1021/acsinfecdis.6c00123

ACS Infect Dis. 2026 May 13. doi: 10.1021/acsinfecdis.6c00136. Online ahead of print.

ABSTRACT

PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets) is an antimicrobial discovery platform based on chemical-genetic interaction profiling of compounds against a pool of Mycobacterium tuberculosis (Mtb) hypomorphs, each depleted of an essential gene. We now report a novel N-oxolan-3-yl pyrazole carboxamide inhibitor, BRD1554, with selective activity against strains depleted of polyketide synthase 13 (Pks13), an essential polyketide synthase in mycolic acid synthesis, and Rv2581c, an uncharacterized protein similar to glyoxylase II enzymes. PCL analysis, our previously described reference-based approach to PROSPECT mechanism of action (MOA) assignment, predicted Pks13 was the likely target, implicating its thioesterase domain. We synthesized a more potent analogue 1554-06 and assigned the absolute stereochemistry of the active 3R,4S diastereomer, which had an MIC₉₀ of 3.0 μM against Mtb H37Rv. Expression profiling and the identification of resistance-conferring mutations in the thioesterase domain of Pks13 were consistent with the PCL prediction. Stereoisomers of 1554-06 inhibited recombinant Pks13 thioesterase domain in a stereospecific manner, consistent with their whole cell activity, and computational docking revealed the structural basis for the observed specificity, thereby confirming Pks13 thioesterase domain as the target. We observed unique chemical-genetic interactions between inhibitors of the different Pks13 domains and different Mtb detoxifying enzymes, including Rv2581c. These results highlight how PROSPECT can not only immediately reveal, with domain-level resolution, the MOA of new inhibitors, allowing the integration of biological insight into early prioritization, but can also illuminate genetic interactions linked to those mechanisms that could inform synergy predictions for combination therapy.

PMID:42126262 | DOI:10.1021/acsinfecdis.6c00136

J Struct Biol X. 2026 Apr 23;13:100145. doi: 10.1016/j.yjsbx.2026.100145. eCollection 2026 Jun.

ABSTRACT

Angiotensin-converting enzyme 2 (ACE2) is a key node in the protective axis of the renin-angiotensin-aldosterone system (RAAS) for blood pressure and hydroelectrolyte regulation and the receptor recognized by the spike glycoproteins of the severe acute respiratory syndrome (SARS) coronaviruses (CoV) SARS-CoV and SARS-CoV-2. We identified the macrocyclic peptide WJL-63 by mRNA display and characterized it biochemically and with respect to ACE2-binding. The crystal structure of the extracellular region of ACE2 in complex with the peptide at 2.2 Å resolution was elucidated. The structure revealed a binding mode in which WJL-63 is accommodated towards one side of the catalytic cleft of the peptidase domain, without contacting the conserved zinc ion site. WJL-63 residues Q4, R7, R11, and R14 anchor the peptide in the binding pocket. The peptide contacts both peptidase subdomains. The upright binding mode requires an open ACE2 conformation, in contrast to small-molecule carboxypeptidase inhibitors, which typically bind to the closed conformation. One side of WJL-63 is accessible for modification such as the herein reported conjugation of a chelator for radiometal labeling. The radiolabeled DOTA-WJL-63 was evaluated on ACE2-transfected HEK cells, where it exhibited binding with a K_D value of 90 ± 28 nM. The ACE2 - WJL-63 complex structure provides a basis for the development of compounds that modulate ACE2 conformation and for the development of imaging agents for visualization of ACE2, including fluorescence or electron microscopy and positron emission tomography (PET).

PMID:42095196 | PMC:PMC13142116 | DOI:10.1016/j.yjsbx.2026.100145

ACS Infect Dis. 2026 May 8. doi: 10.1021/acsinfecdis.6c00092. Online ahead of print.

