Biology subjects

Wolan, D. W.

Publications and source records attributed to Wolan, D. W..

13 recordsLinked to original sources

bioRxiv2025

Inactivation of peptidoglycan remodeling promotes antibiotic susceptibility in vancomycin-resistant Enterococcus faecium

Vancomycin-resistant Enterococcus faecium (VREfm) is a leading cause of healthcare-associated infections globally and demands new approaches for treatment. Here we show that genetic and pharmacological inactivation of a highly conserved NlpC/P60 peptidoglycan hydrolase, secreted antigen A (SagA), enhanced vancomycin susceptibility of VREfm ex vivo and in vivo. Notably, genetic deletion of sagA impaired VREfm peptidoglycan remodeling, growth and increased the activity of vancomycin. We then identified first-in-class covalent NlpC/P60 peptidoglycan hydrolase inhibitors and demonstrated that pharmacological inactivation of SagA activity also impaired peptidoglycan remodeling and increased the efficacy of vancomycin across genetically distinct VREfm clinical isolates. Our study reveals peptidoglycan hydrolases are druggable targets whose inactivation improves the efficacy of vancomycin against VREfm.

microbiology↗

bioRxiv2024

Caspase prime-side active-site characterization with non-hydrolyzable peptides assists in the design of a caspase-7-selective irreversible probe.

Limited structural information and biochemical studies are available that demonstrate how the prime side of cysteine protease active sites, such as for human caspases, are used for substrate recognition and how these binding regions can be exploited in the design of inhibitors and probes. Reported small molecules that interact with, and are optimized for, the prime side of caspase active sites are limited to methylketone leaving groups and other nonpeptidic inhibitor moieties, such as aza-Michael acceptors. We present the design, synthesis, and co-complex x-ray structures of the first prime-side elongated non-hydrolyzable peptidomimetic ketomethylene inhibitors designed to interrogate the S4-S4 active-site binding pockets of the executioner caspases-3 and -7. In addition to our structures depicting the first examples of an active-site cysteine in complex with a P1 residue trapped in a non-covalent tetrahedral intermediate, we elucidated prime-side binding interactions for pockets S1 through S4 with our biologically relevant peptide Ac-DEVD-Propionate-AAA. Despite the substantial homology among the caspase active sites, we identified a key difference in the prime-side architecture within binding distance to the P2 inhibitor alanine whereby caspase-3 F128 is substituted for caspase-7 Y151. We exploited this prime-side difference in side chains and their reactivities in the design of non-hydrolyzable ketomethylene-based probes bearing a C-terminal tyrosine-reactive 4-phenyl-1,2,4-triazole-3,5-dione moiety. Our probe selectively labels caspase-7 over caspase-3 and we posit that further characterization of protease active-site prime sides with similar non-hydrolyzable molecules will yield additional tool-like compounds that will assist in establishing the non-redundant roles of caspase family members and other cysteine proteases.

biophysics↗

bioRxiv2022

PROTAC-mediated selective degradation of cytosolic soluble epoxide hydrolase enhances ER-stress reduction

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme responsible for lipid metabolism and is a promising drug target. Here, we report the first-in-class PROTACs small molecule degraders of sEH. Our optimized PROTAC selectively targets the degradation of cytosolic but not peroxisomal sEH, resulting in exquisite spatiotemporal control. Remarkably, our sEH PROTAC molecule has higher potency in cellular assays compared to the parent sEH inhibitor as measured by significantly reduced ER stress. Interestingly, our mechanistic data indicate that our PROTAC directs degradation of cytosolic sEH via the lysosome, not through the proteasome. The molecules presented here are useful chemical probes to study the biology of sEH with the potential for therapeutic development. Broadly, our results represent a proof-of-concept for the superior cellular potency of sEH degradation over sEH enzymatic inhibition, as well as subcellular compartment-selective modulation of a protein by PROTACs. HighlightsO_LIFirst-in-class soluble epoxide hydrolase (sEH) small-molecule degraders. C_LIO_LISelective degradation of cytosolic but not peroxisomal sEH. C_LIO_LISignificant and stable reduction in sEH protein levels, leading to enhanced cellular efficacy in ER stress reduction relative to the parent inhibitor. C_LI

biochemistry↗

bioRxiv2021

Targeted Neuronal Activation of the Gastrointestinal Tract Shapes the Environment of the Gut in Mice

