Rachita Dash

Cell. 2025 Jul 29:S0092-8674(25)00798-6. doi: 10.1016/j.cell.2025.07.009. Online ahead of print.

ABSTRACT

The rational design of receptor agonists to control cell signaling is an emerging strategy for developing disease therapeutics. Creating a soluble cytokine-like agonist for the Notch receptor, which regulates cell fate in embryonic and adult development, is challenging, as receptor activation requires a mechanical force that is usually mediated by cell-associated transmembrane ligands. Here, we exploit computationally designed protein complexes with precise valencies and geometries to generate soluble cytokine-like Notch agonists. These molecules promote cell-cell bridging, cluster Notch receptors at cell synapses, and activate receptor signaling. We show that these agonists drive T cell differentiation from cord blood progenitors and human induced pluripotent stem cells (iPSCs) and in bioreactor production of T cells in liquid suspension. When delivered intravenously in mice, they stimulate cytokine production, expansion of antigen-specific CD4⁺ T cells, and antibody class switching. These de-novo-designed ligands can be broadly applied to optimize in vitro cell differentiation and advance immunotherapy development.

PMID:40752493 | PMC:PMC12327808 | DOI:10.1016/j.cell.2025.07.009

Nature. 2025 Sep;645(8081):755-763. doi: 10.1038/s41586-025-09286-3. Epub 2025 Jul 30.

ABSTRACT

Mycobacterium tuberculosis (Mtb) remains the world's deadliest bacterial pathogen¹. There is an urgent medical need to develop new drugs that shorten the treatment duration to combat widespread multi-drug-resistant and extensive-drug-resistant Mtb. Here, we present a preclinical covalent compound, CMX410, that contains an aryl fluorosulfate (SuFEx)² warhead and uniquely targets the acyltransferase domain of Pks13, an essential enzyme in cell-wall biosynthesis. CMX410 is equipotent against drug-sensitive and drug-resistant strains of Mtb and efficacious in multiple mouse models of infection. Inhibition by CMX410 is irreversible through a previously undescribed mechanism: CMX410 reacts with the catalytic serine of the AT domain of Pks13, rapidly and irreversibly disabling the active site by forming a β-lactam. CMX410 is highly selective for its target and thus demonstrates excellent pharmacological and safety profiles, including no adverse effects in a 14-day rat toxicity study up to 1,000 mg kg^-1 per day. The distinctive mode of action from current drugs, high potency across all tested clinical isolates, oral bioavailability, favourable performance in drug combination testing and superior pharmacological and safety characteristics make CMX410 a promising first-in-class candidate to replace outdated cell-wall biosynthesis inhibitors, such as isoniazid and ethambutol, in tuberculosis regimens.

PMID:40739353 | PMC:PMC12962449 | DOI:10.1038/s41586-025-09286-3

Nature. 2025 Aug;644(8077):809-817. doi: 10.1038/s41586-025-09248-9. Epub 2025 Jul 30.

ABSTRACT

Proteins that bind to intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) with high affinity and specificity could be useful for therapeutic and diagnostic applications^1-4. However, a general methodology for targeting IDPs or IDRs has yet to be developed. Here we show that starting only from the target sequence of the input, and freely sampling both target and binding protein conformations, RFdiffusion⁵ can generate binders to IDPs and IDRs in a wide range of conformations. We used this approach to generate binders to the IDPs amylin, C-peptide, VP48 and BRCA1_ARATH in diverse conformations with a dissociation constant (K_d) ranging from 3 to 100 nM. For the IDRs G3BP1, common cytokine receptor γ-chain (IL-2RG) and prion protein, we diffused binders to β-strand conformations of the targets, obtaining K_d between 10 and 100 nM. Fluorescence imaging experiments show that the binders bind to their respective targets in cells. The G3BP1 binder disrupts stress granule formation in cells, and the amylin binder inhibits amyloid fibril formation and dissociates existing fibres, enables targeting of both monomeric and fibrillar amylin to lysosomes, and increases the sensitivity of mass spectrometry-based amylin detection. Our approach should be useful for creating binders to flexible IDPs or IDRs spanning a wide range of intrinsic conformational preferences.

PMID:40739343 | PMC:PMC12367549 | DOI:10.1038/s41586-025-09248-9

J Am Chem Soc. 2025 Aug 6;147(31):28303-28312. doi: 10.1021/jacs.5c09059. Epub 2025 Jul 28.

