Rachita Dash

Nat Commun. 2025 Jun 25;16(1):5367. doi: 10.1038/s41467-025-60907-x.

ABSTRACT

Macrocyclic peptides represent an attractive drug modality due to their favourable properties and amenability to in vitro evolution techniques such as phage or mRNA display. Although very powerful, these technologies are not without limitations. In this work, we address some of their drawbacks by developing a yeast display-based strategy to generate, screen and characterise structurally diverse disulfide-cyclised peptides. The use of quantitative flow cytometry enables real-time monitoring of the screening of millions of individual macrocyclic peptides, leading to the identification of ligands with good binding properties to five different protein targets. X-ray analysis of a selected ligand in complex with its target reveals optimal shape complementarity and extensive surface interaction, explaining its exquisite affinity and selectivity. The yeast display-based approach described here offers a facile, quantitative and cost-effective alternative to rapidly and efficiently discover and characterise genetically encoded macrocyclic peptide ligands with sufficiently good binding properties against therapeutically relevant targets.

PMID:40562762 | PMC:PMC12198371 | DOI:10.1038/s41467-025-60907-x

J Am Chem Soc. 2025 Jul 9;147(27):24113-24126. doi: 10.1021/jacs.5c08019. Epub 2025 Jun 25.

ABSTRACT

Liquid-liquid phase separation (LLPS) of proteins can form membraneless organelles in the cell and can lead to pathological aggregation associated with neurodegenerative diseases. However, progress in controlling LLPS has been limited, and there has been no emergence of engineered de novo molecules to induce and modulate LLPS of targeted proteins. Here, we report de novo peptides that efficiently induce the LLPS of α-synuclein (αSyn), a protein involved in Parkinson's disease, discovered by the RaPID (random nonstandard peptides integrated discovery) system. These peptides primarily interact with the C-terminal region of αSyn, leading to the formation of an interaction network with αSyn and resulting in efficient droplet formation. Our study demonstrates the capacity of target-specific peptides to control LLPS and the subsequent liquid-solid phase transition (LSPT). Our novel LLPS-inducing and LSPT-modulating peptides may serve as a promising tool for fundamental investigations of LLPS and potentially for therapeutic intervention in amyloid diseases.

PMID:40560766 | PMC:PMC12257511 | DOI:10.1021/jacs.5c08019

Nat Chem Biol. 2025 Jun 20. doi: 10.1038/s41589-025-01929-w. Online ahead of print.

ABSTRACT

Developing macrocyclic binders to therapeutic proteins typically relies on large-scale screening methods that are resource intensive and provide little control over binding mode. Despite progress in protein design, there are currently no robust approaches for de novo design of protein-binding macrocycles. Here we introduce RFpeptides, a denoising diffusion-based pipeline for designing macrocyclic binders against protein targets of interest. We tested 20 or fewer designed macrocycles against each of four diverse proteins and obtained binders with medium to high affinity against all targets. For one of the targets, Rhombotarget A (RbtA), we designed a high-affinity binder (K_d < 10 nM) despite starting from the predicted target structure. X-ray structures for macrocycle-bound myeloid cell leukemia 1, γ-aminobutyric acid type A receptor-associated protein and RbtA complexes match closely with the computational models, with a Cα root-mean-square deviation < 1.5 Å to the design models. RFpeptides provides a framework for rapid and custom design of macrocyclic peptides for diagnostic and therapeutic applications.

PMID:40542165 | DOI:10.1038/s41589-025-01929-w

bioRxiv [Preprint]. 2025 May 27:2025.05.23.655853. doi: 10.1101/2025.05.23.655853.

ABSTRACT

As drug and natural product libraries expand, assays for assessing mechanisms of action (MoA) are increasingly critical. Performing cytological profiling using the Cell Painting (CP) assay enables image-based profiling of cellular states upon treatment, yet many bioactive compounds remain uncharacterized due to undetectable cellular effects under standard conditions. To address this, we combined drug dosing with cell activation using the protein kinase C (PKC) agonist phorbol myristate acetate (PMA). Profiling A549 lung cancer cells treated with 8,387 compounds at two concentrations (1 and 10 µM) in both resting and PMA-activated states allowed us to detect phenotypic effects for up to 40% of all screened compounds, effectively illuminating new phenotypic "dark space". Over 1,000 compounds exhibited phenotypes exclusively under PMA activation, establishing its advantage for MoA studies. We introduce novel quality control measures for CP screens and demonstrate that integrating phenotypic signatures enhances MoA discovery. Notably, 2-methoxycinnamaldehyde clustered with glucocorticoid receptor modulators and induced nuclear translocation, emphasizing the power of this approach in uncovering novel drug mechanisms and, therefore, aiding in improving therapeutic strategies.

