Rachita Dash

bioRxiv [Preprint]. 2025 Aug 6:2025.08.06.668955. doi: 10.1101/2025.08.06.668955.

ABSTRACT

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the Huntingtin (HTT) gene, with no disease-modifying therapies currently available. The precise molecular function of the HTT protein is unclear, and the lack of selective chemical tools has limited functional studies. We have identified and characterized macrocyclic peptide binders targeting HTT. These binders exhibit low-nanomolar affinity in vitro and engage distinct HTT and HTT-HAP40 interfaces, as revealed by hydrogen-deuterium exchange mass spectrometry and cryo-electron microscopy. Chemoproteomics confirmed selective binding in cell extracts from wildtype but not HTT-null cell lines. HAP40 consistently and stoichiometrically co-purified with HTT across cell lines, including with HTT variants containing different CAG repeat lengths, highlighting the broad presence of the HTT-HAP40 complex.

PMID:41030958 | PMC:PMC12478414 | DOI:10.1101/2025.08.06.668955

bioRxiv [Preprint]. 2025 Sep 19:2025.09.15.676411. doi: 10.1101/2025.09.15.676411.

ABSTRACT

Determining the mechanism of action (MOA) for natural products remains a significant bottleneck in drug discovery, particularly for researchers with limited computational resources or small compound libraries. Traditional approaches require screening large numbers of annotated compounds alongside unknowns, which is cost-prohibitive, or depend on complex machine learning models that need substantial computational resources and large datasets. Here, we present a dissertation chapter excerpt: MOAST (Mechanism of Action Similarity Tool), a BLAST-inspired computational workflow that addresses these limitations by providing rapid MOA hypotheses for newly screened compounds. This chapter investigates two complementary approaches: a kernel density estimation (KDE) method providing statistical significance measures and E-values for MOA class membership, and a CatBoost machine learning classifier for multi-class prediction with ranked outputs. Using cytological profiling data from HeLa and A549 cell lines, MOAST achieved 22% accuracy for the top 5 predictions among ~ 300 MOA classes, with the CatBoost classifier reaching 10% balanced accuracy-significantly better than the ~ 3% reported in literature. The tool suggests a 0.8 prediction probability threshold for trustworthy results and demonstrates robust performance across multiple feature reduction strategies. MOAST provides a practical, accessible solution that bridges traditional phenotypic screening and modern computational approaches, making MOA determination feasible for researchers with limited resources while maintaining statistical rigor and interpretability.

PMID:41000814 | PMC:PMC12458938 | DOI:10.1101/2025.09.15.676411

J Org Chem. 2025 Oct 10;90(40):14074-14080. doi: 10.1021/acs.joc.5c01314. Epub 2025 Sep 25.

ABSTRACT

An improved catalyst system, PdI₂/DiPPF, is described for the silver-mediated Liebeskind-Srogl coupling of thioalkyltetrazines with arylboronic and heteroarylboronic acids. The methodology advances the synthesis of functionalized tetrazines, offering greater efficiency, broader compatibility, and practical utility for accessing tool molecules for bioorthogonal chemistry applications.

PMID:40997917 | PMC:PMC12554015 | DOI:10.1021/acs.joc.5c01314

bioRxiv [Preprint]. 2025 Sep 18:2025.09.18.676967. doi: 10.1101/2025.09.18.676967.

ABSTRACT

Deep learning has accelerated protein design, but most existing methods are restricted to generating protein backbone coordinates and often neglect interactions with other biomolecules. We present RFdiffusion3 (RFD3), a diffusion model that generates protein structures in the context of ligands, nucleic acids and other non-protein constellations of atoms. Because all polymer atoms are modeled explicitly, conditioning the model on complex sets of atom-level constraints for enzyme design and other challenges is both simpler and more effective than previous approaches. RFD3 achieves improved performance compared to prior approaches on a range of in silico benchmarks with one tenth the computational cost. Finally, we demonstrate the broad applicability of RFD3 by designing and experimentally characterizing DNA binding proteins and cysteine hydrolases. The ability to rapidly generate protein structures guided by complex sets of atom-level constraints in the context of arbitrary non-protein atoms should further expand the range of functions attainable through protein design.

