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Petr Kuzmic

Publications and source records attributed to Petr Kuzmic.

10 recordsLinked to original sources
PubMed2006

Self-assembled small-molecule microarrays for protease screening and profiling.

Small-molecule microarrays are attractive for chemical biology as they permit the analysis of hundreds to thousands of interactions in a highly miniaturized format. Methods to prepare small-molecule microarrays from combinatorial libraries by a self-assembly process based on the sequence-specific hybridization of peptide nucleic acid (PNA) encoded libraries to oligonucleotide arrays are presented. A systematic study of the dynamic range for multiple detection agents, including direct fluorescence of attached fluorescein and cyanine-3 dyes, antibody-mediated fluorescence amplification, and biotin-gold nanoparticle detection, demonstrated that individual PNA-encoded probes can be detected to concentrations of 10 pM on the oligonucleotide microarrays. Furthermore, a new method for parallel processing of biological samples by using gel-based separation of probes is presented. The methods presented in this report are exemplified through profiling two closely related cysteine proteases, cathepsin K and cathepsin F, across a 625-member PNA-encoded tetrapeptide acrylate library. A series of the specific cathepsin K and F inhibitors identified from the library were kinetically characterized and shown to correlate with the observed microarray profile, thus validating the described methods. Importantly, it was shown that this method could be used to obtain orthogonal inhibitors that displayed greater than tenfold selectivity for these closely related cathepsins.

Endopeptidases↗
PubMed2006

Mixed-type noncompetitive inhibition of anthrax lethal factor protease by aminoglycosides.

We report a detailed kinetic investigation of the aminoglycosides neomycin B and neamine as inhibitors of the lethal factor protease from Bacillus anthracis. Both inhibitors display a mixed-type, noncompetitive kinetic pattern, which suggests the existence of multiple enzyme-inhibitor binding sites or the involvement of multiple structural binding modes at the same site. Quantitative analysis of the ionic strength effects by using the Debye-Hückel model revealed that the average interionic distance at the point of enzyme-inhibitor attachment is likely to be extremely short, which suggests specific, rather than nonspecific, binding. Only one ion pair seems to be involved in the binding process, which suggests the presence of a single binding site. Combining the results of our substrate competition studies with the ionic strength effects on the apparent inhibition constant, we propose that aminoglycoside inhibitors, such as neomycin B, bind to the lethal factor protease from B. anthracis in two different structural orientations. These results have important implications for the rational design of lethal factor protease inhibitors as possible therapeutic agents against anthrax. The strategies and methods we describe are general and can be employed to investigate in depth the mechanism of inhibition by other bioactive compounds.

Aminoglycosides↗
PubMed2005

A generalized numerical approach to rapid-equilibrium enzyme kinetics: application to 17beta-HSD.

A generalized numerical treatment of rapid-equilibrium enzyme kinetics is presented. This new approach relies on automatic computer derivation of the underlying mathematical model (a system of simultaneous nonlinear algebraic equations) from a symbolic representation of the reaction mechanism (a system of biochemical equations) provided by the researcher. The method allows experimental biochemists to analyze initial-rate enzyme kinetic data without having to use any mathematical equations. An illustrative example is based on the inhibition kinetics of 17beta-hydroxysteroid dehydrogenase type 5 by a class of natural compounds. A computer implementation of the new method, a newly modified software package DYNAFIT [Kuzmic, P., 1996. Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase. Anal. Biochem. 237, 260-273], is freely available to all academic researchers.

17-Hydroxysteroid Dehydrogenases↗
PubMed2004

Molecular characterization of Ancylostoma ceylanicum Kunitz-type serine protease inhibitor: evidence for a role in hookworm-associated growth delay.