ABSTRACT

Ubiquitination is a critical post-translational modification that regulates key cellular processes such as protein degradation and DNA damage repair. Targeting a specific type of ubiquitin chain (e.g., Lys48 or Lys63-linked ubiquitin chain) via cyclic peptides presents a new strategy to modulate biological processes with therapeutic potential for various diseases. However, such a strategy remains challenging due to the obstacles of cell permeability and bioactivity. Here, we present a new method that directly assesses these parameters by integrating palladium-mediated Cys arylation with direct cellular screening. Using CP4, a previously identified cyclic peptide modulator of Lys63-linked ubiquitin chains, we generated a focused library of arylated analogues and optimized the Pd-mediated arylation for direct cellular screening. We discovered a new analog, CP-P12-Ar^H, that demonstrated enhanced binding affinity and robust bioactivity, as evidenced by increased γ-H2AX phosphorylation and apoptosis induction in cancer cells. Furthermore, CP-P12-Ar^H effectively inhibited the in vitro formation of NF-κB essential modulator (NEMO) biomolecular condensates by disrupting the elongation of Lys63-linked ubiquitin chains, offering a novel way to modulate NF-κB signaling. This work establishes a generalizable platform for the rapid optimization of cyclic peptide therapeutics targeting protein-protein interactions.

PMID:40720895 | DOI:10.1021/jacs.5c09059

J Am Chem Soc. 2025 Aug 6;147(31):27494-27505. doi: 10.1021/jacs.5c05004. Epub 2025 Jul 28.

ABSTRACT

Frequent, reliable cortisol measurement is critical for diagnosing and managing adrenal disorders, stress responses, and circadian rhythm disruptions. However, current cortisol assays or detection methods remain confined to laboratory settings, limiting on-site testing. Protein-based biosensors provide a promising point-of-care (POC) solution, yet no robust, field-ready protein-based cortisol biosensor is available. Here, we de novo design cortisol-inducible dimerization modules and systematically sample their fusions with split luciferase reporters by using a protein structure prediction pipeline. The resulting biosensor, designed straight from the computer, yields over 300-fold luminescent response with picomolar sensitivity and can be rapidly imaged by a standard camera or smartphone. This work highlights the power of computational protein design for developing next-generation protein-based biosensors.

PMID:40720516 | DOI:10.1021/jacs.5c05004

Science. 2025 Jul 24;389(6758):386-391. doi: 10.1126/science.adv0185. Epub 2025 Jul 24.

ABSTRACT

Class I major histocompatibility complex (MHC-I) molecules present peptides derived from intracellular antigens on the cell surface for immune surveillance. Proteins that recognize peptide-MHC-I (pMHCI) complexes with specificity for diseased cells could have considerable therapeutic utility. Specificity requires recognition of outward-facing amino acid residues within the disease-associated peptide as well as avoidance of extensive contacts with ubiquitously expressed MHC. We used RFdiffusion to design pMHCI-binding proteins that make extensive contacts with the peptide and identified specific binders for 11 target pMHCs starting from either experimental or predicted pMHCI structures. Upon incorporation into chimeric antigen receptors, designs for eight targets conferred peptide-specific T cell activation. Our approach should have broad utility for both protein- and cell-based pMHCI targeting.

PMID:40705892 | DOI:10.1126/science.adv0185

Biochemistry. 2025 Aug 5;64(15):3382-3392. doi: 10.1021/acs.biochem.5c00068. Epub 2025 Jul 23.

ABSTRACT

A hallmark of neurodegenerative diseases like Alzheimer's Disease (AD) and chronic traumatic encephalopathy (CTE) is the presence of toxic protein aggregates in neurons. In AD and CTE specifically, the protein tau forms insoluble fibrils that are hundreds of nanometers in length. Intriguingly, recent experimental structures suggest that tau ligands like the disaggregator EGCG and positron emission tomography (PET) tracers like GTP-1 and MK-6240 bind to tau fibrils in long stacks reflecting the symmetry of the protein across many binding sites. In these stacks, each ligand makes more contact with its symmetry mates than it does with the protein. To interpret the binding of these molecules and new ligands, we must understand the effects of the cooperativity between sites and the entropy coming from the number of sites. Here, we investigate a nearest-neighbors model of cooperativity and use statistical mechanics to derive binding isotherms for saturation and competition experiments. This model allows us to relate measured EC₅₀ and IC₅₀ values to the intrinsic binding affinity to a single site and to cooperativity across sites in ways resembling the Cheng-Prusoff Equation. Depending on the degree of cooperativity between molecular species, this model permits solutions that lack the steep binding curves expected from cooperative systems and even solutions resembling 2-site systems. We finally consider conditions for a fibril's detection in a PET scan and practical matters of fitting this model's parameters to data.

