Network


Latest external collaboration on country level. Dive into details by clicking on the dots.

Hotspot


Dive into the research topics where Robert G. Stafford is active.

Publication


Featured researches published by Robert G. Stafford.


Applied and Environmental Microbiology | 2003

Fluorigenic substrates for the protease activities of botulinum neurotoxins, serotypes A, B, and F

James J. Schmidt; Robert G. Stafford

ABSTRACT The seven botulinum neurotoxins (BoNTs) are zinc metalloproteases that cleave neuronal proteins involved in neurotransmitter release and are among the most toxic natural products known. High-throughput BoNT assays are needed for use in antibotulinum drug discovery and to characterize BoNT protease activities. Compared to other proteases, BoNTs exhibit unusually stringent substrate requirements with respect to amino acid sequences and polypeptide lengths. Nonetheless, we have devised a strategy for development of fluorigenic BoNT protease assays, based on earlier structure-function studies, that has proven successful for three of the seven serotypes: A, B, and F. In synthetic peptide substrates, the P1 and P3′ residues were substituted with 2,4-dinitrophenyl-lysine and S-(N-[4-methyl-7-dimethylamino-coumarin-3-yl]-carboxamidomethyl)-cysteine, respectively. By monitoring the BoNT-catalyzed increase in fluorescence over time, initial hydrolysis rates could be obtained in 1 to 2 min when BoNT concentrations were 60 ng/ml (about 1 nM) or higher. Each BoNT cleaved its fluorigenic substrate at the same location as in the neuronal target protein, and kinetic constants indicated that the substrates were selective and efficient. The fluorigenic assay for BoNT B was used to characterize a new competitive inhibitor of BoNT B protease activity with a Ki value of 4 μM. In addition to real-time activity measurements, toxin concentration determinations, and kinetic studies, the BoNT substrates described herein may be directly incorporated into automated high-throughput assay systems to screen large numbers of compounds for potential antibotulinum drugs.


Journal of Biological Chemistry | 2007

Inhibition of Metalloprotease Botulinum Serotype A from a Pseudo-peptide Binding Mode to a Small Molecule That Is Active in Primary Neurons

James C. Burnett; Gordon Ruthel; Christian M. Stegmann; Rekha G. Panchal; Tam Luong Nguyen; Ann R. Hermone; Robert G. Stafford; Douglas Lane; Tara Kenny; Connor F. McGrath; Peter Wipf; Andrea M. Stahl; James J. Schmidt; Rick Gussio; Axel T. Brunger; Sina Bavari

An efficient research strategy integrating empirically guided, structure-based modeling and chemoinformatics was used to discover potent small molecule inhibitors of the botulinum neurotoxin serotype A light chain. First, a modeled binding mode for inhibitor 2-mercapto-3-phenylpropionyl-RATKML (Ki = 330 nm) was generated, and required the use of a molecular dynamic conformer of the enzyme displaying the reorientation of surface loops bordering the substrate binding cleft. These flexible loops are conformationally variable in x-ray crystal structures, and the model predicted that they were pivotal for providing complementary binding surfaces and solvent shielding for the pseudo-peptide. The docked conformation of 2-mercapto-3-phenylpropionyl-RATKML was then used to refine our pharmacophore for botulinum serotype A light chain inhibition. Data base search queries derived from the pharmacophore were employed to mine small molecule (non-peptidic) inhibitors from the National Cancer Institutes Open Repository. Four of the inhibitors possess Ki values ranging from 3.0 to 10.0 μm. Of these, NSC 240898 is a promising lead for therapeutic development, as it readily enters neurons, exhibits no neuronal toxicity, and elicits dose-dependent protection of synaptosomal-associated protein (of 25 kDa) in a primary culture of embryonic chicken neurons. Isothermal titration calorimetry showed that the interaction between NSC 240898 and the botulinum A light chain is largely entropy-driven, and occurs with a 1:1 stoichiometry and a dissociation constant of 4.6 μm.