Hookworm infection is a major cause of iron deficiency anemia and malnutrition in developing countries. The Ancylostoma ceylanicum Kunitz-type inhibitor (AceKI) is a 7.9-kDa broad-spectrum inhibitor of trypsin, chymotrypsin, and pancreatic elastase that has previously been isolated from adult hookworms. Site-directed mutagenesis of the predicted P1 inhibitory reactive site amino acid confirmed the role of Met(26) in mediating inhibition of the three target serine proteases. By using reverse transcription-PCR, it was demonstrated that the level of AceKI gene expression increased following activation of third-stage larvae with serum and that the highest level of expression was reached in the adult stage of the parasite. Immunohistochemistry studies performed with polyclonal immunoglobulin G raised against recombinant AceKI showed that the inhibitor localized to the subcuticle of the adult hookworm, suggesting that it has a potential in vivo role in neutralizing intestinal proteases at the surface of the parasite. Immunization with recombinant AceKI was shown to confer partial protection against hookworm-associated growth delay without a measurable effect on anemia. Taken together, the data suggest that AceKI plays a role in the pathogenesis of hookworm-associated malnutrition and growth delay, perhaps through inhibition of nutrient absorption in infected hosts.

Amino Acid Sequence↗
PubMed2003

Mechanism of loading the Escherichia coli DNA polymerase III sliding clamp: I. Two distinct activities for individual ATP sites in the gamma complex.

The Escherichia coli DNA polymerase III gamma complex loads the beta clamp onto DNA, and the clamp tethers the core polymerase to DNA to increase the processivity of synthesis. ATP binding and hydrolysis promote conformational changes within the gamma complex that modulate its affinity for the clamp and DNA, allowing it to accomplish the mechanical task of assembling clamps on DNA. This is the first of two reports (Snyder, A. K., Williams, C. R., Johnson, A., O'Donnell, M., and Bloom, L. B. (2004) J. Biol. Chem. 279, 4386-4393) addressing the question of how ATP binding and hydrolysis modulate specific interactions with DNA and beta. Pre-steady-state rates of ATP hydrolysis were slower when reactions were initiated by addition of ATP than when the gamma complex was equilibrated with ATP and were limited by the rate of an intramolecular reaction, possibly ATP-induced conformational changes. Kinetic modeling of assays in which the gamma complex was incubated with ATP for different periods of time prior to adding DNA to trigger hydrolysis suggests a mechanism in which a relatively slow conformational change step (kforward = 6.5 s(-1)) produces a species of the gamma complex that is activated for DNA (and beta) binding. In the absence of beta, 2 of the 3 molecules of ATP are hydrolyzed rapidly prior to releasing DNA, and the 3rd molecule is hydrolyzed slowly. In the presence of beta, all 3 molecules of ATP are hydrolyzed rapidly. These results suggest that hydrolysis of 2 molecules of ATP may be coupled to conformational changes that reduce interactions with DNA, whereas hydrolysis of the 3rd is coupled to changes that result in release of beta.

Adenosine Triphosphate↗
PubMed2003

Kinetic determination of tight-binding impurities in enzyme inhibitors.

A novel rate equation to characterize the dose-response behavior of a moderately potent ("classical") enzyme inhibitor contaminated with a very potent ("tight-binding") impurity is derived. Mathematical properties of the new rate equation show that, for such contaminated materials, experimentally observed I(50) values are ambiguous. The four-parameter logistic equation, conventionally used to determine I(50) values, cannot be used to detect the presence of tight-binding impurities in inhibitor samples. In contrast, fitting the newly derived rate equation to inhibitor dose- response curves can, in favorable cases, reveal whether the unknown material is chemically homogeneous or whether it is contaminated with a tight-binding impurity. The limitations of our method with respect to the detectable range of inhibition constants (both classical and tight-binding) were examined by using Monte-Carlo simulations. To test the new analytical procedure experimentally, we added a small amount (0.02 mole%) of a tight-binding impurity (K(i)=0.065 nM) to an otherwise weak inhibitor of human mast-cell tryptase (K(i)=50.4 microM). The resulting material was treated as "unknown." Our kinetic equation predicts that such adulterated material should show I(50)=0.40 microM, which was identical to the experimentally observed value. The best-fit value of the apparent inhibition constants for the tight-binding inhibitor was K(i)=(0.107+/-0.035)nM, close to the true value of 0.065 nM.

