Agency: Department of Health and Human Services. Branch: National Institutes of Health. Contract: 1R43AI Agency Tracking Number: AI Phase: Phase I. Program: SBIR. Solicitation Number: PHS Solicitation Year: When Peet had the time he and his wife Pat would often spend their leisure time ice skating, playing bridge, putting together jigsaw puzzles, and seeing an occasional movie when the budget would allow. However, during his grad school years there were some challenges. Peet recalls that he and his wife had to live on a tight budget while both were attending UNL.
Sound familiar? He stayed with this progression until the company name was Aventis, where he was Head of Medicinal Chemistry and Distinguished Scientist. Peet realized that his major responsibility had evolved into the management of mergers rather than science, so he left big pharma in to become a VP at ArQule, where he built an integrated Drug Discovery Group for the company. He built and staffed a small laboratory in Boston and a large laboratory in Bangalore, India. In , Peet became an International Consultant to the Pharmaceutical Industry and shortly after became the Director of Chemistry at Microbiotix in Worcester, MA, where he is presently one of the executives that manages this anti-infective company.
Peet is also very active in the scientific community with several professional affiliations, including the American Chemical Society and the Industrial Advisory Board for the Chemistry Department at UNL. Reactions were stopped by addition of acetic acid to 0.
Any compound that possessed intrinsic fluorescence was not analyzed further and was rejected to maintain screening efficiency.
Hit confirmations, IC 50 determinations, and kinetic analysis for inhibitor mechanism of action were carried out utilizing a well FRET assay microplate format. The reactions were stopped by the addition of acetic acid to 0. For kinetic analysis of compounds, substrate concentration was varied in the presence of increasing inhibitor concentrations, and the results were analyzed utilizing Eadie-Hofstee transformations Atkins and Nimmo, The extent of hydrolysis of the peptide substrate was determined by HPLC separation of the products from the substrate, followed by measurement of the peak areas Figure 2.
The upper lines represent the programmed and actual gradients and the lower line the absorption at nm of the HPLC effluent. Inactivation of the enzyme was achieved by addition of acetic acid to 0. The fluorescent signal of the cleaved substrate was measured at nm after excitation at nm.
The dissociated cell population was enhanced for neuronal cells by briefly plating to attach non-neuronal cells and then treating with a mixture of the mitotic inhibitor, 5-fluorodeoxyuridine As described previously Burnett et al. Likewise, N-Hydroxy-2,4-dichlorocinnanamide, a hydroxamate zinc non-chelator Eubanks et al. Compounds were classified as zinc chelators if they displayed a zinc concentration-dependent decrease in potency.
FRET-based assay results were confirmed in an HPLC-based assay using the same substrate and enzyme to ensure that compounds with intrinsic quenching capability did not interfere with the activity measurements. The most potent screening hits consisted of a variety of structural types, including, 8-hydroxyquinolines, benzimidazole acrylonitriles, arylamides, hydroxypyrazoles, vinylbenzothiazoles, and vinylbenzimidazoles Figure 3.
Two benzimidazole acrylonitrile compounds were identified as hits, and the most potent and selective one, MSL, was selected for further studies See Figure 4 for the IC 50 plot for MSL The benzimidazole acrylonitrile, MSL, was characterized further for specificity of inhibition and for zinc chelation potential. Roughly 50 compounds were synthesized manuscript in preparation and one, MBX Figure 5 , appeared to exhibit improved selectivity Table 2. MSL exhibited little protection in the assay at the concentrations tested.
MSL exhibited a noncompetitive mechanism of inhibition Figure 7A , as evidenced by a decrease in V max y intercept and no change in K m line slope with increasing inhibitor concentrations, thus yielding parallel lines. MBX also appeared to act by a noncompetitive mechanism since the Eadie-Hofstee plots revealed a series of parallel lines, indicating no change in the K m values and decreasing V max values with increasing inhibitor concentrations Figure 7B.
The results observed for both compounds indicate that excess peptide substrate cannot compete out the effects of inhibitor. Analyses of the data using both Lineweaver-Burk and Hanes-Woolf plots yielded the same results i.
Inhibitor enzyme kinetics — linear transformation using Eadie-Hofstee plots. The method employed was a well automated high-throughput screen, followed by confirmation and characterization utilizing a 96 well format. There are several sources for this variation. Finally, although strong quenchers were dropped from the screen, some inherent mild quenching activity for true hits may also be a source of variation. We focused on the benzimidazole acrylonitrile scaffold for further characterization, in particular compounds MSL and MBX , due to their novelty and a large body of synthetic chemistry knowledge of this particular class of compounds.
Kinetic studies indicate that mechanism of inhibition is of a noncompetitive nature. Shin et al. An accurate understanding of the actual toxicity of the compound must await the completion of appropriate animal toxicity studies. Chemical modification of this inhibitor scaffold yielded MBX , a benzimidazole acrylonitrile that not only exhibited improved enzyme specificity over that of its parent compound, but also demonstrated better potency in the chick neuronal cell assay.
However, the results cannot clearly distinguish between the possible mechanisms of inhibition in the cellular assay, i. In summary, we describe a novel benzimidazole scaffold that demonstrates notable specificity and a noncompetitive mechanism of action, both desirable characteristics when designing drugs to inactivate toxins.
The authors wish to acknowledge Donald T. Moir for extensive editorial assistance. At Microbiotix, he is participating in the small molecule inhibitor discovery efforts for BoNT Light Chain A and B and supporting in house Medicinal Chemistry efforts for inhibitor characterization. Michelle M. Butler received her Ph. She currently serves as a Senior Research Scientist at Microbiotix.
Gordon Ruthel received his Ph. He currently works as a consultant contracted by Akimeka Technologies to the U. Jonathan E. He currently works at the US Army Medical Research Institute for Infectious Diseases where he works on the development of small molecules to counteract botulinum neurotoxins and the development of broad-spectrum anti-viral compounds.
Laura M. Wanner Laura M. She currently serves as a research associate at the U. Army Medical Research Institute for Infectious Diseases working on botulinum neurotoxin countermeasures. Bing Li received his Ph. He currently serves as a Senior Research Scientist at Microbiotix. His research interest is focused on discovery and development of antiviral and antibacterial agents as well as development of small molecule inhibitors of botulinum neurotoxins.
Ramdas P. Pai currently works at Microbiotix Inc. Norton P. He currently is the Director of Chemistry at Microbiotix, Inc. The Chemistry Department at Microbiotix is designing new antiviral and antibacterial agents using structure-based drug design techniques with those projects where structural information is available.
Sina Bavari received his Ph. In addition, Dr. Bavari is a leading expert in bacterial toxins. Terry L. Bowlin received his Ph. He has published 85 peer reviewed papers and book chapters and has 14 awarded patents to date. Steven C. Cardinale, Microbiotix, Inc.
Butler, Microbiotix, Inc. Bing Li, Microbiotix, Inc. Ramdas Pai, Microbiotix, Inc. Peet, Microbiotix, Inc. Bowlin, Microbiotix, Inc. Read article at publisher's site DOI : J Med Chem , 63 19 , 18 Sep Sci Rep , 8 1 , 11 Jun Molecules , 16 1 , 30 Dec To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation.
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