PMID:40700656 | PMC:PMC12918765 | DOI:10.1021/acs.biochem.5c00068

Cell. 2025 Aug 7;188(16):4470. doi: 10.1016/j.cell.2025.07.019. Epub 2025 Jul 22.

NO ABSTRACT

PMID:40695275 | PMC:PMC12369702 | DOI:10.1016/j.cell.2025.07.019

Science. 2025 Jul 17;389(6757):eadr8063. doi: 10.1126/science.adr8063. Epub 2025 Jul 17.

ABSTRACT

Intrinsically disordered proteins and peptides play key roles in biology, but a lack of defined structures and high variability in sequence and conformational preferences have made targeting such systems challenging. We describe a general approach for designing proteins that bind intrinsically disordered protein regions in diverse extended conformations with side chains fitting into complementary binding pockets. We used the approach to design binders for 39 highly diverse unstructured targets, including polar targets, and obtained designs with 100-picomolar to 100-nanomolar affinities in 34 cases, testing ~22 designs per target. The designs function in cells and as detection reagents and are specific for their intended targets in all-by-all binding experiments. Our approach is a major step toward a general solution to the intrinsically disordered protein and peptide recognition problem.

PMID:40674483 | DOI:10.1126/science.adr8063

bioRxiv [Preprint]. 2025 Jul 7:2025.07.03.661155. doi: 10.1101/2025.07.03.661155.

ABSTRACT

Emerging evidence has demonstrated the importance of pattern recognition receptors (PRRs), including the nucleotide-binding and oligomerization domain receptor 2 (NOD2), in human health and disease states. NOD2 activation has shown promise with aiding malnutrition recovery, lessening irritable bowel disease (IBD) symptoms, and increasing the efficacy of cancer immunotherapy. Currently, most NOD2 agonists are derivatives or analogs of the endogenous agonist derived from bacterial peptidoglycan, muramyl dipeptide (MDP). These MDP-based agonists can suffer from low oral bioavailability and cause significant adverse side effects. With the goal of broadly improving NOD2 therapeutic interventions, we sought to discover a novel small molecule capable of activating NOD2 by screening a library of total 1917 FDA approved drugs in a phenotypic assay. We identified a class of compounds, benzimidazoles, that act as NOD2 agonists, with the most potent member of this class being nocodazole. Nocodazole activates NOD2 with nanomolar potency and causes the release of cytokines canonically associated with MDP-induced NOD2 activation, suggesting its potential to elicit similar therapeutic immune effects as MDP and potentially offer improved pharmacological properties.

PMID:40672166 | PMC:PMC12265511 | DOI:10.1101/2025.07.03.661155

Angew Chem Int Ed Engl. 2025 Aug 18;64(34):e202506802. doi: 10.1002/anie.202506802. Epub 2025 Jul 9.

ABSTRACT

Protein-based tools are emerging as innovative solutions to interfere with biological pathways in molecular biology and medicine. They offer advantages over traditional small molecules due to their adaptable structural diversity and their ability to engage previously inaccessible cellular targets. However, most proteins do not penetrate the lipid bilayer of mammalian cells and are therefore restricted to extracellular targets. Despite recent advances, a general method for the delivery of functional proteins into human cells remains a significant challenge. In this study, we present a bioreversible protein modification strategy of amines using short arginine-containing peptides (termed BioRAM) that enables cytosolic delivery starting from genetically non-engineered proteins. We optimized the bioconjugation strategy to achieve fast intracellular cleavage and complete recovery of the native protein. In combination with our previously established cell-penetrating peptide (CPP)-additive protocol, we show superior delivery of fluorescent protein and functional RNase A into the cytosol, achieving physiological response. Moreover, we are able to demonstrate the excellent performance of BioRAM in the presence of serum, thereby broadening the scope for intracellular applications of functional proteins.

PMID:40452583 | PMC:PMC12363615 | DOI:10.1002/anie.202506802

PLoS Pathog. 2025 Jul 7;21(7):e1013324. doi: 10.1371/journal.ppat.1013324. eCollection 2025 Jul.