ABSTRACT

Previous work suggests that Enterococcus faecalis uses exogenous oleic acid (FA 18:1(9z)) to increase its tolerance against the membrane-targeting antimicrobial, daptomycin. However, the specific roles of OA and the endogenous positional isomer, cis-vaccenic acid (FA 18:1(11z)), in both daptomycin-susceptible (Dap-S) and daptomycin-resistant (Dap-R) strains have not been fully explored. We performed lipidomics using hydrophilic interaction liquid chromatography and reversed-phase liquid chromatography-ion mobility-mass spectrometry to identify alterations in the lipid composition of Dap-S (S613) and Dap-R (R712) strain pairs of E. faecalis following OA or CV supplementation. Lipidomics showed that total PG intensity in Dap-S was only impacted by OA, while total DGDG intensity was impacted by both OA and CV. However, total PG and DGDG intensities in Dap-R were not impacted by either FA. Both strains produced significantly more lipids containing two 18:1 acyl tails following OA supplementation, while CV had a similar effect on Dap-S only, indicating that Dap-S and Dap-R respond to OA and CV differently. Additionally, the preferences for OA vs CV, alone and in mixtures, were distinguished using ozonolysis and deuterium-labeling techniques that can resolve C═C positional isomers. Growth and survival assays show that Dap-S responds differently to daptomycin when it is cultured with OA or CV. Together, these results reveal that E. faecalis strains with genetic determinants for daptomycin resistance respond differently, in membrane lipid composition and overall growth, to biologically relevant FA 18:1 isomers compared to strains that acquire daptomycin tolerance solely through exogenous fatty acid uptake.

PMID:42100965 | DOI:10.1021/acsinfecdis.6c00092

Bioconjug Chem. 2026 Apr 29. doi: 10.1021/acs.bioconjchem.5c00627. Online ahead of print.

ABSTRACT

One prominent open question in the field of antisense oligonucleotide remains the intracellular delivery of active sequences. While different strategies have been developed for addressing this issue, including formulation and/or conjugation approaches, multiconjugations with both sugars and nucleolipids have not been investigated so far. In this contribution, we are interested in a new hypothesis that focuses on the double conjugation of oligonucleotides with both glucose-thymine and nucleolipid moieties. The lipid carbohydrate modified antisense oligonucleotides were the most efficient in down regulating the expression of KRAS in pancreatic cancer cells.

PMID:42055799 | DOI:10.1021/acs.bioconjchem.5c00627

Bioconjug Chem. 2026 May 20;37(5):846-874. doi: 10.1021/acs.bioconjchem.6c00123. Epub 2026 May 3.

ABSTRACT

Neoplasms of the brain remain challenging to treat due to their complex heterogeneity and the presence of physiological barriers that confer the brain its immune privileged status. Extensive drug discovery efforts have focused on small molecule inhibitors targeting dysregulated cellular pathways to improve patient survival and quality of life. However, these approaches often face limitations, including incomplete efficacy arising from pathway redundancy and compensatory mechanisms. Induced protein degradation has emerged as a novel therapeutic strategy with a distinct mechanism of action, offering the potential to overcome many of the shortcomings associated with conventional inhibition. Several classes of degraders have now advanced into clinical trials for various diseases including cancers. This review provides an overview of the current state of protein degradation technologies as applied to brain cancer therapy, with a particular focus on three modalities: PROTACs, molecular glues, and LYTACs. Key aspects discussed include their synthesis, biological evaluation, and translational potential. The field offers an opportunity to develop meaningful therapies and stands at the precipice of breakthroughs that could transform the treatment landscape for brain cancer patients.

PMID:42076869 | DOI:10.1021/acs.bioconjchem.6c00123

ACS Infect Dis. 2026 Apr 17. doi: 10.1021/acsinfecdis.6c00011. Online ahead of print.

ABSTRACT

The pathogen Mycobacterium abscessus (Mab) can cause severe and difficult-to-treat chronic lung infections. Despite the rising incidence and clinical concern of Mab infections, treatment options are limited and often ineffective. Treatment is complicated by Mab's ability to persist in a nonreplicating, drug-resistant state. Several β-lactam antibiotics are potently bactericidal against Mab but are underutilized because their molecular mechanisms of action against Mab are incompletely understood. In the current study, we used β-lactam-derived activity-based probes and chemoproteomics to report the first comprehensive list of Mab enzymes targeted by β-lactams. We compared β-lactam targets across two Mab subspecies in actively replicating and nonreplicating cultures, using a new carbon starvation model of persistence. We identified 17 targets that were active in every condition tested, seven of which were previously unknown to bind β-lactams. Lastly, we characterized the β-lactamase activity and β-lactam inhibition profiles of nine Mab enzymes, demonstrating that imipenem inhibits these targets more effectively than cefoxitin. These findings demonstrate β-lactam target engagement in persistent Mab and provide clarity on the mechanisms of action of clinically relevant β-lactams in Mab, crucial steps toward fully realizing their potential for treating infections caused by this opportunistic pathogen.