The gastrointestinal (GI) tract is extensively innervated by intrinsic neurons of the enteric nervous system (ENS) and extrinsic neurons of the central nervous system and peripheral ganglia, which together regulate gut physiology. The GI tract also harbors a diverse microbiome, but interactions between the ENS and the microbiome remain poorly understood. Herein, we activate choline acetyltransferase (ChAT)-expressing or tyrosine hydroxylase (TH)-expressing gut-associated neurons in mice to determine effects on intestinal microbial communities and their metabolites, as well as on host physiology. The resulting multi-omics datasets support broad roles for discrete peripheral neuronal subtypes in shaping microbiome structure, including modulating bile acid profiles and fungal colonization. Physiologically, activation of either ChAT+ or TH+ neurons increases fecal output, while only ChAT+ activation results in increased colonic migrating motor complexes and diarrhea-like fluid secretion. These findings suggest that specific subsets of peripherally-activated ENS neurons differentially regulate the gut microbiome and GI physiology in mice, without involvement of signals from the brain.

physiology↗

bioRxiv2020

Chemical inhibition of ENL/AF9 YEATS domains in acute leukemia

Transcriptional co-regulators, which mediate chromatin-dependent transcriptional signaling, represent tractable targets to modulate tumorigenic gene expression programs with small molecules. Genetic loss-of-function studies have recently implicated the transcriptional co-activator, ENL, as a selective requirement for the survival of acute leukemia and highlighted an essential role for its chromatin reader YEATS domain. Motivated by these discoveries, we executed a screen of nearly 300,000 small molecules and identified an amido-imidazopyridine inhibitor of the ENL YEATS domain (IC50 = 7 {micro}M). Leveraging a SuFEx-based high-throughput approach to medicinal chemistry optimization, we discovered SR-0813 (IC50 = 25 nM), a potent and selective ENL/AF9 YEATS domain inhibitor that exclusively inhibits the growth of ENL-dependent leukemia cell lines. Armed with this tool and a first-in-class ENL PROTAC, SR-1114, we detailed the response of AML cells to pharmacological ENL disruption for the first time. Most notably, displacement of ENL from chromatin by SR-0813 elicited a strikingly selective suppression of ENL target genes, including HOXA9/10, MYB, MYC and a number of other leukemia proto-oncogenes. Our study reproduces a number of key observations previously made by CRISPR/Cas9 loss of function and dTAG-mediated degradation, and therefore, both reinforces ENL as an emerging leukemia target and validates SR-0813 as a high-quality chemical probe.

cancer biology↗

bioRxiv2020

Quantitative metaproteomics of patient fecal microbiota identifies host and microbial proteins associated with ulcerative colitis

Mass spectrometry-based metaproteomics technologies enable the direct observation of proteins within complex multi-organism environments. A major hurdle in mapping metaproteomic fragmentation spectra to their corresponding peptides is the need for large peptide databases encompassing all anticipated species contained within a biological sample. As we cannot predict the taxonomic composition of microbiomes a priori, we developed the ComPIL database which contains a comprehensive collection of 4.8 billion unique peptides from public sequencing repositories to enable our proteomics analyses. We analyzed fecal samples from ulcerative colitis (UC) patients using a tandem mass spectrometry (LC-MS/MS) workflow coupled to ComPIL in search of aberrant UC-associated proteins. We found 176 host and microbial protein groups differentially enriched between the healthy (control) or UC volunteer groups. Notably, gene ontology (GO) enrichment analysis revealed that serine-type endopeptidases are overrepresented in UC compared to healthy volunteers. Additionally, we demonstrate the feasibility of serine hydrolase chemical enrichment from fecal samples using a biotinylated fluorophosphate (FP) probe. Our findings illustrate that probe-susceptible hydrolases from hosts and microbes are likely active in the distal gut. Finally, we applied de novo peptide sequencing methods to our metaproteomics data to estimate the size of the "dark peptidome," the complement of peptides unidentified using ComPIL. We posit that our metaproteomics methods are generally applicable to future microbiota analyses and that our list of FP probe-enriched hydrolases may represent an important functionality to understanding the etiology of UC.

microbiology↗

bioRxiv2020

Identification of an N-acetylneuraminic acid-presenting bacteria isolated from a healthy human microbiome

N-acetylneuraminic acid is the most abundant sialic acid in humans and is generally expressed as the terminal sugar on intestinal mucus glycans. Several pathogenic bacterial species harvest sialic acid from the mucus, diet, as well as other intestinal sources and display this sugar on their own surface to evade sialic acid-binding immunoglobulin-type lectin (Siglec)-mediated host immune surveillance. While previous studies have identified bacterial enzymes associated with sialic acid catabolism, no reported methods permit the selective labeling, tracking, and quantitation of sialic acid-presenting microbes within complex multi-microbial systems. Here, we apply an interdisciplinary approach combining metabolic labeling, click chemistry, metagenomic, and whole-genome sequencing to selectively track and identify sialic acid-presenting microbes from a cultured healthy human fecal microbiome. We isolated and identified a new strain of Escherichia coli that incorporates sialic acid onto its own surface. Analysis of the sequence data reveals that this E. coli strain encodes for the NanT, NeuA, and NeuS genes necessary for harvesting environmental sialic acid and generating the capsular polysialic acid. We envision that this method is applicable to the detection and quantitation of sialic acid-presenting bacteria from human, animal, and environmental microbiomes, as well as investigating the importance of other carbohydrates to commensal and pathogenic bacteria.

