Biology subjects

Talbot, K. M.

Publications and source records attributed to Talbot, K. M..

8 recordsLinked to original sources

bioRxiv2026

The small molecule regacin binds a conserved pocket in RegA to inhibit the expression of bacterial virulence factors

Antivirulence therapeutics are a promising strategy to combat rising antimicrobial resistance. Transcription factor proteins have shown merit as antivirulence drug targets, as demonstrated by the small molecule Savirin. Regacin was identified as a potent inhibitor of the AraC-family virulence regulator (AraC-VR) RegA of Citrobacter rodentium, providing proof-of-concept for targeting AraC-VR family members in related human enteric pathogens. The structural basis of RegA inhibition by regacin was previously undefined and could prove essential for future drug design campaigns targeting AraC-VR family members. This work reveals that RegA is structurally similar to its homologs ToxT and Rns, including a conserved ligand-binding pocket in the N-terminal domain. A regacin-RegA co-crystal structure shows regacin binds within this pocket, with some heterogeneity in binding pose. Finally, structure-guided mutagenesis of key pocket residues, validated using a {beta}-galactosidase reporter assay, confirmed their role in regacin-mediated inhibition. Together, these findings define the regacin binding site, suggest a molecular basis of RegA inhibition, and provide a framework for future inhibitor design targeting this conserved pocket in other virulence regulators in human enteric pathogens.

microbiology↗

bioRxiv2026

Regulation and Characterization of the Cex system of enteric pathogens: a pathway for the lipidation and secretion of noncanoncial bacterial lipoproteins

CexE is an outer membrane lipoprotein of enterotoxigenic Escherichia coli (ETEC); one of the most prevalent etiological agents of diarrheal disease. Homologs of CexE are present in enteroaggregative E. coli, Citrobacter rodentium, and other enteropathogens. We have previously shown that expression of CexE is dependent upon Rns, the master virulence regulator of ETEC and a member of the AraC/XylS superfamily of transcription factors. In this study we report that the expression of cexD and cexPABC, the genes required for lipidation and delivery of CexE to the outer leaflet of the outer membrane, are also Rns-dependent. Although cexE and cexPABC are arranged in the same orientation, they are separated by an intergenic region sufficiently large to accommodate a promoter and transcription factor binding sites. Nevertheless, expression of the four gene cluster does not originate within the intergenic region. Rather, cexPABC are expressed from the Rns-dependent cexE promoter. The remaining gene of the system, cexD, is monocistronic and expressed from its own Rns-dependent promoter. Mutagenesis of a predicted Rns binding site upstream of cexD abolished Rns binding to a cexD promoter fragment in vitro and activation of the promoter in vivo. CexD is a novel bacterial acyltransferase. Unlike canonical bacterial acyltransferases that lipidate amino-terminal cysteines, CexD attaches a lipid to the amino-terminal glycine of CexE. Lipidation must occur in the periplasm because topological mapping revealed that CexD has six transmembrane domains with both its amino-terminus and enzymatic domain in the periplasm. We further show that CexD is required for CexE localization to the outer leaflet of the outer membrane and that a cexD mutant of C. rodentium is attenuated in a murine model. These findings further define the Rns virulence regulon, the enzymology of a noncanonical acyltranferase and its contributions to pathogenicity.

microbiology↗

bioRxiv2026

Linking the kinetic mechanism to structural dynamics required for nucleotide hydrolysis by an alphavirus nsP2 RNA helicase