PMID:40501955 | PMC:PMC12154737 | DOI:10.1101/2025.05.23.655853

ACS Med Chem Lett. 2025 Jun 2;16(6):1017-1023. doi: 10.1021/acsmedchemlett.5c00078. eCollection 2025 Jun 12.

ABSTRACT

A library of triazine macrocycles was obtained to evaluate strategies for predicting lipophilicity using additive algorithms. Two synthetic routes were examined. While both were successful, one proved amenable to solution-phase library synthesis. The octanol-water partition coefficients (logP) were measured using reverse-phase HPLC at pH 10. When experimental and computed values (AlogP) are compared, a linear correlation is observed. That is, while additive algorithms underestimate hydrophobicity by a factor of 100, a simple correction yields accurate predictions. Two macrocycles showed anomalous hydrophobicities at high pH that were borne out in membrane transit (PAMPA) studies. Homodimers containing two primary amines were more hydrophobic than the corresponding heterodimers containing a single amine and a hydrophobic group. Structural analysis and computation provide a rationale for this behavior: the amines engage in an intramolecular hydrogen bond.

PMID:40529054 | PMC:PMC12169478 | DOI:10.1021/acsmedchemlett.5c00078

Elife. 2025 Jun 16;14:RP106844. doi: 10.7554/eLife.106844.

ABSTRACT

Proteolysis-targeting chimeras (PROTACs) enable the selective and sub-stoichiometric elimination of pathological proteins, yet only two E3 ligases are routinely used for this purpose. Here, we expand the repertoire of PROTAC-compatible E3 ligases by identifying a novel small molecule scaffold targeting the ubiquitin E3 ligase KLHDC2 using a fluorescence polarization-based high-throughput screen. We highlight the utility of this ligand with the synthesis of PROTACs capable of potently degrading BRD4 in cells. This work affords additional chemical matter for targeting KLHDC2 and suggests a practical approach for identifying novel E3 binders by high-throughput screening.

PMID:40522120 | PMC:PMC12169847 | DOI:10.7554/eLife.106844

ACS Infect Dis. 2025 Jul 11;11(7):1893-1906. doi: 10.1021/acsinfecdis.4c01062. Epub 2025 Jun 5.

ABSTRACT

The recalcitrance of Mycobacterium tuberculosis to antibiotic treatment has been broadly attributed to the impermeability of the organism's outer mycomembrane. However, the studies that support this inference have been indirect or reliant on bulk population measurements. We previously developed the Peptidoglycan Accessibility Click-Mediated AssessmeNt (PAC-MAN) method to covalently trap azide-modified small molecules in the peptidoglycan cell wall of live mycobacteria after they have traversed the mycomembrane. Using PAC-MAN, we now show that the mycomembrane differentially restricts access to fluorophores and antibiotic derivatives. Mycomembranes of both M. tuberculosis and the model organism Mycobacterium smegmatis discriminate between divergent classes of antibiotics as well as between antibiotics within a single family, the fluoroquinolones. By analyzing subpopulations of M. tuberculosis and M. smegmatis, we also found that some fluorophores and vancomycin are heterogeneously restricted by the mycomembrane. Our data indicate that the mycomembrane is a molecule- and cell-specific barrier to antibiotic permeation.

PMID:40471697 | DOI:10.1021/acsinfecdis.4c01062

Microb Biotechnol. 2025 Jun;18(6):e70158. doi: 10.1111/1751-7915.70158.

ABSTRACT

New therapeutics are urgently needed to curb the spread of drug-resistant diseases. Bioactive peptides (BAPs), including antimicrobial peptides, are emerging as an exciting new class of compounds with advantages over current drug modalities, especially small molecule drugs that are prone to resistance development. Here, we evaluated a bacteriophage P22 virus-like particle (VLP) system where BAPs are encapsulated as fusion proteins with the P22 scaffold protein (SP) within self-assembling protein cages in Escherichia coli. Representative peptides from three structurally distinct classes of BAPs were successfully encapsulated into P22 VLPs at high cargo to VLP coat protein (CP) ratios that corresponded to interactions between the compact electropositive structures of the SP-BAPs and electronegative regions on the inward facing surface of CP subunits. However, high loading densities did not correspond to improved SP-BAP yields. An unexpected finding of this study was that while encapsulation alleviated negative effects of SP-BAPs on E. coli growth, the P22 scaffold protein acted as a sufficient fusion partner for accumulating BAPs, and co-expression of the CP did not further improve SP-BAP yields. Nevertheless, encapsulation in VLPs provided a useful first step in the purification pipeline for producing both linear and cyclic recombinant (r)BAPs that were functionally equivalent to their synthetic counterparts. Further efforts to optimise expression ratios of CP to SP-BAP fusions will be required to realise the full potential of encapsulation for protecting expression hosts and maximising rBAP yields.