microbiology↗

bioRxiv2020

An influenza A hemagglutinin small-molecule fusion inhibitor identified by a new high-throughput fluorescence polarization screen.

Influenza hemagglutinin (HA) glycoprotein is the primary surface antigen targeted by the host immune response and a focus for development of novel vaccines, broadly neutralizing antibodies (bnAbs) and therapeutics. HA enables viral entry into host cells via receptor binding and membrane fusion and is a validated target for drug discovery. However, to date, only a very few bona fide small molecules have been reported against the HA. To identity new antiviral lead candidates against the highly conserved fusion machinery in the HA stem, we synthesized a fluorescence-polarization probe based on a recently described neutralizing cyclic peptide P7 derived from the complementarity-determining region loops of human bnAbs FI6v3 and CR9114 against the HA stem. We then designed a robust binding assay compatible with high-throughput screening to identify molecules with low M to nM affinity to influenza A group 1 HAs. Our simple, low-cost, and efficient in vitro assay was used to screen H1/Puerto Rico/8/1934 HA trimer against approximately 72,000 compounds. The crystal structure of H1/Puerto Rico/8/1934 HA in complex with our best hit compound F0045(S) confirmed that it binds to pockets in the HA stem similar to bnAbs FI6v3 and CR9114, cyclic peptide P7, and small molecule inhibitor JNJ4796. F0045 is enantioselective against a panel of group 1 HAs and F0045(S) exhibits in vitro neutralization activity against multiple H1N1 and H5N1 strains. Our assay, compound characterization, and small-molecule candidate should further stimulate the discovery and development of new compounds with unique chemical scaffolds and enhanced influenza antiviral capabilities. SummaryInfluenza hemagglutinin (HA) glycoprotein enables viral entry into host cells and is the main target for antibodies in our immune system. While HA has now been established as a validated target for drug discovery, no FDA-approved small molecules are available that specifically prevent HA from binding host receptors or inhibit its membrane fusion activity and thus prevent infection. We therefore designed a fluorescence polarization probe to enable rapid identification of small molecules that bind to the stem fusion machinery of group 1 HAs. Application of our assay yielded a small molecule to the influenza A group 1 HA stem with antiviral efficacy.

biophysics↗

bioRxiv2019

Integrative x-ray structure and molecular modeling for the rationalization of procaspase-8 inhibitor potency and selectivity

Caspases are a critical class of proteases involved in regulating programmed cell death and other biological processes. Selective inhibitors of individual caspases, however, are lacking, due in large part to the high structural similarity found in the active sites of these enzymes. We recently discovered a small-molecule inhibitor, 63-R, that covalently binds the zymogen, or inactive precursor (pro-form), of caspase-8, but not other caspases, pointing to an untapped potential of procaspases as targets for chemical probes. Realizing this goal would benefit from a structural understanding of how small molecules bind to and inhibit caspase zymogens. There have, however, been very few reported procaspase structures. Here, we employ x-ray crystallography to elucidate a procaspase-8 crystal structure in complex with 63-R, which reveals large conformational changes in active-site loops that accommodate the intramolecular cleavage events required for protease activation. Combining these structural insights with molecular modeling and mutagenesis-based biochemical assays, we elucidate key interactions required for 63-R inhibition of procaspase-8. Our findings inform the mechanism of caspase activation and its disruption by small molecules, and, more generally, have implications for the development of small molecule inhibitors and/or activators that target alternative (e.g., inactive precursor) protein states to ultimately expand the druggable proteome.

biochemistry↗

bioRxiv2019

X-ray structures of two active secreted Bacteroides thetaiotaomicron C11 proteases in complex with peptide-based inhibitors