FEBS Letters | 1998

Type A botulinum neurotoxin proteolytic activity: development of competitive inhibitors and implications for substrate specificity at the S1' binding subsite.

James J. Schmidt; Robert G. Stafford; Karen A. Bostian

Type A botulinum neurotoxin (botox A) is a zinc metalloprotease that cleaves only one peptide bond in the synaptosomal protein, SNAP‐25. Single‐residue changes in a 17‐residue substrate peptide were used to develop the first specific, competitive inhibitors of its proteolytic activity. Substrate analog peptides with P4, P3, P2′ or P3′ cysteine were readily hydrolyzed by the toxin, but those with P1 or P2 cysteine were not cleaved and were inhibitors. Peptides with either d‐ or l‐cysteine as the N‐terminus, followed by the last six residues of the substrate, were the most effective inhibitors, each with a K i value of 2 μM. Elimination of the cysteine sulfhydryl group yielded much less effective inhibitors, suggesting that inhibition was primarily due to binding of the active‐site zinc by the sulfhydryl group. Botox A displayed an unusual requirement for arginine as the P1′ inhibitor residue, demonstrating that the S1′ binding subsite of botox A is dissimilar to those of most other zinc metalloproteases. This characteristic is an important element in shaping the substrate specificity of botox A.


FEBS Letters | 2002

A high-affinity competitive inhibitor of type A botulinum neurotoxin protease activity

James J. Schmidt; Robert G. Stafford

The peptide N‐acetyl‐CRATKML‐amide is an effective inhibitor of type A botulinum neurotoxin (BoNT A) protease activity [Schmidt et al., FEBS Lett. 435 (1998) 61–64]. To improve inhibitor binding, the peptide was modified by replacing cysteine with other sulfhydryl‐containing compounds. Ten peptides were synthesized. One peptide adapted the structure of captopril to the binding requirements of BoNT A, but it was a weak inhibitor, suggesting that angiotensin‐converting enzyme is not a good model for BoNT A inhibitor development. However, replacing cysteine with 2‐mercapto‐3‐phenylpropionyl yielded a peptide with K i of 330 nM, the best inhibitor of BoNT A protease activity reported to date. Additional modifications of the inhibitor revealed structural elements important for binding and supported our earlier findings that, with the exception of P1′ arginine, subsites on BoNT A are not highly specific for particular amino acid side chains.


Nature Structural & Molecular Biology | 2009

Mode of VAMP substrate recognition and inhibition of Clostridium botulinum neurotoxin F

Rakhi Agarwal; James J. Schmidt; Robert G. Stafford; Subramanyam Swaminathan

Clostridium botulinum neurotoxins (BoNTs) cleave neuronal proteins responsible for neurotransmitter release, causing the neuroparalytic disease botulism. BoNT serotypes B, D, F and G cleave and inactivate vesicle-associated membrane protein (VAMP), each at a unique peptide bond. The specificity of BoNTs depends on the mode of substrate recognition. We have investigated the mechanism of substrate recognition of BoNT F by determining the crystal structures of its complex with two substrate-based inhibitors, VAMP 22-58/Gln58D-cysteine and 27-58/Gln58D-cysteine. The inhibitors bind to BoNT F in the canonical direction (as seen for BoNTs A and E substrates) but are positioned specifically via three major exosites away from the active site. The cysteine sulfur of the inhibitors interacts with the zinc and exists as sulfinic acid in the inhibitor VAMP 27-58/Gln58D-cysteine. Arg133 and Arg171, which form part of two separate exosites, are crucial for substrate binding and catalysis.