RNA helicases encoded by positive-strand RNA viruses are essential for genome replication, yet the specific biological functions and mechanochemical basis underlying these functions remain poorly defined. Progress has been limited by the difficulty of resolving individual catalytic steps under single-turnover conditions, which are often experimentally inaccessible for viral enzymes. Alphaviruses replicate within membrane-bound spherules that may alter local metabolite concentrations, raising the possibility that the enzymatic properties of alphaviral proteins differ from those of viruses with greater cytosolic exposure. Here, we present a kinetic and binding analysis of full-length non-structural protein 2 (nsP2) from Chikungunya virus, a multifunctional superfamily 1B NTPase and RNA helicase. Purified nsP2 binds nucleoside triphosphates with high affinity, exhibiting equilibrium dissociation constants in the single digit micromolar range. This property enabled single-turnover, pre-steady-state, and isotope-trapping experiments that are rarely feasible for viral helicases. These analyses identified two sequential conformational-change steps required for nucleotide hydrolysis. Molecular dynamics simulations suggest tightening of the RecA1 and RecA2 domains upon ATP binding followed by compaction of the enzyme mediated by interactions between the 1B subdomain and RecA2 domain. Product inhibition patterns support random release of ADP and inorganic phosphate, with relative binding affinities indicating that ADP dissociates first. The reaction is irreversible. Although nsP2 binds RNA tightly, strand separation under single-turnover conditions is too slow to represent ATP-driven unwinding, instead likely reflecting formation of an unwinding-competent nsP2-RNA complex. Together, these findings establish a quantitative framework for nsP2 function and provide a roadmap for mechanistic studies of alphaviral helicases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/723793v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@13899a1org.highwire.dtl.DTLVardef@ee1aadorg.highwire.dtl.DTLVardef@1991e1org.highwire.dtl.DTLVardef@b877f6_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Identification of a Broadly Acting Inhibitor of the Alphavirus Non-Structural Protein 2 Helicase

Alphaviruses are mosquito-borne viruses that have caused significant outbreaks in the 21st century. Despite multiple recent outbreaks, there are no approved antiviral drugs to treat any alphavirus infection. Therefore, developing broadly acting antiviral drugs effective against multiple alphaviruses is necessary and could provide protection from both current and emerging alphavirus threats. A critical component of the alphavirus replication complex is non-structural protein 2 (nsP2), which is a multifunctional enzyme containing a helicase domain connected to a protease domain by a flexible linker. nsP2 functions as an ATP-dependent helicase, is conserved across the alphavirus genus, and is essential for virus replication, making it a promising target for development of alphavirus broad-acting antiviral drugs. Previous studies identified an enantioselective compound RA-0025298 that inhibited nsP2 ATPase activity and chikungunya virus CHIKV replication. Antiviral testing of RA-25298. against a diverse group of alphaviruses found broad activity except for Sindbis-like viruses. Using this information along with mutational profiling of virus passaged with RA-0025298 we identified the site of RA-0025298 action and confirmed the binding site via biophysical analyses. Finally, we found that the active enantiomer of RA-0025298 (SGC-NSP2hel-1) reduced viral loads in vivo and protected mice from tissue damage and disease caused by CHIKV infection. These findings further describe the mechanism of action of a first-in-class nsP2 helicase inhibitor with the potential for development as a broad spectrum drug for treating or preventing disease caused by current and emerging alphaviruses. One Sentence SummaryThis study describes the mechanism of action and in vivo efficacy of a first in class broadly acting inhibitor of alphavirus nsP2 helicase activity.

microbiology↗

bioRxiv2025

An Enantioselective Chemical Probe for Chikungunya nsP2 Helicase with Antialphaviral Activity

Chikungunya virus (CHIKV) replication relies on the multifunctional nsP2 protein, making it an attractive target for antiviral drug discovery. Here, we report the resolution of oxaspiropiperidine 1, a first-in-class inhibitor of the CHIKV nsP2 RNA helicase (nsP2hel), into its constitutive enantiomers and characterization of their antiviral activity. The enantiomer (R)-1 exhibited potent inhibition of viral replication, nsP2hel ATPase activity, and dsRNA unwinding, while the (S)-1 enantiomer was >100-fold less active. The (R)-1 enantiomer also demonstrated high selectivity for CHIKV over other RNA viruses and for nsP2hel over other RNA helicases. Direct binding of (R)-1 to nsP2hel protein was confirmed by 19F NMR. Biophysical and structural studies revealed conformational polymorphism in the spirocyclic scaffold of (R)-1, suggesting a potential role of thermal mobility of the ligand in allosteric inhibition of nsP2hel. Collectively, these findings designate (R)-1 (RA-NSP2-1) as a high-quality chemical probe and (S)-1 (RA-NSP2-1N) as a negative control for probing the biology of alphavirus RNA helicases.

pharmacology and toxicology↗

bioRxiv2025

Identification of Direct-acting nsP2 Helicase Inhibitors with Anti-alphaviral Activity

