Alternate substrate inhibitors

SCHEME 11.3 Postulated mechanisms for the inhibition of serine proteases by coumarin derivatives. NuH nucleophile. Pathway a suicide-type inactivation (suicide substrate). Pathway b transient inactivation by formation of a stable acyl-enzyme (alternate substrate-inhibitor). 

Several aryl esters of 6-chloromethyl-2-oxo-2//-l -benzopyran-3-carboxylic acid act as human Lon protease inhibitors (alternate substrate inhibitors)46 without having any effect on the 20S proteasome. Proteasomes are the major agents of protein turnover and the breakdown of oxidized proteins in the cytosol and nucleus of eukaryotic cells,47 whereas Lon protease seems to play a major role in the elimination of oxidatively modified proteins in the mitochondrial matrix. The coumarin derivatives are potentially useful tools for investigating the various biological roles of Lon protease without interfering with the proteasome inhibition. 

A procedure that utilizes quantitative structure-activity relationships to assist in the characterization and design of enzyme alternative substrates, inhibitors, and effectors. In this procedure, the inhibition (or activity) results of a series of structurally related compounds are analyzed to determine the coefficients in an equation of the form ... 

Measurement of the in vitro efficacy of compounds as substrates is usually deduced by comparison of their k JK ratios where is the first-order rate constant for product formation and is the Michaelis equilibrium constant [38]. For those compounds which are classical, reversible inhibitors, K, the dissociation (or inhibition) equilibrium constant, and (kassoc) the rate constant for enzyme inhibition, are the most commonly reported kinetic values. These values may be measured while using either a high-molecular-weight natural substrate or a low-molecular-weight synthetic substrate. For alternate-substrate inhibitors, that is, compounds which form a stable complex (an acyl-enzyme ) that dissociates to enzyme and intact inhibitor or to enzyme and an altered form of the inhibitor, the usually reported value is K, the apparent K. For compounds which irreversibly inactivate the enzyme, the kinetics are usually measured under conditions such that the initial enzyme concentration [E] is much lower than the inhibitor concentration [I] which in turn is much lower than the Ky Under these conditions the commonly reported value is obs/[I]> the apparent... 

For alternate-substrate inhibitors, the enzyme (E) and inhibitor (I) first interact to give a reversible Michaelis complex (E I). This complex rapidly progresses to a relatively stable acyl-enzyme (E I) which may slowly either revert back to the active enzyme (E) and the intact inhibitor (I) or continue on via the normal catalytic machinery to the active enzyme (E) and the modified inhibitor (I ). With dual-acting inhibitors the acyl-enzyme contains a second reactive functionality which acylates or alkylates a second amino acid residue in the enzyme active-site, while the compound is still tethered to Ser-195 [177], resulting in inactivated enzyme (E I). 

An alternate approach, which also uses enzyme-catalyzed ring-opening of a lactone to generate a mechanism-activated inhibitor, was developed by Katzenellenbogen and his co-workers [183], who found enol lactones, exemplified by (13-8) and (13-9), to be potent, selective inhibitors of HLE. The haloenol lactone (13-9) was an irreversible inactivator of HLE and chymotrypsin, and after exposure to (13-9), active enzyme could not be regenerated even upon treatment with hydrazine. Enol lactone (13-8), on the other hand, was an alternate-substrate inhibitor, which produced only transient inhibition of HLE and chymotrypsin. These results have been interpreted to mean that, with the halo-substituted compounds, ring opening results in formation of an acyl-enzyme that contains a reactive halomethyl ketone, which then alkylates His-57. That these compounds... 

In the early studies exploring the interaction of lactams with mammalian serine proteinases, a single -lactam derivative (11a) had been reported to react with a-chymotrypsin to form a very stable acyl-enzyme, that is, it was an alternate-substrate inhibitor [204]. However, after this report more than... 

Sem, D.S. and C.B. Kasper (1993). Enzyme-substrate binding interactions of NADPH-cytochrome P-450 oxidoreductase characterized with pH and alternate substrate/inhibitor studies. Biochemistry 32, 11539-11547. 

Seah VM, Wong KP. 2,6-Dichloro-4-nitrophenol (DCNP), an alternate-substrate inhibitor of phenolsulfotransferase. Biochem Pharmacol 1994 47(10) 1743-1749. 

An alternate substrate inhibitor produces a stable intermediate during the normal course of catalysis, tying up the enzyme in its E-1 form. Although there can be many steps during the process, and more than one product may be formed. Scheme 13.1 shows the essential steps of the mechanism of inhibition. To fully characterize alternate substrate inhibition, the... 

The on rate, kon, is equivalent to k, and the off rate, off> is equivalent to the sum of all pathways of E-I breakdown, in this case, A i - - k2. It is possible that multiple products are formed, and the rates of formation of these should be included in the koff term. A progress curve or continuous assay is the best way to determine the kon and Ki of an alternate substrate. Addition of an alternate substrate inhibitor to an enzyme assay results in an exponential decrease in rate to some final steady-state turnover of substrate (Fig. 13.1). In an individual assay, both the rate of inhibition (kobs) and the final steady-state rate (C) will depend on the concentration of inhibitor. Care must be taken to have a sufficient excess of inhibitor over enzyme concentration present, since the inhibitor is consumed during the process. Where possible, working at assay conditions well below the of the assay substrate simplifies the kinetics, as the substrate will not interfere in the inhibition. If the... 

