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Biomedical subjects

C S Cassidy

Publications and source records attributed to C S Cassidy.

9 recordsLinked to original sources
PubMed2001

Biflavonoids as novel antituberculosis agents.

A series of naturally occurring and synthetic biflavonoids was evaluated for inhibitory activity against Mycobacterium tuberculosis H37Rv (Mtb). Compounds 6, 24, and 25 demonstrated 96, 95, and 87% inhibition, respectively, at a screening concentration of 12.5 microg/mL. The type of linkage and the presence of methoxy- and nitro-substituents in biflavonoids may contribute to the observed inhibitory activity. The results of this study represent the discovery of biflavonoids as a potential new class of antituberculosis agent.

Antitubercular Agents↗
PubMed2001

Enzymes from extremophiles.

The industrial application of enzymes that can withstand harsh conditions has greatly increased over the past decade. This is mainly a result of the discovery of novel enzymes from extremophilic microorganisms. Recent advances in the study of extremozymes point to the acceleration of this trend. In particular, enzymes from thermophilic organisms have found the most practical commercial use to date because of their overall inherent stability. This has also led to a greater understanding of stability factors involved in adaptation of these enzymes to their unusual environments.

Amino Acid Substitution↗
PubMed2001

Amyloid fibril formation in microwell plates for screening of inhibitors.

Fibril formation is the basis of amyloid production in a number of disease states, such as Alzheimer's disease, diabetes and immunocytic dyscrasias. Compounds that inhibit fibril formation could be directly relevant to the treatment of amyloid diseases, and may also provide a foundation for the development of interventions in other molecular condensation diseases ranging from sickle cell anemia to atherosclerosis. We developed an economical and convenient high-throughput method for screening compounds against fibril formation in microwell plates. Chalcones, flavonoids and biflavonoids were screened against fibril formation by a recombinant antibody variable domain (V1). Chalcones 6 and 14 were found to demonstrate inhibition at 0.1 microM in 79 microM of protein solution in both test tube and microwell plate assays. The concentration of protein in the microwell plate assay could be as low as 5 microM using ThT as a monitoring agent. Molecular modeling studies indicated that both compounds could be individually docked into a binding site at the monomer-monomer interface of the V(L) protein dimer. These studies suggested that these compounds could potentially stabilize the VL dimer and therefore reduce its tendency to form fibrils. These findings may provide the basis for a new therapeutic approach to prevent or treat amyloid diseases.

Amyloid beta-Peptides↗
PubMed2000

The deuterium isotope effect on the NMR signal of the low-barrier hydrogen bond in a transition-state analog complex of chymotrypsin.

The participation of a low-barrier hydrogen bond (LBHB) in the mechanism of action of chymotrypsin introduces a new role for Asp 102 and His 57 in catalysis [C. S. Cassidy, J. Lin, and P. A. Frey (1997) Biochemistry 36, 4576-4584]. It is postulated that the LBHB increases the basicity of His 57-N(epsilon2) in the transition state, thereby facilitating the abstraction of a proton from Ser 195, and stabilizes the tetrahedral intermediate in the acylation step. Evidence for this mechanism includes the downfield chemical shift of the proton bridging His 57 and Asp 102 in transition-state analog complexes and the low deuterium fractionation factors for this proton in the same complexes. We present additional spectroscopic evidence supporting the assignment of an LBHB between His 57 and Asp 102. The tetrahedral addition complex between Ser 195 of chymotrypsin and N-acetyl-l-leucyl-l-phenylalanyl trifluoromethylketone is regarded as a close structural analog of a tetrahedral intermediate. The deuterium NMR signal for the downfield deuteron bridging His 57 and Asp 102 in D(2)O has now been observed as a broad band centered at 17.8 +/- 0.5 ppm. The proton NMR signal in H(2)O is centered at 18.9 +/- 0.05 ppm. The two signals are clearly separated corresponding to a deuterium isotope effect of Delta[delta(H) - delta(D)] = 1.1 +/- 0.5 ppm. Deuterium isotope effects in this range are characteristic of LBHBs, and this observation provides further support for the assignment of the proton bridging His 57 and Asp 102 in transition-state analog complexes as an LBHB.