Commensal bacteria secrete proteins and metabolites to influence host intestinal homeostasis and proteases represent a significant constituent of the components at the host:microbiome interface. Here, we determined the structures of the two secreted C11 cysteine proteases encoded by the established gut commensal Bacteroides thetaiotaomicron. We employed mutational analysis to demonstrate the two proteases, termed \"thetapain\" and \"iotapain\", undergo in trans self-maturation after lysine and/or arginine residues, as observed for other C11 proteases. We determined the structures of the active forms of thetapain and iotapain in complex with irreversible peptide inhibitors, Ac-VLTK-AOMK and biotin-VLTK-AOMK, respectively. Structural comparisons revealed key active-site interactions important for peptide recognition are more extensive for thetapain; however, both proteases employ a glutamate residue to preferentially bind small polar residues at the P2 position. Our results will aid in the design of protease-specific probes to ultimately understand the biological role of C11 proteases in bacterial fitness, elucidate their host and/or microbial substrates, and interrogate their involvement in microbiome-related diseases.

biochemistry↗

bioRxiv2018

Triflic acid treatment enables LC-MS/MS analysis of insoluble bacterial biomass

The lysis and extraction of soluble bacterial proteins from cells is a common practice for proteomics analyses, but insoluble bacterial biomasses are often left behind. Here, we show that with triflic acid treatment, the insoluble bacterial biomass of Gram- and Gram+ bacteria can be rendered soluble. We use LC-MS/MS shotgun proteomics to show that bacterial proteins in the soluble and insoluble post-lysis fractions differ significantly. Additionally, in the case of Gram- Pseudomonas aeruginosa, triflic acid treatment enables the enrichment of cell envelope-associated proteins. Finally, we apply triflic acid to a human microbiome sample to show that this treatment is robust and enables the identification of a new, complementary subset of proteins from a complex microbial mixture.

systems biology↗

bioRxiv2018

The Genetic Basis for the Cooperative Bioactivation of Plant Lignans by a Human Gut Bacterial Consortium

Plant-derived lignans, consumed daily by most individuals, are inversely associated with breast cancer; however, their bioactivity is only exerted following gut bacterial conversion to enterolignans. Here, we dissect a four-species bacterial consortium sufficient for all four chemical reactions in this pathway. Comparative genomics and heterologous expression experiments identified the first enzyme in the pathway. Transcriptional profiling (RNAseq) independently identified the same gene and linked a single genomic locus to each of the remaining biotransformations. Remarkably, we detected the complete bacterial lignan metabolism pathway in the majority of human gut microbiomes. Together, these results are an important step towards a molecular genetic understanding of the gut bacterial bioactivation of lignans and other plant secondary metabolites to downstream metabolites relevant to human disease.\n\nOne Sentence SummaryBess et al. provide a first step towards elucidating the molecular genetic basis for the cooperative gut bacterial bioactivation of plant lignans, consumed daily by most individuals, to phytoestrogenic enterolignans.

microbiology↗

bioRxiv2017

Metaproteomics of colonic microbiota unveils discrete protein functions among colitic mice and control groups

Metaproteomics can greatly assist established high-throughput sequencing methodologies to provide systems biological insights into the alterations of microbial protein functionalities correlated with disease-associated dysbiosis of the intestinal microbiota. Here, we utilized the well-characterized murine T cell transfer model of colitis to find specific changes within the intestinal luminal proteome associated with inflammation. MS proteomic analysis of colonic samples permitted the identification of {bsim}10,000-12,000 unique peptides that corresponded to 5,610 protein clusters identified across three groups, including the colitic Rag1-/- T cell recipients, isogenic Rag1-/- controls, and wild-type mice. We demonstrate that the colitic mice exhibited a significant increase in Proteobacteria and Verrucomicrobia and show that such alterations in the microbial communities contributed to the enrichment of specific proteins with transcription and translation gene ontology terms. In combination with 16S sequencing, our metaproteomics-based microbiome studies provide a foundation for assessing alterations in intestinal luminal protein functionalities in a robust and well-characterized mouse model of colitis, and set the stage for future studies to further explore the functional mechanisms of altered protein functionalities associated with dysbiosis and inflammation.\n\nStatement of significance of the studyThe commensal gut microbiota is essential to maintaining health and has a primary role in digestion/metabolism, homeostasis, and protection from pathogenic bacteria. High-throughput sequencing has established Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria as the four major bacterial phyla that comprise the ecological makeup of the intestinal microbiota. However, the tremendous inter-/intra-variability in microbial composition across individuals, as well as along the length of the intestinal tract has made it difficult to definitively ascertain specific bacterial species associated with health or as drivers of disease states, including inflammatory bowel disease. In this study, we expanded upon the current metaproteomics techniques and use the robust and highly reproducible murine T cell transfer model of colitis as well as a comprehensive database of mouse, human, plant, and all microbial genomes sequenced to date to elucidate alterations in both host and gut microbial proteins associated with intestinal inflammation. Our results show that host genetics, gut microbiota, and inflammation have tremendous influences on the intestinal luminal proteomic landscape.

bioinformatics↗