Biology subjects

Robbins, N.

Publications and source records attributed to Robbins, N..

7 recordsLinked to original sources

bioRxiv2026

Extracellular vesicle packaging of a supercluster-encoded antimicrobial combination with enhanced potency against fungal biofilms

Bacterial specialized metabolites are bioactive compounds that have been mined extensively as antibacterials, antifungals, and other drugs. They are widely used as experimental probes of complex biological systems. Since their discovery, their functional characteristics have typically been investigated as single, soluble compounds. The discoveries in this work expand this model. First, we report the co-packaging in extracellular vesicles (EVs) of two specialized metabolite complexes, candicidin and antimycin A, the products of a supercluster of biosynthetic genes, by a Streptomyces soil isolate. Secondly, we report that the products of this supercluster have synergistic antimicrobial activity against fungal biofilms, whereas biofilms are tolerant to the individual compounds. Finally, we show that co-packaging these compounds in EVs ensures simultaneous delivery with enhanced potency. This research advances understanding of the elegant complexity that shapes the evolution and function of bacterial specialized metabolites. In particular, the enhanced antibiofilm activity of co-packaged metabolites compared to individual soluble compounds is notable given the relevance of biofilms to microbial ecology and to pathogenesis. Our work supports the evolution of coordinated activity of specialized metabolites to counter conditional environmental challenges, and highlights co-packaging in EVs as one mechanism for secretion and co-delivery of combinations of bioactive molecules.

microbiology↗

bioRxiv2025

Transcriptomic analysis of 40 human and rodent skeletal muscle exerkines

Animal and human studies show that exercise induces organism wide molecular adaptations, many of which are mediated by exerkines which are secreted factors that enable communication between tissues such as skeletal muscle, adipose tissue, liver and the brain. However, the tissue specific responsiveness of individual exerkines and how these responses differ across species, exercise conditions and sexes remain poorly understood. To address this gap, we systematically analyzed skeletal muscle transcriptomic responses of 40 exerkines using three publicly available datasets which include MetaMEx, Extrameta and the MoTrPAC rat training study. We analyzed exerkine specific regulation in humans, mice and rats across acute and chronic exercise as well as inactivity, and determined which responses were conserved, species specific, sex dependent or dependent on exercise duration. Our analysis reveals substantial heterogeneity in skeletal muscle exerkine regulation with only a small subset showing conserved changes across species, while many exerkines exhibited human exclusive, rodent exclusive, acute specific or chronic specific patterns. These results provide a ranked overview of the most exercise responsive skeletal muscle exerkines and highlight the need for multi species and multi condition approaches when selecting exerkines as biomarkers or therapeutic targets.

physiology↗

bioRxiv2025

Structure-activity Relationship for Diarylpyrazoles as Inhibitors of the Fungal Kinase Yck2

Candida albicans is a growing global health threat, causing 1.5 million invasive infections and 1 million deaths annually. Yeast casein kinase 2 (Yck2) in C. albicans has emerged as an antifungal target of the kinase inhibitor LY364947 (LY). Herein, we report Yck2 structure-activity relationships for 3,4- and 3,4,5-substituted pyrazole analogs of LY. X-ray crystallography and in vitro profiling revealed the importance of the hinge-binding heterocycle for Yck2 inhibition and fungal kinome selectivity. A hydrogen-bond network between the inhibitor, a bound water molecule, and catalytic residues within the ATP pocket was identified as a key determinant of selectivity over other fungal and human kinases. Phenol analog 11 showed remarkable selectivity for Yck2 and Yck22 over all other C. albicans protein kinases. Several of the LY analogs, including 11, demonstrated improved antifungal activity. These findings provide a framework for translating human kinase inhibitors into highly selective antifungal Yck2 inhibitors. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/664496v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@ef6a04org.highwire.dtl.DTLVardef@193b73org.highwire.dtl.DTLVardef@8e109corg.highwire.dtl.DTLVardef@e4a243_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Chemoproteomic Profiling of C. albicans for Characterization of Anti-fungal Kinase Inhibitors