PMID:41996672 | DOI:10.1021/acsinfecdis.6c00011

ACS Infect Dis. 2026 May 8;12(5):1600-1610. doi: 10.1021/acsinfecdis.5c00701. Epub 2026 Apr 28.

ABSTRACT

Tuberculosis (TB) remains a major global health threat, largely due to Mycobacterium tuberculosis (Mtb) resilience, which can be exacerbated by exposure to sublethal antibiotic concentrations arising from factors such as patient nonadherence and the granuloma structure which limits drug penetration. Within host granulomas, Mtb can exhibit both phenotypic tolerance and genotypic resistance, complicating curative treatments. This study aimed to determine whether sublethal rifampicin acts as a signaling molecule in Mycobacterium smegmatis (Msm) and the attenuated Mtb H37Ra strain, triggering phenotypic changes that promote tolerance to lethal drug levels. Msm exposed to half-MIC rifampicin showed an initial transient growth deceleration followed by a resumption of proliferation, indicating the acquisition of phenotypic tolerance. Deep data-independent acquisition (DIA) mass spectrometry-based proteomic profiling revealed that the early response (45 min) involved the upregulation of ribosomal proteins, DNA replication, and de novo purine biosynthesis. Proteins associated with phenotypic resistance (e.g., RpoZ, GidB, WhiB2) and efflux transporters were also upregulated. As Msm recovered (180 min), its proteome largely returned to baseline, but key resistance-associated pathways, including the Rifampin ADP-ribosyltransferase superfamily and certain efflux systems, remained dysregulated. Parallel studies on Mtb H37Ra also demonstrated a distinct proteomic shift, comprising conserved adaptive responses such as ribosomal perturbation and compensatory transcriptional activity, as well as species-specific dysregulation of drug influx/efflux pumps and cell envelope remodelling via the polyketide synthase family. These findings demonstrate that sublethal rifampicin exposure primes mycobacteria for enhanced tolerance to lethal drug concentrations, underscoring a significant challenge in current TB therapy.

PMID:42048180 | PMC:PMC13162311 | DOI:10.1021/acsinfecdis.5c00701

bioRxiv [Preprint]. 2026 Apr 26:2026.04.24.720480. doi: 10.64898/2026.04.24.720480.

ABSTRACT

Investigating and manipulating cellular events requires precise control of protein function. To enable control over cellular processes, we set out to design a chemically induced dimerization (CID) system consisting of a de novo designed ligand and protein pair. Here we describe the design of a C2 symmetric membrane permeable macrocyclic peptide and a cognate protein homodimer which binds the macrocycle through a large interface with both chains. The designed homodimer binds the macrocycle with a K D of 36 nM, and the x-ray crystal structure of the protein homodimer-macrocycle complex is very close to the computational design model, with the C2 axis of the macrocycle aligned with the homodimer C2 axis. Transcriptional and split luciferase assays in mammalian cells demonstrates conditional control over both a reporter gene expression and luciferase reconstitution.

PMID:42079119 | PMC:PMC13131926 | DOI:10.64898/2026.04.24.720480

Yi Chuan. 2026 Apr 20;48(4):345-354. doi: 10.16288/j.yczz.25-186.

ABSTRACT

Cyclic peptides have emerged as a promising therapeutic modality for targeting classically deemed "undruggable" protein that defy conventional small-molecule intervention. Their rigid conformations improve binding affinity, selectivity and proteolytic stability, bridging the gap between low-molecular-weight drugs and large biologics. Early discovery of cyclic peptide relied on natural product isolation. However, display technologies now enable the generation and screening of libraries containing up to 10¹⁵ unique sequences. Among these, mRNA display technology offers a uniquely powerful in vitro platform that covalently links genotype (mRNA) to phenotype (encoded peptide), facilitating the ultrahigh-throughput interrogation of trillions of variants in a single selection cycle. Recent clinical translation of de novo mRNA display-derived cyclic peptides signals the approach's maturation. In this review, we systematically evaluate mRNA display technology by outlining its methodological framework and highlighting its unique advantages for engineering macrocyclic therapeutics. We critically examine its transformative potential as well as its inherent limitations in identifying bioactive cyclic peptides, with particular attention to library diversity and the precision of on-resin screening. Through this analysis, we aim to provide insightful perspectives and strategic recommendations to guide future efforts in exploiting mRNA display for the development of innovative cyclic peptide-based drugs.

