Ian B. Seiple

Covalent molecules have emerged as next-generation therapeutics and as powerful tools for perturbing fundamental biological processes. Chemical proteomic methods to screen for reactive proteinaceous amino acids have transformed small-molecule discovery pipelines, but their application remains mostly limited to sites where reactive cysteines and lysines are present. Here we report a ninhydrin-based warhead that selectively modifies arginine residues, thus expanding the repertoire of amino acids targetable by covalent molecules. Specifically, we developed alkyne-functionalized variants of ninhydrin to establish an arginine-specific chemical proteomics platform, enabling the classification of more than 6,800 unique reactive arginines from one cell line. These studies uncovered potential modification sites on disease-relevant proteins, including reactive arginines within catalytic sites that are essential for function and reactive arginines on proteins lacking reactive cysteines. By endowing a reversible small-molecule binder of cyclophilin A with a ninhydrin warhead, we achieved selective, covalent engagement in cell lysates and attenuation of enzymatic activity in vitro. These findings establish ninhydrin as a warhead for studying arginine reactivity and identifying at scale arginines amenable to covalent modification.

Macrolide antibiotics are a class of structurally diverse polyketides including essential medicines like erythromycin and azithromycin. Structurally minimal macrolides such as narbomycin lack much of the functionality common to other members of the class and exhibit a distinct mode of action wherein they completely inhibit bacterial growth despite only moderate inhibition of global protein synthesis. Unfortunately, lack of availability of these natural products has prevented a thorough investigation of their unusual mechanisms of action. Here we report a 10-step synthesis of narbomycin enabled by a Liebeskind-Srogl fragment coupling, a novel macrolactonization by direct displacement of an Evans auxiliary, and a stereoselective late-stage enone reduction. In addition to accessing the natural product, the route enables access to C8,19-unsaturated variants which could in principle serve as useful precursors for late-stage functionalization by conjugate addition.

Macrolide antibiotics are a class of structurally diverse polyketides including essential medicines like erythromycin and azithromycin. Structurally minimal macrolides such as narbomycin lack much of the functionality common to other members of the class and exhibit a distinct mode of action wherein they completely inhibit bacterial growth despite only moderate (~50%) inhibition of global protein synthesis. Unfortunately, lack of availability of these natural products has prevented a thorough investigation of their unusual mechanisms of action. Here we report a 10-step synthesis of narbomycin enabled by a Liebeskind-Srogl fragment coupling, a novel macrolactonization by direct displacement of an Evans auxiliary, and a stereoselective late-stage enone reduction. In addition to accessing the natural product, the route enables access to C8,19-unsaturated variants which could serve as useful precursors for late-stage functionalization by conjugate addition.

Antimicrobial resistance remains a formidable challenge to modern medicine, with bacterial resistance mechanisms increasingly eroding the utility of clinically important antibiotics. While recent efforts have expanded the antibacterial pipeline, the development of resistance in priority pathogens continues to exceed the pace of new drug development. One emerging strategy to overcome resistance is the rational design of hybrid antibiotics that engage multiple binding sites. Here we describe the design, synthesis, and microbiological and structural characterization of hybrid antibiotics of azithromycin, tedizolid, and chloramphenicol that span the peptidyltransferase center (PTC) and nascent peptide exit tunnel (NPET) in the bacterial ribosome. We characterize the binding of four such hybrids by cryo-electron microscopy, granting insight into their molecular mechanisms of action. We identify a hybrid of azithromycin and tedizolid that is active against a diverse panel of multidrug-resistant Gram-positive bacteria and is minimally affected by ribosomal protection (ABC-F) resistance mechanisms. These results extend our understanding of ribosome inhibition and provide a pipeline for the rational design of dual-action antibiotics that target the ribosome. In a broader context, this work offers a framework for developing bifunctional inhibitors that engage adjacent binding sites by means of a rational cycle of synthetic optimization, biological evaluation, and structural characterization.

Trichothecenes are a structurally diverse family of fungal natural products whose potent cytotoxic, antifungal, and immunomodulatory effects make them both a threat to food security and a scaffold for therapeutic development. These sesquiterpenes share a conserved tricyclic core decorated with diverse oxidative and functional modifications, yielding hundreds of known congeners. However, the lack of concise synthetic access to key trichothecenes has limited efforts to systematically interrogate their structure–activity relationships. Here we developed concise, fully synthetic routes to the minimal trichothecenes verrucarol, diacetylverrucarol, and trichodermin. Acetylation of verrucarol markedly enhanced its inhibition of protein synthesis in vitro and increased cytotoxicity toward cancer and non-cancer cell lines, underscoring the role of peripheral modifications in modulating biological activity. Cryo-EM characterization of diacetylverrucarol bound to the human 50S ribosomal subunit at 2.7 Å revealed previously unresolved binding determinants, including bridging water interactions with the G4451 ribose. These results establish a synthetic and structural platform for probing trichothecene structure–function relationships and guiding their optimization for therapeutic applications.