PMID:41000976 | PMC:PMC12458353 | DOI:10.1101/2025.09.18.676967

bioRxiv [Preprint]. 2025 Sep 8:2025.09.08.674823. doi: 10.1101/2025.09.08.674823.

ABSTRACT

Protein nanopores are essential components of single-molecule oligonucleotide sequencing and sensing devices. Here, we demonstrate that installing additional de novo subunits enables large-scale architectural changes of nanopore complexes. We design de novo proteins that integrate seamlessly with the CsgG pore to form 18-subunit, 315-kilodalton complexes with precisely sculpted pore architectures and tailored ion conduction, opening new possibilities for engineering enhanced nanopores with customized structural and functional properties.

PMID:40964308 | PMC:PMC12439879 | DOI:10.1101/2025.09.08.674823

Nat Chem. 2025 Dec;17(12):1928-1940. doi: 10.1038/s41557-025-01931-8. Epub 2025 Sep 17.

ABSTRACT

Photocatalytic proximity labelling has emerged as a powerful tool to resolve a variety of biomolecular and cellular interactions. Although the use of high-resolution probes, such as diazirines, enables cell-surface protein labelling with nanometre precision, intracellular applications are limited by either the intrinsic toxicity of metal-based photocatalysts or by the lower resolution when long-lived reactive intermediates are used. Here we describe the discovery, characterization and application of an organic flavin cofactor derivative, deazaflavin, that activates diazirine to generate carbenes via triplet energy transfer and offers excellent biocompatibility. We demonstrate deazaflavin-diazirine energy-transfer labelling (DarT labelling) for cell surfaceome mapping and, most importantly, for intracellular interactome mapping as exemplified for cell-penetrating peptides. We successfully map the localization of linear and cyclic polyarginine cell-penetrating peptides, identifying putative membrane interactors. Furthermore, we show the applicability of DarT labelling over an extended time period by mapping the intracellular trafficking of a stable cyclic derivative to reveal its eventual exocytosis from the cell. We anticipate that DarT labelling could be used to profile intracellular dynamics across diverse biological systems with high spatio-temporal control.

PMID:40962911 | PMC:PMC12669049 | DOI:10.1038/s41557-025-01931-8

Antimicrob Agents Chemother. 2025 Oct;69(10):e0079525. doi: 10.1128/aac.00795-25. Epub 2025 Sep 11.

ABSTRACT

Development of new and improved tuberculosis (TB) chemotherapies is hampered by antibiotic resistance and drug tolerance by Mycobacterium tuberculosis (Mtb). Phenotypic drug tolerance, a phenomenon where Mtb populations can temporarily survive therapeutic antibiotic concentrations, represents a significant hurdle to TB treatment and is indeed one of the factors responsible for prolonged TB therapy. Assays that can identify compounds with improved efficacy against drug-tolerant Mtb are urgently required to improve TB treatment regimens. Here, we report the development of a 96-well plate assay capable of identifying anti-Mtb drugs with activity against drug-tolerant Mtb in physiologically relevant intracellular environments within macrophages. Primary murine macrophages, modified either by immunological activation or specific CRISPR/Cas9 gene knockouts to generate tolerance-inducing environments, were infected with an Mtb strain constitutively expressing luciferase. Following drug exposure, differences in bacterial survival were measured by bacterial outgrowth after lysis of the host macrophages. By monitoring Mtb luciferase in infected macrophages before, during, and after drug treatment, we confirmed earlier observations that host immune stresses trigger induction of drug tolerance. However, while host stresses induced tolerance against some anti-TB compounds, the same host stresses were synergistic with other anti-TB drugs. Our assay provides the ability to profile the activities of anti-TB drugs on bacteria in intracellular host environments, which is critical to the rational design of drug combinations that provide optimal coverage of the Mtb sub-populations in the infected host.