ABSTRACT

The spirochete Borrelia burgdorferi causes Lyme disease. In some patients, an excessive, dysregulated proinflammatory immune response can develop in joints leading to persistent arthritis even after antibiotic therapy. In such patients, persistence of antigenic B. burgdorferi peptidoglycan (PGBb) fragments within joint tissues may contribute to immunopathogenesis pre- and post-antibiotic treatment. In live B. burgdorferi cells, the outer membrane shields the polymeric PGBb sacculus from exposure to the immune system. However, unlike most diderm bacteria, B. burgdorferi releases PGBb turnover products into its environment due to the absence of recycling activity. In this study, we identified the released PGBb fragments using a mass spectrometry-based approach. By characterizing the l,d-carboxypeptidase activity of B. burgdorferi protein BB0605 (renamed DacA), we found that PGBb turnover largely occurs at sites of PGBb synthesis. In parallel, we demonstrated that the lytic transglycosylase activity associated with BB0259 (renamed MltS) releases PGBb fragments with 1,6-anhydro bond on their N-acetylmuramyl residues. Stimulation of human cell lines with various synthetic PGBb fragments revealed that 1,6-anhydromuramyl-containing PGBb fragments are poor inducers of a NOD2-dependent immune response relative to their hydrated counterparts found in the polymeric PGBb isolated from dead bacteria. We also showed that the activity of the human N-acetylmuramyl-l-alanine amidase PGLYRP2, which reduces the immunogenicity of PGBb material, is low in joint (synovial) fluids relative to serum. Altogether, our findings suggest that MltS activity helps B. burgdorferi evade PG-based immune detection by NOD2 during growth despite shedding PGBb fragments and that PGBb-induced immunopathology likely results from host sensing of PGBb material from dead (lysed) spirochetes. Additionally, our results suggest the possibility that natural variation in PGLYRP2 activity may contribute to differences in susceptibility to PG-induced inflammation across tissues and individuals.

PMID:40623106 | PMC:PMC12279116 | DOI:10.1371/journal.ppat.1013324

Macromolecules. 2024 Oct 22;57(20):9585-9594. doi: 10.1021/acs.macromol.4c01866. Epub 2024 Oct 7.

ABSTRACT

Peptides capable of forming homotetrameric coiled-coil bundles are utilized as the monomeric building blocks ("bundlemers") to synthesize protein-like hybrid polymers consisting of covalently linked coiled-coil microdomains with regularly spaced ethylene glycol repeats via step-growth polymerization employing the highly efficient, bioorthogonal tetrazine (Tz) ligation with trans-cyclooctene (TCO). Polymerization of Tz and TCO-functionalized peptides in aqueous media under strict stoichiometry at Tz or TCO concentrations of 0.1 to 4.5 mM leads to the establishment of exceptionally long, semiflexible polymer chains with a Kuhn length of 6-7 nm and an apparent molecular weight up to 3 MDa. Bioorthogonal polymerization at bundlemer concentrations above 5 mM gives rise to physical gels through interchain entanglements. Hydrogels prepared at 10 mM exhibit an average elastic modulus of 400 Pa and a strain to failure of 300%. Copolymerization of coiled-coil peptides with distinct composition and thermal stability results in hydrogels that are thermally tunable. Solid-to-fluid transition occurs when one of the coiled-coil repeats melts. Upon cooling, solid-like properties are partially recovered through intermolecular association of the helical peptides. Overall, tetrazine ligation has enabled the covalent polymerization of self-assembled coiled-coil motifs for the establishment of protein-like linear polymers with unprecedented molecular weight.

PMID:40599260 | PMC:PMC12208629 | DOI:10.1021/acs.macromol.4c01866

J Am Chem Soc. 2025 Jul 16;147(28):24628-24642. doi: 10.1021/jacs.5c05536. Epub 2025 Jul 1.

ABSTRACT

Obesity stands as a global epidemic and is the primary risk factor for type 2 diabetes, ranking as the fifth leading cause of death worldwide. While lifestyle changes can address body fat accumulation, pharmacotherapies can also assist in sustained weight loss. Here, we report the design of a new generation of prohibitin peptide-based therapeutics engineered to target white adipose tissues. These peptides demonstrate significant reduction of body weight in a high-fat diet-induced obesity mouse model and represent a paradigm shift in approaches to the treatment of obesity by inducing mitochondrial uncoupling. The most potent compound, PTP-r, was prohibitin-TP01 substituted with d-arginine. Overall, the study reveals the promising development of next-generation adipose-targeting prohibitin peptides, capable of curbing adipocyte expansion and body weight, with favorable preclinical safety profiles. These peptides hold immense potential for developing new treatments to address obesity and metabolic syndrome.

PMID:40591817 | DOI:10.1021/jacs.5c05536