The resorcinolic macrolide (RM) family comprises over 50 natural products with diverse biological activities, including the potent natural Hsp90 inhibitor radicicol. Here, we report a modular and enantioselective synthetic strategy featuring a biomimetic macrocyclization–aromatization cascade that enables rapid access to natural and unnatural RMs. Using this route, we generated a small panel of minimal RMs, introducing covalent warheads, double bonds for macrocycle rigidification, and modifications at the C15 position, a position that has not been modified before. We also synthesized macrocyclic peptide‐RM hybrids, offering a platform for rapid diversification. We evaluated all analogs for inhibitory activity against Hsp90α and Hsp90β and for disruption of the KRas^G12C–CRAF protein–protein interaction (PPI). We found that introduction of polar groups at C15 improved Hsp90 activity relative to the representative RM de‐O‐methyllasiodiplodin, dependent on stereochemistry, whereas introduction of larger groups at C15 or introduction of a trans double bond into the macrocycle reduced activity. Two analogs with poor Hsp90 inhibitory activity exhibited dose‐dependent inhibition of the KRas^G12C–CRAF PPI. This work demonstrates that strategic modifications of a minimal RM scaffold through modular chemical insight can provide foundational structure–activity relationships for the class.

Antimicrobial resistance remains a formidable challenge to modern medicine, with bacterial resistance mechanisms increasingly eroding the utility of clinically important antibiotics. While recent efforts have expanded the antibacterial pipeline, the development of resistance in priority pathogens continues to exceed the pace of new drug development. One emerging strategy to overcome resistance is the rational design of hybrid antibiotics that engage multiple binding sites. Here we describe the design, synthesis, and microbiological and structural characterization of hybrid antibiotics of azithromycin, tedizolid, and chloramphenicol that span the peptidyltransferase center (PTC) and nascent peptide exit tunnel (NPET). We characterize the binding of five such hybrids to the bacterial ribosome by cryo-electron microscopy, granting insight into their molecular mechanisms of action. We identify a hybrid of azithromycin and tedizolid that is active against a diverse panel of Gram-positive bacteria, including strains with both macrolide and oxazolidinone resistance mechanisms. These results extend our understanding of ribosome inhibition and demonstrate that the rational design of dual-action antibiotics can restore or even enhance antibacterial efficacy against resistant strains. In a broader context, this work offers a promising framework for developing next-generation therapeutics to combat multidrug-resistant pathogens and reinvigorate the dwindling antibiotic pipeline.

The resorcinolic macrolide (RM) class of natural products is characterized by a resorcinolic ester core linked to a 10-14-membered macrocyclic lactone, encompassing over 50 members with diverse biological activities. Among the most notable is radicicol, a potent natural inhibitor of the 90-kDa heat-shock protein (Hsp90). Here we present an enantioselective, modular synthetic strategy to access diverse RMs, focusing on the modification of the C15 position, covalent warheads, and rigidity of the macrocycle to explore their effects on Hsp90 inhibitory activity and isoform selectivity. We synthesized 27 RM derivatives and evaluated their inhibitory activity against Hsp90𝛼 and Hsp90β isoforms and KRas. Compound 12A exhibited the highest potency with IC50 values of 14 µM and 37 µM against Hsp90𝛼 and Hsp90β, respectively. Additionally, compound 1C displayed high selectivity for Hsp90𝛼. These findings indicate that targeted modifications at C15 can yield novel RMs with potential as biological probes and therapeutic leads, offering a new avenue for the development of Hsp90 or KRas inhibitors with reduced resistance and improved specificity.

Summary: Streptogramins are potent antibiotics targeting bacterial ribosome. The synergistic binding of group A and B streptogramins to 50S-ribosome, yields bactericidal effects. However, their efficacy is compromised by resistance mechanisms, including enzymatic acetylation of group A streptogramins by Virginiamycin Acetyl Transferase (Vat) enzymes, which reduces their affinity for ribosomes. Using fragment-based drug discovery we identified starting points for development of VatD inhibitors. X-ray crystallography screening revealed three primary fragment-binding sites on VatD. In the acetyl binding subsite, fragments stabilized distinct conformational states in critical residues, His82 and Trp121. In the antibiotic binding site, two fragments formed interactions that could be leveraged for competitive inhibition. Elaborations of these fragments showed weak inhibition of VatD activity, indicating potential for further optimization. These findings establish initial hits that could restore streptogramin efficacy by targeting VatD directly, providing a structural foundation for inhibitor development against resistant bacterial strains.