PMID:40934365 | PMC:PMC12486812 | DOI:10.1128/aac.00795-25

bioRxiv [Preprint]. 2025 Aug 18:2025.08.18.670751. doi: 10.1101/2025.08.18.670751.

ABSTRACT

The rise of multidrug-resistant bacterial infections necessitates the discovery of novel antimicrobial strategies. Here, we show that protein design provides a generalizable means of generating new antimicrobials by neutralizing the function of bacterial adhesins, which are virulence factors critical in host-pathogen interactions. We de novo designed high-affinity miniprotein binders to FimH and Abp1D/Abp2D chaperone usher pili adhesins from uropathogenic Escherichia coli and Acinetobacter baumannii, respectively, which are implicated in mediating both uncomplicated and catheter-associated urinary tract infections (UTI) responsible for significant morbidity and mortality worldwide. The designed antagonists have high specificity and stability, disrupt bacterial recognition of host receptors, block biofilm formation, and are effective in treating and preventing uncomplicated and catheter-associated UTIs in vivo. Targeting virulence factors outside the cell membrane with protein design provides a rapid route to next-generation therapeutics that can disrupt pathogenesis and thus are capable of treating and preventing disease in an antibiotic-sparing manner.

PMID:40894640 | PMC:PMC12393372 | DOI:10.1101/2025.08.18.670751

Proc Natl Acad Sci U S A. 2025 Sep 9;122(36):e2421336122. doi: 10.1073/pnas.2421336122. Epub 2025 Sep 2.

ABSTRACT

Isoniazid (INH) inhibits mycolic acid synthesis in Mycobacterium tuberculosis (Mtb) and is a cornerstone of treatment regimens against this deadly pathogen. However, over 10% of Mtb infections are INH-resistant. The compound C10 can sensitize clinically relevant INH-resistant mutants to killing by INH. Thus, understanding the mechanism of action for C10 could aid in designing new strategies for circumventing drug resistance. We find that C10 treatment reroutes carbon flux toward valine, drawing carbon away from gluconeogenesis and the TCA cycle. As a result, C10 decreases cell envelope capsule thickness and blocks an accumulation of peptidoglycan precursors that occurs in response to INH treatment in an INH-resistant Mtb katG mutant. In this altered metabolic state induced by C10, INH treatment of the INH-resistant Mtb katG mutant inhibits peptidoglycan synthesis, precipitating collapse of cell envelope integrity. Pyruvate supplementation relieves the C10-induced requirement for carbon flux toward valine, enhancing carbon assimilation into cell envelope precursors and restoring resistance to INH. In addition, we identify the formation of isoniazid-pyruvate in INH-treated katG^W328L Mtb, where pyruvate sequesters INH, lowering the concentration of INH available to inhibit Mtb. Together, our findings reveal a bactericidal activity for INH in Mtb that can function in INH-resistant mutants independently of INH-mediated inhibition of mycolic acid synthesis. This activity for INH can be elicited by shifting carbon flux toward valine and away from cell envelope precursor synthesis, highlighting a metabolic vulnerability that can be exploited to kill INH-resistant Mtb.

PMID:40892921 | DOI:10.1073/pnas.2421336122

J Am Chem Soc. 2025 Aug 27. doi: 10.1021/jacs.5c12291. Online ahead of print.