PMID:40457540 | PMC:PMC12129825 | DOI:10.1111/1751-7915.70158

Nat Biomed Eng. 2025 Oct;9(10):1775-1787. doi: 10.1038/s41551-025-01404-w. Epub 2025 May 29.

ABSTRACT

Small-molecule-binding proteins can neutralize toxins and cytotoxic drugs, but their development is hindered by unpredictable in vivo behaviour and the poor immunogenicity of small molecules, which limits antibody-based approaches. Here we present a strategy to engineer de novo-designed proteins for the effective clearance of Food and Drug Administration-approved drugs in healthy mice. As a proof of concept, we designed proteins that bind to the anticoagulant apixaban. Since apixaban lacks chirality, we used mirror-image synthesis and cyclization to enhance the designed protein's in vivo stability. Both protein variants demonstrated effective activity, facilitating the rapid clearance of apixaban within 30 min. To assess the broader applicability of this approach, we extended our analysis to a de novo-designed protein targeting the anticancer drug rucaparib, further confirming its potential for small-molecule clearance. Our study shows that de novo-designed small-molecule-binding proteins can be used as antidotes in vivo, and that computational tools can be integrated with medicinal chemistry strategies for precise pharmacological interventions.

PMID:40442481 | DOI:10.1038/s41551-025-01404-w

ACS Chem Biol. 2025 Jun 20;20(6):1288-1297. doi: 10.1021/acschembio.5c00121. Epub 2025 May 26.

ABSTRACT

Affinity selections by phage display or other display techniques are typically performed against single targets immobilized as a purified protein. In order to develop cross-specific binders that engage with multiple proteins, such as members of a related family, we herein propose to perform selections against mixtures of proteins as bait. Combined with follow-up selection rounds against the individual proteins, deep sequencing, and single clone enrichment analysis, we expected to distinguish binders that are cross-specific from those that are not. Indeed, applying the strategy to human and mouse coagulation factor XI (hFXI and mFXI), and thus to a situation with limited complexity due to a mixture of only two targets, allowed rapid identification of peptide-based binders along with precise information about their specificity. The study also provided insights into the dynamics and challenges of multitarget affinity selections, showing that one target can easily dominate the selection process and hinder the enrichment of binders to other proteins in a mixture.

PMID:40418758 | DOI:10.1021/acschembio.5c00121

Chemistry. 2025 May 13:e202501298. doi: 10.1002/chem.202501298. Online ahead of print.

ABSTRACT

Naturally occurring peptides are almost exclusively composed of L-amino acids, and the incorporation of D-amino acids can profoundly alter their ability to fold and self-assemble. Here we explore the effects of chirality on the formation of disulfide dynamic combinatorial libraries generated by short cysteine-rich peptides. Our findings consistently show that heterochiral tripeptides form more diverse dynamic combinatorial libraries than their homochiral counterparts. The most complex library appears to encompass all possible cyclic species up to 19mers. Given that each of these species exists as a mixture of parallel and antiparallel isomers, we estimate this library to contain a total of 2,045 distinct compounds - a remarkable result considering that the library generated by the analogous homochiral peptide predominantly contains two dimers. In certain situations, peptide chirality also affects the relative stability of parallel and antiparallel isomers. Taken together, these results show that small changes in peptide chirality can be dramatically amplified through the formation of cyclic species.

PMID:40356358 | DOI:10.1002/chem.202501298

Chem Asian J. 2025 May 6:e202500534. doi: 10.1002/asia.202500534. Online ahead of print.

ABSTRACT

In this work, 13 nonsymmetric cyclic peptides with a thieno[2,3-c]pyrrole bridge were efficiently and regioselectively synthesized by a stapling reaction of the primary amine (from lysine (Lys) side chain and peptide N-terminus), thiol (from cysteine (Cys) side chain), and 2-acetyl-thiophene-3-carboxaldehyde (ATA). A single regioisomeric thieno[2,3-c]pyrrole staple was formed, generating the thieno[2,3-c]pyrrole-bridged peptide. The stapling reaction can be carried out in various solvents. Moreover, the obtained nonsymmetric cyclic peptides were stable in PBS solution. Furthermore, three bicyclic peptides containing both an acetone-like bridge and the thieno[2,3-c]pyrrole bridge were synthesized via two stapling strategies: (1) a symmetric stapling reaction of 1,3-dichloroacetone (DCA) with two Cys residues, and (2) a nonsymmetric stapling reaction of ATA with Lys and Cys residues. Satisfied yields of the bicyclic peptides were achieved. The positions of the two bridges determined the synthetic accessiblity of the bicyclic peptides. Certainly, a wide variety of thieno[2,3-c]pyrrole-bridged nonsymmetric cyclic peptides can be designed, thus entrenching the methodologies in peptide cyclization. The strategy is applied conveniently to the synthesis of nonsymmetric cyclic peptides with a large structural diversity.

PMID:40326470 | DOI:10.1002/asia.202500534