Enzymes inhibitor detection

One approach to combating antibiotic resistance caused by P-lactamase is to inhibit the enzyme (see Enzyme inhibition). Effective combinations of enzyme inhibitors with P-lactam antibiotics such as penicillins or cephalosporins, result in a synergistic response, lowering the minimal inhibitory concentration (MIC) by a factor of four or more for each component. However, inhibition of P-lactamases alone is not sufficient. Pharmacokinetics, stability, ability to penetrate bacteria, cost, and other factors are also important in determining whether an inhibitor is suitable for therapeutic use. Almost any class of P-lactam is capable of producing P-lactamase inhibitors. Several reviews have been pubUshed on P-lactamase inhibitors, detection, and properties (8—15). 

Enzymes can be used not only for the determination of substrates but also for the analysis of enzyme inhibitors. In this type of sensors the response of the detectable species will decrease in the presence of the analyte. The inhibitor may affect the vmax or KM values. Competitive inhibitors, which bind to the same active site than the substrate, will increase the KM value, reflected by a change on the slope of the Lineweaver-Burke plot but will not change vmax. Non-competitive inhibitors, i.e. those that bind to another site of the protein, do not affect KM but produce a decrease in vmax. For instance, the acetylcholinesterase enzyme is inhibited by carbamate and organophosphate pesticides and has been widely used for the development of optical fiber sensors for these compounds based on different chemical transduction schemes (hydrolysis of a colored substrate, pH changes). 

As angiotensin-converting enzyme inhibitors influence protein excretion in renal disease, Gansevoort et al. (G2) and Keilani et al. (K10) investigated serum Lp(a) concentrations in patients treated with Lisinopril resp. fosinopril and detected a reduction. 

All these advances have resulted not only in increases in resolution but have also alleviated the detection problems to a considerable extent. As a result, the last decade has seen a dramatic growth in 15N- and 170-NMR spectroscopy as a versatile method for studying molecular structure, both in isotropic (liquid) and anisotropic (solid) phases. Studies at a natural abundance level of the nucleides are now commonplace. The scope of chemical applications extends from inorganic, organometallic and organic chemistry to biochemistry and molecular biology, and includes the study of reactive intermediates, biopolymers and enzyme-inhibitor complexes. 

J. van der Greef Rapid detection and identification of angiotensin-converting enzyme inhibitors by on-line liquid chromatography-biochemical detection, coupled to electrospray mass spectrometry. 

Inhibitors are substances that tend to decrease the rate of an enzyme-catalysed reaction. Although some act on the substrate, the discussion here will be restricted to those inhibitors which combine directly with the enzyme. Inhibitors have many uses, not only in the determination of the characteristics of enzymes, but also in aiding research into metabolic pathways where an inhibited enzyme will allow metabolites to build up so that they are present in detectable levels. Another important use is in the control of infection where drugs such as sulphanilamides competitively inhibit the synthesis of tetrahydrofolates which are vitamins essential to the growth of some bacteria. Many antibiotics are inhibitors of bacterial protein synthesis (e.g. tetracyclin) and cell-wall synthesis (e.g. penicillin). 

G. Palleschi, Extraction of enzyme inhibitors using a mixture of organic solvent and aqueous solution and their detection with electrochemical biosensors. The Eighth World Congress on Biosensors. P 3.7.64, Abstract Book. Granada, Spain, 2004. 

R Hyde-DeRuyscher, LA Paige, DJ Christensen, N Hyde-DeRuyscher, A Lim, ZL Fredericks, J Kranz, P Gallant, J Zhang, SM Rocklage, DM Fowlkes, PA Wendler, PT Hamilton. Detection of small-molecule enzyme inhibitors with peptides isolated from phage-displayed combinatorial peptide libraries. Chem Biol 7 17-25, 2000. 

It should be noted that the mechanism depicted in Scheme 1 is the simplest that is consistent with mechanism-based inhibition. The mechanism for a given inhibitor and enzyme may be considerably more complex due to (a) multiple intermediates [e.g., MIC formation often involves four or more intermediates (29)], (b) detectable metabolite that may be produced from more than one intermediate, and (c) the fact that enzyme-inhibitor complex may produce a metabolite that is mechanistically unrelated to the inactivation pathway. Events such as these will necessitate alternate definitions for Z inact, Kh and r in terms of the microrate constants of the appropriate model. The hyperbolic relationship between rate of inactivation and inhibitor concentration will, however, remain, unless nonhyperbolic kinetics characterize this interaction. Silverman discussed this possibility from the perspective of an allosteric interaction between inhibitor and enzyme (5). Nonhyperbolic kinetics has been observed for the interaction of several drugs with members of the CYPs (30). 

As was shown experimentally, micro-metabolites that are capable of acting as enzyme inhibitors may greatly affect product distribution. A biosimulation model can considerably assist drug approval by identifying physiological effects of drug metabolites too dilute in mammalian systems to be detected analytically. These results also show that the prediction of approximate product distribution requires implementation of a much more complex metabolic network, which is currently being done. 

Bonazzi et al. used an HPLC method and a second derivative ultraviolet spectroscopy method for the analysis of benazepril and other angio-tensen-converting enzyme inhibitors [17]. For HPLC, 20 pL sample solutions containing the drug and an internal standard dissolved in 1 1 acetonitrile/20 mM sodium heptanesulfonate (pH 2.5) were used. HPLC was performed on a 5 pm Hypersil ODS column (25 cm x 4.5 mm) with a mobile phase mixture consisting of (A) 20 mM sodium heptanesulfonate (pH 2.5) and (B) 19 1 acetonitrile-tetrahydrofuran, eluted at a flow rate of 1 mL/min, and with detection at 215 nm. The A/B mixture used was 52 48 for benazepril. A low pH of 2.5 was essential to avoid peak splitting and band broadening. 

Sioufi A, Pommier F, Kaiser G, Dubois JP (1988) Determination of benazepril, a new angiotensin-converting enzyme inhibitor and its active metabolite, benazeprilat, in plasma and urine by capillary gas chromatography-mass-selective detection. J Chromatogr 434 239-246... 

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