PMID:41992883 | DOI:10.16288/j.yczz.25-186

Front Pharmacol. 2026 Apr 10;17:1699987. doi: 10.3389/fphar.2026.1699987. eCollection 2026.

ABSTRACT

Antimicrobial resistance (AMR) remains one of the most serious global threats to public health, driven by the rapid emergence and dissemination of multidrug-resistant bacterial pathogens that compromise existing antibiotic therapies. In response, the World Health Organization (WHO) has defined priority lists of antibiotic-resistant bacteria to guide research, innovation, and drug development efforts. This narrative review synthesizes current knowledge on the molecular mechanisms underlying resistance in WHO-priority pathogens, including reduced membrane permeability, efflux pump overexpression, enzymatic drug inactivation, target modification, biofilm formation, and horizontal gene transfer. Beyond mechanistic insights, we critically evaluate the therapeutic limitations of conventional antibiotics, the failure of traditional discovery pipelines, and the growing clinical and economic burden of resistant infections. Emerging strategies, including artificial intelligence-assisted drug discovery, phage therapy, antimicrobial peptides, CRISPR-based systems, resistance-modifying combinations, and natural product-derived compounds and plant compounds, are assessed with emphasis on pharmacological feasibility, translational challenges, and clinical relevance. Particular attention is given to issues of delivery, toxicity, dosing optimization, resistance emergence, regulatory barriers, and real-world implementation. Finally, we highlight the central role of antimicrobial stewardship, surveillance, and a One Health framework integrating human, animal, and environmental sectors in mitigating resistance and sustaining therapeutic effectiveness. Collectively, this review underscores that addressing WHO-priority pathogens will require integrated, multidisciplinary strategies that bridge molecular biology, pharmacology, clinical translation, and public health.

PMID:42038316 | PMC:PMC13106162 | DOI:10.3389/fphar.2026.1699987

Bioconjug Chem. 2026 Apr 23. doi: 10.1021/acs.bioconjchem.5c00653. Online ahead of print.

ABSTRACT

Aminoglycosides have long served as indispensable antibacterial agents, yet their clinical utility has diminished due to toxicity and pervasive resistance with the advent of superbugs. Over the past decade, however, a renewed chemical interest transformed these classical antibiotics into versatile molecular scaffolds through various covalent modifications. Among these the lipidation, a strategy that fundamentally reprograms their biological behavior is of particular interest. By installing hydrophobic or amphiphilic domains onto the polycationic 2-deoxystreptamine framework, aminoglycosides acquire a new mode of action that diverges strikingly from ribosomal targeting. Lipidated aminoglycosides emerge as potent membrane-active agents capable of overcoming multidrug resistance, penetrating biofilms, eradicating persister cells, and displaying broad-spectrum antifungal activity. In parallel, their intrinsic RNA affinity and enriched cationic functionality enable efficient condensation with nucleic acids, endosomal escape, and the formation of self-assembled nanostructures. This positions lipidated aminoglycosides as promising candidates for nonviral DNA, siRNA, and mRNA delivery. This review focuses on the chemical logic, methods, and mechanistic insights that underpin the evolution of lipidated aminoglycosides from early acylated derivatives to modern amphiphilic, guanidinium-linked, sterol conjugated, and ionizable aminoglycoside lipids.

PMID:42024108 | DOI:10.1021/acs.bioconjchem.5c00653

Bioconjug Chem. 2026 Apr 24. doi: 10.1021/acs.bioconjchem.5c00645. Online ahead of print.