Toxicon | 1995

Urinary elimination of saxitoxin after intravenous injection

Robert G. Stafford; Harry B. Hines

Paralytic shellfish poisoning is a serious public health concern throughout the world. An analytical method with diagnostic potential was used to isolate and measure saxitoxin, the most potent and studied paralytic shellfish poisoning toxin, in the urine of rats injected i.v. with sublethal doses (2 micrograms/kg) of saxitoxin. Urine was collected at intervals between 4 and 144 hr after injection. Saxitoxin was isolated from urine with an ion-exchange procedure, identified, and measured with a precolumn-oxidation-HPLC procedure coupled with fluorescence detection. The identity of oxidized saxitoxin was confirmed with electrospray ionization mass spectrometry. Four hours after injection, approximately 19% of the injected saxitoxin dose was excreted. By 24 hr, approximately 58% of the administered dose was excreted. Average total urinary excretion of administered saxitoxin was approximately 68% for the full study period. These results demonstrate that small quantities of unmetabolized saxitoxin can be detected in rat urine up to 144 hr after i.v. administration, and that the analytical method may have diagnostic potential for saxitoxin intoxication and paralytic shellfish poisoning.


Journal of Chromatography B: Biomedical Sciences and Applications | 1994

Method for the identification of saxitoxin in rat urine

Robert G. Stafford; Harry B. Hines

Saxitoxin (STX) is one of several related toxins that cause paralytic shellfish poisoning. We used solid-phase extraction (SPE) and prechromatographic oxidation/HPLC with fluorescence detection to isolate, identify, and quantify STX in rat urine. STX recovery from urine with the SPE procedure was approximately 76 +/- 6.5%. The standard curve was linear between 2 and 50 ng/ml. The lower limit of quantification with the method was 2 ng STX/ml of rat urine. Preliminary results with i.v. administration of STX to rats demonstrated that this method can detect and quantify STX in urine.


PLOS ONE | 2014

Cleavage of SNAP25 and its shorter versions by the protease domain of serotype A botulinum neurotoxin.

Rahman M. Mizanur; Robert G. Stafford; S. Ashraf Ahmed

Various substrates, catalysts, and assay methods are currently used to screen inhibitors for their effect on the proteolytic activity of botulinum neurotoxin. As a result, significant variation exists in the reported results. Recently, we found that one source of variation was the use of various catalysts, and have therefore evaluated its three forms. In this paper, we characterize three substrates under near uniform reaction conditions using the most active catalytic form of the toxin. Bovine serum albumin at varying optimum concentrations stimulated enzymatic activity with all three substrates. Sodium chloride had a stimulating effect on the full length synaptosomal-associated protein of 25 kDa (SNAP25) and its 66-mer substrates but had an inhibitory effect on the 17-mer substrate. We found that under optimum conditions, full length SNAP25 was a better substrate than its shorter 66-mer or 17-mer forms both in terms of kcat, Km, and catalytic efficiency kcat/Km. Assay times greater than 15 min introduced large variations and significantly reduced the catalytic efficiency. In addition to characterizing the three substrates, our results identify potential sources of variations in previous published results, and underscore the importance of using well-defined reaction components and assay conditions.


Toxicon | 2015

Ex vivo inhibition of Clostridium botulinum neurotoxin types B, C, E, and F by small molecular weight inhibitors

Vicki A. Montgomery; S. Ashraf Ahmed; Mark A. Olson; Rahman M. Mizanur; Robert G. Stafford; Virginia Roxas-Duncan; Leonard A. Smith

