Biology subjects

Newkirk, S. E.

Publications and source records attributed to Newkirk, S. E..

3 recordsLinked to original sources

bioRxiv2026

A Luciferase Reporter of Ester Prodrug Delivery to the Cytosol of Intracellular Mycobacteria

The bacterial cell envelope restricts access to cytoplasmic targets and is a principal reason that otherwise potent antibacterial compounds fail in cells. Masking an anionic carboxylic acid as an ester is a common way to cross that barrier, but the strategy succeeds only if the target organism also hydrolyzes the ester once the compound arrives. These two requirements, permeation and intracellular unmasking, are rarely measured together in live bacterial cells, and existing reporters cannot specify where unmasking occurred. Here we describe a bioluminescent platform that resolves both steps in real time in live mycobacteria using carboxylate esters of D-luciferin. Luciferase requires that an ester both reach the bacterial cytoplasm and be hydrolyzed there, which makes light a compartment-specific readout of productive delivery. Across a series of alkyl esters in Mycobacterium smegmatis, chain length governed permeation while branching adjacent to the carbonyl governed esterase processing, indicating that the two parameters can be tuned independently. The n-butyl ester gave the strongest signal in broth, in infected macrophages, and in the pathogen Mycobacterium avium, whereas the tert-butyl ester was inert in Mycobacterium avium and unmasking of the whole series was attenuated at phagosomal pH. Ester activation is therefore both species- and compartment-dependent, and masking groups are best selected in the organism and environment in which they must act.

microbiology↗

bioRxiv2026

Mapping the Modification Landscape of MHC-I Epitopes: A Framework for Immunogenic Peptidomimetic Antigen Design

Peptide-based cancer vaccines offer a promising strategy to target tumor-specific neoantigens. This approach is increasingly critical as post-translationally modified peptides, driven by altered tumor metabolism, emerge as a unique class of neoantigens. Because these chemically distinct epitopes cannot be genetically encoded by mRNA or viral platforms, synthetic peptide vaccines are poised to be the primary route to target these types of neoantigens. Yet, their clinical translation is restricted by poor metabolic stability, limited intracellular permeability, and structural requirements for MHC-I binding and T cell receptor (TCR) recognition. Although peptidomimetic modifications have been widely explored to improve pharmacokinetics, their impact on antigen presentation and immune recognition remains poorly understood. Here, we undertook a comprehensive evaluation of peptidomimetics geared at MHC-I neoantigens and generated a diverse library of systematically modified peptides that incorporate backbone N-methylation, peptoid substitution, and stereochemical inversion. Integrated assays revealed a highly position-dependent tolerance to peptidomimetic modifications, while subsequent combinatorial designs demonstrated non-additive effects on the balance between immunogenicity and pharmacokinetics. Collectively, these findings establish design principles and provide a framework for balancing immune recognition with enhanced stability and permeability in peptidomimetic antigen design.

immunology↗

bioRxiv2025

Evaluation and In Situ Library Expansion of Small Molecule MHC-I Inducers

Immunotherapy has emerged as a powerful strategy for combating cancer by harnessing the patients immune system to recognize and eliminate malignant cells. Major histocompatibility complex class I (MHC-I) plays a pivotal role by presenting neoantigens to CD8+ T cells, triggering T cell-mediated killing. However, cancer cells often evade detection by downregulating MHC-I surface expression, hindering the immune response. This resistance mechanism offers an opportunity to bolster MHC-I surface expression via therapeutic interventions. We conducted a comprehensive evaluation of previously purported small molecule MHC-I inducers and identified heat shock protein 90 (Hsp90) inhibitors as privileged enhancers. Using a core scaffold, we employed an in situ click chemistry-based derivatization strategy to generate 380 novel compounds. New agents showed high induction levels, with one triazole-based analog, CliMB-325, also enhancing T cell activation and exhibiting lower toxicity. Altogether, we demonstrated the potential of click chemistry-based diversification for discovering small molecules to counter immune evasion.

immunology↗