ABSTRACT

Phage display is an ideal platform for selecting peptide hits and offers a diverse array of cyclic binders with high affinity. While many recently developed phage display platforms incorporate chemical strategies, the vast majority of these are detrimental to the phage life cycle due to cross-reactivity with the capsid protein. In contrast, enzyme catalysis, which combines high efficiency and biocompatibility, offers a promising approach for phage-based cyclic peptide display. However, enzyme-mediated cyclization approaches remain underexplored. Here, we present a tyrosinase-mediated phage display platform that enables one-step cyclization via o-quinone-cysteine coupling, which is a simple and efficient strategy that does not compromise phage infectivity. Importantly, the catalytic property of tyrosinase is highly selective and spatially constrained, allowing it to bypass native tyrosine residues in the phage and selectively recognize only the engineered tyrosine residues. Using this platform, we constructed a macrocyclic peptide library that facilitated the discovery of macrocyclic peptide inhibitors targeting therapeutically relevant proteins. Notably, the cyclic peptide ACI1 demonstrated potent inhibition of PIP4K2A kinase activity with an IC₅₀ value of 0.93 ± 0.05 μM, while ACP1 effectively inhibited the dephosphorylation activity of PTP1B with an IC₅₀ of 1.06 ± 0.25 μM. The generality and efficiency of this strategy highlight its potential as a valuable tool for the development of bioactive cyclic peptides.

PMID:40859814 | DOI:10.1021/jacs.5c12291

Proc Natl Acad Sci U S A. 2025 Sep 2;122(35):e2504264122. doi: 10.1073/pnas.2504264122. Epub 2025 Aug 26.

ABSTRACT

Light scattering in biological tissue presents a significant challenge for deep in vivo imaging. Our previous work demonstrated the ability to achieve optical transparency in live mice using intensely absorbing dye molecules, which created transparency in the red spectrum while blocking shorter-wavelength photons. In this paper, we extend this capability to achieve optical transparency across the entire visible spectrum by employing molecules with strong absorption in the ultraviolet spectrum and sharp absorption edges that rapidly decline upon entering the visible spectrum. This color-neutral and reversible tissue transparency method enables optical transparency for imaging commonly used fluorophores in the green and yellow spectra. Notably, this approach facilitates tissue transparency for structural and functional imaging of the live mouse brain labeled with yellow fluorescent protein and GCaMP through the scalp and skull. We show that this method enables longitudinal imaging of the same brain regions in awake mice over multiple days during development. Histological analyses of the skin and systemic toxicology studies indicate minimal acute or chronic damage to the skin or body using this approach. This color-neutral and reversible tissue transparency technique opens opportunities for noninvasive deep-tissue optical imaging, enabling long-term visualization of cellular structures and dynamic activity with high spatiotemporal resolution and chronic tracking capabilities.

PMID:40857313 | PMC:PMC12415250 | DOI:10.1073/pnas.2504264122

Nat Chem. 2025 Oct;17(10):1565-1575. doi: 10.1038/s41557-025-01889-7. Epub 2025 Aug 14.

ABSTRACT

Aggregation of microtubule-associated protein tau into conformationally distinct fibrils underpins neurodegenerative tauopathies. Fluorescent probes (fluoroprobes) such as thioflavin T have been essential tools for studying tau aggregation; however, most of them do not discriminate between amyloid fibril conformations (polymorphs). This gap is due, in part, to a lack of high-throughput methods for screening large, diverse chemical collections. Here we leverage advances in protein-adaptive differential scanning fluorimetry to screen the Aurora collection of 300+ fluoroprobes against multiple synthetic fibril polymorphs, including those formed from tau, α-synuclein and islet amyloid polypeptide. This screen-coupled with excitation-multiplexed bright-emission recording (EMBER) imaging and orthogonal secondary assays-revealed pan-fibril-binding chemotypes, as well as fluoroprobes selective for fibril subsets. One fluoroprobe recognized tau pathology in ex vivo brain slices from Alzheimer's disease and rodent models. We propose that these scaffolds represent entry points for developing fibril-selective ligands.

PMID:40813616 | PMC:PMC12491066 | DOI:10.1038/s41557-025-01889-7

Angew Chem Int Ed Engl. 2025 Sep 15;64(38):e202508656. doi: 10.1002/anie.202508656. Epub 2025 Aug 8.