Alphaviruses are mosquito-borne RNA viruses that pose a significant public health threat, with no FDA-approved antiviral therapeutics available. The non-structural protein 2 helicase (nsP2hel) is an enzyme involved in unwinding dsRNA essential for alphavirus replication. This study reports the discovery and optimization of first-in-class oxaspiropiperidine inhibitors targeting nsP2hel. Structure-activity relationship (SAR) studies identified potent cyclic sulfonamide analogs with nanomolar antiviral activity against chikungunya virus (CHIKV). Biochemical analyses of nsP2hel ATPase and RNA unwindase activities showed these compounds act by a non-competitive mode suggesting that they are allosteric inhibitors. Viral resistance mutations mapped to nsP2hel and a fluorine-labeled analog exhibited direct binding to the protein by 19F NMR. The lead inhibitor, 2o, demonstrated broad-spectrum antialphaviral activity, reducing titers of CHIKV, Mayaro virus (MAYV), and Venezuelan equine encephalitis virus (VEEV). These findings support nsP2hel as a viable target for development of broad-spectrum direct-acting antialphaviral drugs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/641060v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@856df5org.highwire.dtl.DTLVardef@1f6225borg.highwire.dtl.DTLVardef@4997d5org.highwire.dtl.DTLVardef@18f42f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2025

Characterization of the ligand binding pocket of the virulence regulator Rns, a Member of the AraC/XylS family of transcription factors

Diarrheal disease caused by Gram-negative enteric pathogens such as enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae, Shigella spp., and, Salmonella spp. are a leading cause of morbidity and mortality of children; especially, in low resource nations. While progress has been made in reducing this burden, there remains a need to develop efficacious therapies. Recently, we determined the structure of Rns, a member of the AraC/XylS family that regulates the expression of pili and other virulence factors in ETEC. The structure revealed decanoic acid bound between the N- and C-terminal domains. To test the hypothesis that bound decanoic acid directly inhibits Rns, we used its structure to identify residues that are necessary for ligand binding. Removal of the positive side chains of R75 and H20 rendered Rns insensitive to fatty acid inhibition. Additionally, mutations designed to occlude decanoic acid binding also produced a variant that was fatty acid insensitive. We also observed that this variant is structurally more flexible than wildtype Rns with decanoic acid; suggesting that bound fatty acid contributes to structural rigidity. These studies precisely demonstrate Rns binding pocket residues critical for binding fatty acids and inhibition of DNA binding. This supports our hypothesis that fatty acids must bind in the binding pocket to inhibit AraC regulators. Further work by us and others suggests inhibition of AraC virulence regulators by fatty acids is a common paradigm among many bacterial pathogens. Therefore, understanding the molecular basis of this inhibition lays the groundwork for the development of small molecule therapeutics targeting enteric disease.

microbiology↗

bioRxiv2020

The structure of Rns from enterotoxigenic Escherichia coli reveals a small molecule inhibitor

Enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrheal disease worldwide and a frequent cause of travelers diarrhea. In addition to the production of enterotoxins, studies with human volunteers established ETEC virulence is dependent upon the production of proteinaceous adhesive pili for attaching to the intestinal wall. Although pilins are highly immunogenic, vaccines incorporating them have yet to be proven efficacious. An additional challenge for vaccines is the heterogeneity of ETEC pili, as 20 different pilus types have been identified. However, the expression of a significant number of pilus types is dependent upon Rns, an AraC family transcription factor. Furthermore, Rns also regulates the expression of the virulence factor CexE, an outer membrane coat protein. To determine how Rns functions and is regulated we solved its structure by X-ray crystallography to 3 [A] resolution. Rns forms a dimer via its N-terminal domain and its structure is consistent with the dimer binding looped DNA. Our analyses also revealed a fatty acid, decanoic acid, bound within the Rns structure. Although Rns was not known to specifically bind small molecule ligands, biochemical analysis showed decanoic acid specifically stabilized Rns in a dose dependent manner. Lac reporter assays further showed that decanoic acid inhibits Rns function at both activated and repressed promoters. In situ, exogenous decanoic acid inhibited the expression of Rns-dependent CFA/I pili and CexE in different ETEC strains. Thus, our study reveals for the first time a naturally occurring small molecule ligand specifically inhibits Rns activity and potently suppresses the expression of ETEC virulence factors. Our findings provide an alternative approach to vaccines for inhibiting ETEC pathogenesis by using the Rns structure as a framework for rational drug design.

biochemistry↗