ABSTRACT

Understanding the spatiotemporal dynamics of protein synthesis and degradation is important for establishing how cells maintain protein homeostasis. Conventional methods for detecting newly synthesized proteins include metabolic labeling with radioactive [³⁵S]methionine (Met) or the incorporation of l-azidohomoalanine (AHA) or l-homopropargylglycine followed by fluorescent labeling via copper(I)-catalyzed click chemistry. However, these methods typically require cell fixation, making them unsuitable for live-cell imaging. Here, we describe a fluorescence imaging technique to monitor newly synthesized proteins in living cells by utilizing a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction, in which l-AHA-containing proteins are labeled with fluorescent dyes conjugated to dibenzocyclooctyne (DBCO). We synthesized orange-emitting tetramethylrhodamine (TAMRA)-DBCO and far-red-emitting silicon rhodamine (SiR)-DBCO. TAMRA-DBCO enabled the visualization of newly synthesized proteins and their time-dependent degradation throughout the entire cell. SiR-DBCO was similarly effective, but was mainly distributed to the cytoplasm. The time-dependent decrease of TAMRA-DBCO fluorescence intensity in living cells was suppressed by lysosomal enzyme inhibitors and a proteasome inhibitor, suggesting that newly synthesized proteins are degraded via both pathways. Moreover, imaging of drug-induced senescent cells with TAMRA-DBCO suggested that senescent cells have a lower protein degradation ability than nonsenescent cells. These methods should be useful for investigating protein homeostasis in living cells.

PMID:42030520 | DOI:10.1021/acs.bioconjchem.5c00645

ACS Infect Dis. 2026 Apr 20. doi: 10.1021/acsinfecdis.5c01091. Online ahead of print.

ABSTRACT

The Mycobacterium avium complex (MAC) comprises opportunistic pathogens of growing clinical significance, particularly in immunocompromised individuals and aging populations. Advances in molecular genomics have refined MAC taxonomy, enabling more precise diagnosis and informing therapeutic strategies. Whole-genome sequencing has revealed substantial genomic diversity within MAC, particularly in M. avium subsp. hominissuis (MAH), the predominant subspecies isolated from patients, and has helped identifying genetic determinants of strain-specific adaptation, virulence, and environmental persistence. The escalating incidence of MAH infections represents a pressing public health concern, further complicated by intrinsic and acquired antimicrobial resistance and the absence of standardized susceptibility testing. This review integrates recent developments in MAC taxonomy and epidemiology, with a particular focus on MAH pathogenesis, and provides an updated overview of the drug discovery landscape. We highlight innovative strategies, including novel antimicrobials and host-directed therapies, with the goal of developing safer, more effective medication. By linking mechanistic insights to translational applications, this work underscores the potential to improve clinical outcomes in patients affected by MAH infections and provides a framework for future precision-guided therapies.

PMID:42009313 | DOI:10.1021/acsinfecdis.5c01091

Chem Sci. 2026 Apr 9. doi: 10.1039/d6sc01722c. Online ahead of print.

ABSTRACT

Macrocyclic peptides represent a promising therapeutic modality for challenging targets, such as protein-protein interactions. However, their clinical utility is often limited by inadequate membrane permeability, which restricts both intracellular target access and oral bioavailability. Existing structure-based generative methods for cyclic peptide design prioritize structural validity and binding affinity, yet lack mechanisms to co-optimize membrane permeability. Here we present CycDiff-DPO, a preference-aligned diffusion framework for designing target-specific macrocyclic peptide binders with optimized membrane permeability. By ranking sampled candidates with a Caco-2 permeability predictor and constructing preference pairs, CycDiff-DPO aligns the generative distribution toward permeability-favorable chemical space while preserving target binding competence. We benchmarked CycDiff-DPO across 56 protein targets, finding higher predicted Caco-2 and PAMPA permeability across multiple independent predictors, alongside superior binding energetics and comparable stereochemical quality relative to baseline methods. Case studies on Keap1-Nrf2 and SPSB2-iNOS confirm that top designs recapitulate hot-spot interactions and maintain stable bound poses in molecular dynamics simulations. CycDiff-DPO provides a framework for permeability-enhanced macrocyclic peptide design with broad therapeutic applications.

PMID:41982931 | PMC:PMC13074632 | DOI:10.1039/d6sc01722c