Binding, Competitive↗
PubMed2003

Effect of ribavirin and amantadine on early hepatitis C virus RNA rebound and clearance in serum during daily high-dose interferon.

The early rebound in serum HCV RNA during HCV dynamic studies with high-dose interferon may be due to de novo infection with interferon escape quasispecies. We simultaneously measured serum alanine aminotransferase (ALT) and HCV RNA at rapid intervals in chronic HCV liver disease patients during interferon therapy alone or in combination with ribavirin and amantadine. HCV RNA declined rapidly between 0 and 48 hr in all patients (phase 1). Ribavirin and amantadine significantly increased this phase 1 decline. In all four monotherapy patients with viral rebound, the increasing levels of HCV RNA were associated with a parallel increase in serum ALT, consistent with a hepatitis flare or de novo infection. By contrast, in the four monotherapy patients without viral rebound, and all eight patients receiving combination therapy, the slow progressive phase two decay was associated with declining serum ALT levels. Ribavirin or ribavirin and amantadine significantly and incrementally increased the phase two HCV RNA clearance. Dynamic sequencing in the HVR1 region in one rebound patient confirmed the potential for rapid evolutionary changes during interferon therapy. These preliminary data suggest that early viral rebound might be associated with de novo infection with interferon escape HCV variants, which in turn are attenuated by ribavirin and amantadine.

Adult↗
PubMed2002

A detailed physical map of the 6p reading disability locus, including new markers and confirmation of recombination suppression.

Loci for several complex disorders have been genetically linked to markers located telomeric of the HLA class I region of the major histocompatibility complex on 6p21.3-22. However, this same region has been characterized by a large interval of recombination suppression with the potential to greatly complicate precise localization of these risk loci. Furthermore, a paucity of markers and physical mapping data has confounded precise localization of the boundaries of linkage and recombination suppression. In order to create a more detailed marker map of this region and define these boundaries we generated a minimal tiling pathway of BACs, PACs, and cosmids and a multiplexed panel of 29 short tandem repeat markers spanning 10 Mb. In addition to providing precise marker order and distances, the pathway and marker panel frame an inversion of recombination frequency that has distorted the resolution of linkage studies for 6p loci such as reading disability and others, and that should be accounted for in the design of future studies.

Base Sequence↗
PubMed2001

Molecular characterization of Ancylostoma inhibitors of coagulation factor Xa. Hookworm anticoagulant activity in vitro predicts parasite bloodfeeding in vivo.

Bloodfeeding hookworms, which currently infect over a billion people in the developing world, are a leading cause of gastrointestinal hemorrhage and iron deficiency anemia. The major anticoagulant inhibitor of coagulation factor Xa has been identified from the hookworm parasite Ancylostoma ceylanicum using reverse transcription PCR and 3'-rapid amplification of cDNA ends. This is the first anticoagulant cloned from a hookworm species for which humans are recognized permissive hosts. Despite approximately 50% amino acid similarity, A. ceylanicum anticoagulant peptide 1 (AceAP1) is both immunologically and mechanistically distinct from AcAP5, its homologue isolated from the dog hookworm Ancylostoma caninum. Studies using plasma clotting times and single stage chromogenic assays of factor Xa activity have demonstrated that the recombinant AceAP1 protein is substantially less potent than AcAP5 and that soluble whole worm protein extracts of adult A. ceylanicum possess less anticoagulant activity than extracts of A. caninum. These values correlate with previously reported differences in bloodfeeding capabilities between these two species of hookworm, suggesting that factor Xa inhibitory activity is predictive of hookworm bloodfeeding capabilities in vivo. These fundamental differences in the mechanism of action and immunoreactivity of the major anticoagulant virulence factors from related Ancylostoma hookworm species may have significant implications for human vaccine development.

Amino Acid Sequence↗