Binding Sites↗
PubMed1998

Correlations of the basicity of His 57 with transition state analogue binding, substrate reactivity, and the strength of the low-barrier hydrogen bond in chymotrypsin.

The basicity of His 57-Nepsilon2 within the low-barrier hydrogen-bonded (LBHB) diad His 57-Asp 102 and the 1H NMR chemical shift of the LBHB proton in tetrahedral, hemiketal complexes of chymotrypsin with peptidyl trifluoromethyl ketones (peptidyl-TFKs) have been studied. The following results were obtained with various peptidyl-TFKs at 5 degrees C: N-Ac-Gly-DL-Phe-CF3, pKa = 11.1 and deltaLBHB = 18.7 ppm; N-Ac-L-Val-DL-Phe-CF3, pKa = 11.8 and deltaLBHB = 18.9 ppm; N-Ac-L-Leu-DL-Val-CF3, pKa = 10.3 and deltaLBHB = 18.9 ppm; and N-Ac-L-Leu-DL-naphthyl-CF3, pKa = 10.9 and deltaLBHB = 19.0 ppm. Results for peptidyl-TFKs with Phe in the P1 position and N-Ac, N-Ac-Gly, N-Ac-L-Val, and N-Ac-L-Leu in the P2 position were well correlated with literature values for inhibition constants Ki and kcat/Km for the corresponding peptidyl methyl esters. The plot of log Ki versus the apparent pKa of His 57-Nepsilon2 displayed a slope of -0.77, and that of log kcat/Km for peptidyl methyl esters versus the pKa of His 57-Nepsilon2 in corresponding peptidyl-TFK complexes gave a slope of 0.68. The slope of a plot of pKa versus deltaLBHB was 3.7, and that of log kcat/Km for peptidyl methyl ester substrates versus deltaLBHB for the corresponding peptidyl-TFK-chymotrypsin complexes was 2.7. A plot of log Ki versus deltaLBHB displayed a slope of -3.0. These plots indicated that the pKa of His 57 and substrate reactivity were correlated with increasing strength of the low-barrier hydrogen bond. The apparent pKa of His 57-Nepsilon2 for the chymotrypsin-N-Ac-L-Leu-DL-Phe-CF3 complex is 10.6 at 25 degrees C, whereas it is 12.0 at 5 degrees C [Cassidy, C. S., Lin, J. L., and Frey, P. A. (1997) Biochemistry 36, 4576-4584]. The apparent discrepancy is likely to be due to a temperature dependence in the cooperative ionization of His 57 in peptidyl-TFK complexes, which appears to be coupled to inhibitor dissociation, hydration and ionization of free peptidyl-TFK, ionization of Ile 16, and a conformational change.

Animals↗
PubMed1997

A new concept for the mechanism of action of chymotrypsin: the role of the low-barrier hydrogen bond.