Candida albicans is a growing health concern as the leading causal agent of systemic candidiasis, a life-threatening fungal infection with a mortality rate of [~]40% despite best available therapy. Yck2, a fungal casein kinase 1 (CK1) family member, is the cellular target of inhibitors YK-I-02 (YK) and MN-I-157 (MN). Here, multiplexed inhibitor beads paired with mass spectrometry (MIB/MS) employing ATP-competitive kinase inhibitors were used to define the selectivity of these Yck2 inhibitors across the global C. albicans proteome. The MIB matrix captured 89% of the known and predicted C. albicans protein kinases present in cell lysate. In MIB/MS competition assays, YK and MN demonstrated exquisite selectivity across the C. albicans fungal kinome with target engagement of only three CK1 homologs (Yck2, Yck22, and Hrr25) and a homolog of human p38 (Hog1). Additional chemoproteomics using a custom MN-kinobead identified only one additional C. albicans protein, confirming its remarkable fungal proteome-wide selectivity. To identify new Yck2 inhibitors with selectivity over Hog1, thirteen human CK1 kinase inhibitors were profiled for fungal kinase-binding activity using MIB/MS competition assays and in-cell NanoBRET target engagement assays. A new chemotype of family-selective Yck2 inhibitors with antifungal activity was identified. Together, these findings expand the application of MIB/MS proteomic profiling for non-human kinomes and demonstrate its utility in the discovery and development of selective inhibitors of fungal kinases with potential antimicrobial activity. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/632200v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1606f86org.highwire.dtl.DTLVardef@39c92borg.highwire.dtl.DTLVardef@1c666cborg.highwire.dtl.DTLVardef@869b5c_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Butyrolactol A is a phospholipid flippase inhibitor that potentiates the bioactivity of caspofungin against resistant fungi

Fungal infections cause millions of deaths annually and are challenging to treat due to limited therapeutic options and rising resistance. Cryptococci are intrinsically resistant to the latest generation of antifungals, echinocandins, while Candida auris, a notorious global threat, is also increasingly resistant. We performed a natural product extract screen to rescue caspofungin fungicidal activity against Cryptococcus neoformans H99 and identified butyrolactol A, which restores echinocandin efficacy against resistant fungal pathogens, including multidrug-resistant C. auris. Mode of action studies revealed that butyrolactol A inhibits the phospholipid flippase Apt1-Cdc50, blocking phospholipid transport. Cryo-electron microscopy analysis of the Apt1*butyrolactol A complex revealed that the flippase is trapped in a dead-end state. Apt1 inhibition disrupts membrane asymmetry, vesicular trafficking, and cytoskeletal organization, thereby enhancing echinocandin uptake and potency. This study identifies lipid flippases as promising antifungal targets and demonstrates the potential of revisiting natural products to expand the antifungal arsenal and combat resistance.

biochemistry↗

bioRxiv2024

Adaptation of the tetracycline-repressible system for modulating the expression of essential genes in Cryptococcus neoformans

The opportunistic human fungal pathogen Cryptococcus neoformans has an enormous impact on human health as the causative agent of cryptococcal meningitis, an AIDS-defining illness that results in significant human mortality. To combat C. neoformans infections, there is a dire need to expand our current antifungal arsenal. Essential gene products often serve as ideal targets for antimicrobials, and thus identifying and characterizing essential genes in a pathogen of interest is critical for drug development. Unfortunately, characterization of essential genes in C. neoformans is limited due to its haploid nature and lack of genetic tools for generating effective conditional-expression mutants. To date, the copper-repressible promoter pCTR4 is the mostly widely used system to regulate essential gene expression, however, its expression is leaky and copper has pleiotropic effects. In diverse fungal species, including Saccharomyces cerevisiae, Candida albicans, and Candida auris, the tetracycline-repressible promoter system is a powerful tool to regulate gene expression, however, it has yet to be adapted for C. neoformans. In this study, we successfully implemented the tetracycline-repressible system in C. neoformans to regulate the expression of the essential gene HSP90. Supplementation of cultures with the tetracycline analog doxycycline efficiently depleted HSP90 at both transcript and protein levels and inhibited C. neoformans growth and viability. Thus, this work unveils a novel approach to generate conditional-expression mutants in C. neoformans, providing unprecedented potential to systematically study essential gene function in this important human fungal pathogen.

molecular biology↗

bioRxiv2021

A functionally divergent intrinsically disordered region underlying the conservation of stochastic signaling

Stochastic signaling dynamics expand living cells information processing capabilities. An increasing number of studies report that regulators encode information in their pulsatile dynamics. The evolutionary mechanisms that lead to complex signaling dynamics remain uncharacterized, perhaps because key interactions of signaling proteins are encoded in intrinsically disordered regions (IDRs), whose evolution is difficult to analyze. Here we focused on the stochastic pulsing dynamics of Crz1, a transcription factor in fungi downstream of the widely conserved calcium signaling pathway. We find that Crz1 IDRs from anciently diverged fungi can all respond transiently to calcium stress; however, only Crz1 IDRs from the Saccharomyces clade support pulsatility, encode extra information, and rescue fitness, while the Crz1 IDRs from distantly related fungi do none of the three. On the other hand, we find that Crz1 pulsing is conserved in the distantly related fungi, consistent with the evolutionary model of stabilizing selection. Further, we show that a calcineurin docking site in a specific part of the IDRs appears to be sufficient for pulsing and show evidence for a beneficial increase in the relative calcineurin affinity of this docking site. We propose that evolutionary flexibility of functionally divergent IDRs underlies the conservation of stochastic signaling by stabilizing selection.

evolutionary biology↗