Two small molecular weight inhibitors, compounds CB7969312 and CB7967495, that displayed inhibition of botulinum neurotoxin serotype A in a previous study, were evaluated for inhibition of botulinum neurotoxin serotypes B, C, E, and F. The small molecular weight inhibitors were assessed by molecular modeling, UPLC-based peptide cleavage assay; and an ex vivo assay, the mouse phrenic nerve - hemidiaphragm assay (MPNHDA). While both compounds were inhibitors of botulinum neurotoxin (BoNT) serotypes B, C, and F in the MPNHDA, compound CB7969312 was effective at lower molar concentrations than compound CB7967495. However, compound CB7967495 was significantly more effective at preventing BoNTE intoxication than compound CB7969312. In the UPLC-based peptide cleavage assay, CB7969312 was also more effective against LcC. Both compounds inhibited BoNTE, but not BoNTF, LcE, or LcF in the UPLC-based peptide cleavage assay. Molecular modeling studies predicted that both compounds would be effective inhibitors of BoNTs B, C, E, and F. But CB7967495 was predicted to be a more effective inhibitor of the four serotypes (B, C, E, and F) than CB7969312. This is the first report of a small molecular weight compound that inhibits serotypes B, C, E, and F in the ex vivo assay.


Scientific Reports | 2018

Bioengineering of bacterial pathogens for noninvasive imaging and in vivo evaluation of therapeutics

Sathish Rajamani; Kyle Kuszpit; Jennifer M. Scarff; Linnea Lundh; Maisha Khan; Jennifer Brown; Robert G. Stafford; Lisa H. Cazares; Rekha G. Panchal; Thomas Bocan

Critical bacterial pathogens of public health and biodefense concerns were engineered to constitutively express Escherichia coli enzyme thymidine kinase (TK) that allows for noninvasive nuclear imaging via phosphorylation and entrapment of radiolabeled nucleoside analog 1-(2′deoxy-2′-fluoro-β-D-arabinofuranosyl)-5-iodouracil (FIAU). Expression of functional TK was established using a nucleoside analog Zidovudine that impeded the growth of tk-engineered bacteria. Significantly, no observable growth differences were detected for FIAU. High resolution mass spectrometry with Pseudomonas aeruginosa PAO1 and its tk variant (PAO1TK) confirmed FIAU phosphorylation and retention only in PAO1TK. In vitro gamma counting with wild-type PAO1, Acinetobacter baumannii and Burkholderia pseudomallei Bp82 and their tk derivatives with [18F]FIAU further confirmed that tk variants selectively incorporated the radiotracer, albeit with varying efficiencies. In vitro [18F]FIAU labeling coupled with in vivo Positron Emission Tomography/Computed Tomography (PET/CT) imaging of PAO1 and PAO1TK confirmed that only PAO1TK can be imaged in mice at sensitivities ≥107 bacteria per infection site. This was further verified by administering [18F]FIAU to animals infected with PAO1 and PAO1TK. Utility of tk-engineered P. aeruginosa in noninvasive PET/CT imaging for bacterial therapeutic evaluation in animals was demonstrated employing antibiotic ciprofloxacin, underscoring the immediate use of PAO1TK and potentially other engineered pathogens for evaluating experimental therapeutics.

Collaboration


Dive into the Robert G. Stafford's collaboration.

Top Co-Authors

Avatar

James J. Schmidt

United States Army Medical Research Institute of Infectious Diseases

View shared research outputs
Top Co-Authors

Avatar

Rick Gussio

National Institutes of Health

View shared research outputs
Top Co-Authors

Avatar

Sina Bavari

United States Army Medical Research Institute of Infectious Diseases

View shared research outputs
Top Co-Authors

Avatar
Top Co-Authors

Avatar

Rekha G. Panchal

United States Army Medical Research Institute of Infectious Diseases

View shared research outputs
Top Co-Authors

Avatar

Andrea M. Stahl

United States Army Medical Research Institute of Infectious Diseases

View shared research outputs
Top Co-Authors

Avatar

Ann R. Hermone

Science Applications International Corporation

View shared research outputs
Top Co-Authors

Avatar

Connor F. McGrath

Science Applications International Corporation

View shared research outputs
Top Co-Authors

Avatar

Douglas Lane

Science Applications International Corporation

View shared research outputs
Top Co-Authors

Avatar

James C. Burnett

Science Applications International Corporation

View shared research outputs
Researchain Logo
Decentralizing Knowledge