The basicities of the diad H57-D102 at N(epsilon)2 in the tetrahedral complexes of chymotrypsin with the peptidyl trifluoromethyl ketones (TFK) N-acetyl-L-Leu-DL-Phe-CF3 and N-acetyl-DL-Phe-CF3 have been studied by 1H-NMR. The protons bridging His 57 and Asp 102 in these complexes are engaged in low-barrier hydrogen bonds (LBHBs). In 1H-NMR spectra at pH 7.0, these protons appear at delta 18.9 and 18.6 ppm, and the pK(a)s of the diads are 12.0 +/- 0.2 and 10.8 +/- 0.1, respectively. The difference indicates that removal of leucine from the second aminoacyl site S2 of chymotrypsin weakens the LBHB and decreases the basicity of the H57-D102 diad relative to the case in which S2 is occupied by leucine. Consideration of the available structural data on chymotrypsin and other serine proteases, together with the high pK(a)s of the hemiketals formed with TFKs, suggests that LBHB formation in catalysis arises through a substrate-induced conformational transition leading to steric compression between His 57 and Asp 102. Because the N-O distance in the LBHB is shorter than the Van der Waals contact distance, the LBHB is proposed to stabilize the tetrahedral intermediate through relief of steric strain between these residues. In this mechanism, substrate-induced steric compression within the diad increases the basicity of N(epsilon)2 in His 57, making it a more effective base for abstracting a proton from Ser 195 in the formation of the tetrahedral intermediate. The values of pK(a) for N(epsilon)2 in TFK adducts lie between those of Ser 195 (pK(a) approximately 14) and the leaving group in tetrahedral adducts (pK(a) approximately 9), making N(epsilon)2 of the H57-D102 diad strong enough as a base to abstract the proton from Ser 195 in tetrahedral adduct formation but not so strong that its conjugate acid cannot protonate the leaving group. According to this theory, the "normal" pK(a) of His 57 in free chymotrypsin arises from the use of part of the stabilization energy provided by the LBHB to drive the conformational compression required for its formation. In catalysis, the energy for conformational compression is supplied by the binding of remote portions of the substrate, including the side chains of P1 and P2.

Animals↗
PubMed1995

Low-barrier hydrogen bonding in molecular complexes analogous to histidine and aspartate in the catalytic triad of serine proteases.

We present spectroscopic evidence for the presence of low-barrier hydrogen bonds (LBHBs) in molecular complexes composed of carboxylic acids and 1-methylimidazole (1-MeIm) dissolved in aprotic organic solvents. A plot of the values of the low-field proton NMR chemical shifts versus the aqueous pKa of the carboxylic acid exhibits a positive slope for pKa values below 2.1 and a negative slope for higher pKa values. The chemical shifts for protons near the maximum in this plot are 18 ppm, similar to that of 18.3 ppm for His57-Asp102 in the protonated catalytic triad of chymotrypsin. The chemical shifts for the proton bonded to C2 of 1-MeIm in these complexes also vary with the pKa of the carboxylic acid and reveal a gradual change from neutral, hydrogen-bonded 1-MeIm in complexes of weaker acids to hydrogen-bonded 1-methylimidazolium ion in complexes of stronger acids. The midpoint chemical shift for the C2 proton corresponds to a carboxylic aqueous pKa of about 2.1. FTIR spectra of the 1-MeIm-carboxylic acid complexes in CHCl3 indicate that hydrogen bonding is strong and that the complexes are of three types: (a) neutral complexes with the weaker acids (pKa > or = 2.2) in which the antisymmetric carbonyl stretching frequencies are lowered relative to the free acids and the ethyl esters of the same acids; (b) ionic complexes of stronger acids (pKa < or = 2.1) in which the carbonyl stretching frequencies are slightly lower than those for the tetrabutylammonium salts of the same acids; (c) ionic complexes of the same acids (pKa < or = 2.1) coexisting with type b, in which the carbonyl stretching frequencies are intermediate between those for the tetrabutylammonium salts (bond order 1.5) and those of the same acids or their esters (bond order 2.0). The latter complexes appear to incorporate a low-barrier hydrogen bond and are presented as models for the protonated triad of chymotrypsin and other serine proteases. These enzymes have been postulated to utilize a low-barrier hydrogen bond between His57 and Asp102 to facilitate the abstraction of the beta-OH proton from Ser195 in the course of catalysis [Frey, P.A., Whitt, S.A., & Tobin, J.B. (1994) Science (Washington, D.C.)264,1927-1930].

Aspartic Acid↗
PubMedDate not supplied

New structural and chemical insight into the catalytic mechanism of epoxide hydrolases.

New structural and mechanistic data on epoxide hydrolases provide additional insight into the details of how these enzymes function. The data reveal that, in addition to the catalytic triad located in the core domain that carries out the hydrolytic reaction, there are two tyrosine residues, located in the cap domain, which assist in the initial alkylation event.

Binding, Competitive↗