ABSTRACT

Achieving modular, selective and homogeneous protein modifications is of utmost importance for the design of next generation biopharmaceuticals, especially in the context of antibody-drug conjugates (ADCs). Here, we introduce unsaturated phosphine oxides as versatile triple-reactive reagents, allowing orthogonal chemoselective bioconjugation schemes. Starting from triethynyl-phosphine oxide, a variety of functionalized diethynyl-triazolyl-phosphine oxides (DTPOs) could be accessed by using Cu^I-catalyzed azide-alkyne cycloaddition (CuAAC). We showcase DTPO-reagents in the fast and selective generation of various highly stable antibody-conjugates via antibody disulfide rebridging. A highlight from this methodology is the synthesis of a DAR 4 ADC following a modular 2-step strategy using bioorthogonal tetrazine-labeling with bicyclo-[6.1.0]non-4-yne (BCN) or trans-cyclooctene (TCO) containing payloads. Notably, the DTPO-rebridged ADC exhibited potent cytotoxicity against Her2⁺ cancer cells. Moreover, we utilize triethynyl-phosphine oxide to obtain ethynyl-ditriazolyl-phosphine oxides (EDPOs) which enable a unique, single-reagent peptide-cyclization-bioconjugation protocol resulting in functional cyclic peptide-protein conjugates. Overall, our work provides versatile and powerful chemoselective modalities for the controlled modification of antibodies, peptide-cyclization and peptide-protein conjugation, expanding the toolkit for chemical biology and therapeutic development.

PMID:40586855 | PMC:PMC12435405 | DOI:10.1002/anie.202508656

Science. 2025 Aug 7;389(6760):618-622. doi: 10.1126/science.adp9583. Epub 2025 Aug 7.

ABSTRACT

Systems that perform continuous hypermutation of designated genes without compromising the integrity of the host genome can substantially accelerate the evolution of new or enhanced protein functions. We describe an orthogonal DNA replication system in Escherichia coli based on the controlled expression of the replisome of bacteriophage T7 (T7-ORACLE). The system replicates circular plasmids that enable high transformation efficiencies and seamless integration into standard molecular biology workflows. Engineering of T7 DNA polymerase yielded variant proteins with mutation rates of 1.7 × 10^-5 substitutions per base in vivo-100,000-fold above the genomic mutation rate. We demonstrated continuous evolution using the T7 replisome by expanding the substrate scope of TEM-1 β-lactamase and increasing activity 5000-fold against clinically relevant monobactam and cephalosporin antibiotics in less than 1 week.

PMID:40773556 | DOI:10.1126/science.adp9583

Nat Commun. 2025 Aug 1;16(1):7055. doi: 10.1038/s41467-025-62200-3.

ABSTRACT

RNA medicine is an emerging groundbreaking technology for the prevention and treatment of disease. However, tools to deliver messenger RNA (mRNA) and other polyanions (circRNA, saRNA, pDNA, CRISPR-Cas, reprogramming factors) are required to advance current RNA therapies and address next generation challenges. Existing delivery systems often suffer from laborious syntheses, limited organ selectivity, formulation complexity, and undesired inflammatory responses. Here, we report novel mRNA delivery systems termed Discrete Immolative Guanidinium Transporters (DIGITs), which are synthesized convergently in as few as 4 steps. Unlike most cationic (ammonium) delivery systems, DIGITs are based on cationic guanidinium moieties, which complex mRNA at acidic pH and undergo irreversible neutralization at physiological pH to enable efficient RNA release. Systematic evaluation of structural variations and formulations have led to DIGIT/mRNA complexes that selectively target lung, spleen, and immature red blood cells in peripheral blood in female mice model. DIGIT/mRNA delivery systems show minimal toxicity based on cell viability and biochemical assays, supporting their future utility in biomedical applications.

PMID:40750796 | PMC:PMC12316917 | DOI:10.1038/s41467-025-62200-3