As with alternate substrate inhibitors, a progress curve or continuous enzyme assay is the most useful to begin to characterize the kinetics of inhibition. There can be immediate, or diffusion-limited inhibition of the... 

Full kinetic characterization for mechanism-based inhibition can be a challenge. Not only are there multiple rates to determine, but the mechanism of inhibition is often a combination of several different steps. The dividing line between alternate substrate inhibitors and the more eom-plex suicide inhibitors is often blurred, with some alternate substrates being virtually irreversible and some suicide substrates with high partition ratios and a significant alternate substrate element of inhibition. The following examples describe the characterization of an alternate substrate inhibitor and a suicide inhibitor of the serine protease human leukocyte elastase. 

Using the enzyme inhibition kinetics and product identification and model studies of alkaline hydrolysis of the compounds, stmcture-activity relationships of the enzyme inhibitor interactions could be understood and predicted. With this knowledge the authors were able to design alternate substrate inhibitors with reasonable chemical stability, inhibition constants in the nanomolar range, and very slow deacylation rates (fcoff), resulting in virtually irreversible inhibition. 

A series of ynenol lactones (stmcture 2) were studied as inhibitors of human leukocyte elastase (Tam et al., 1984 Spencer et al., 1986 Copp et al., 1987). Some of the compounds were alternate substrate inhibitors, being hydrolyzed by the enzyme to the reactive I but then deacylat-ing without an inactivation step. However, with the compound 3-benzyl ynenol butyrolactone (stmcture 2, where R = benzyl, R = H), the acyl-enzyme (E-I ) was stable enough to allow the second alkylation step, resulting in inactivated enzyme. All kinetic constants were determined. Continuous assays gave biphasic kinetics, the second minor phase possibly due to the presence of isozymes or enantiomers of the inhibitor. Immediate diffusion-limited inhibition was observed and gave a competitive Ki value of 4.3 0.7 xM. The first phase of inhibition was saturable, and analysis of the rates gave = 0.090 0.007 s , and... 

AH 2/3 -dideoxynucleoside analogues are assumed to be intraceUularly phosphorylated to thek active form (5 -triphosphate), and then targeted at the vims-associated reverse transcriptase. The rate and extent of the 2 /3 -dideoxynucleosides phosphorylate to the 5 -triphosphates may be of equal or greater importance than the differences in the relative abiUties of these 5 -triphosphates to inhibit the vkal reverse transcriptase (171). At the level of vkal reverse transcriptase, the 5 -triphosphate of AZT and other dideoxynucleosides may either serve as a competitive inhibitor with respect to the natural substrates or may act as an alternate substrate, thus leading to chain termination (172). 

While the ddNs and ANPs must be converted intracellularly to their 5 -triphosphates (ddNTPs) or diphosphate derivatives before they can interact as competitive inhibitors/alternate substrates with regard to the natural substrates (dNTPs), the NNRTIs do not need any metabolic conversion to interact, noncompetitively with respect to the dNTPs, at an allosteric, non-substrate binding site of the HIV-1 RT. Through the analysis of NNRTI-resistant mutants, combined with site-directed mutagenesis studies, it has become increasingly clear which amino acid residues are involved in the interaction of the NNRTIs with HIV-1 RT, and, since the conformation of the HIV-1 RT has been resolved at 3.0 A resolution [73], it is now possible to visualize the binding site of the NNRTIs [74],... 

Zinc protoporphyrin IX is a normal metabolite that is formed in trace amounts during haem biosynthesis. However, in iron deficiency or in impaired iron utilization, zinc becomes an alternative substrate for ferrochelatase and elevated levels of zinc protoporphyrin IX, which has a known low affinity for oxygen, are formed. This zinc-for-iron substitution is one of the first biochemical responses to iron depletion, and erythrocyte zinc protoporphyrin is therefore a very sensitive index of bone-marrow iron status (Labbe et ah, 1999). In addition, zinc protoporphyrin may regulate haem catabolism by acting as a competitive inhibitor of haem oxygenase, the key enzyme of the haem degradation pathway. However, it has been reported... 

Conformationally-restricted arginine analogues as alternative substrates and inhibitors of nitric oxide synthases, Bioorg. Med. Chem. 7 (1999), p. 1097-1104... 

Screening of over 66,000 compounds from the MLSMR by scientists at the PCMD for inhibitors of Cathepsin B resulted in the identification and characterization of an alternate substrate, SID 16952359 [29]. This study also describes issues relating to the nucleophilicity of dithiothreitol (DTT) and cysteine, reductants frequently used in HTS protocols, and the potential for reactivity with electrophilic sites of probe molecules. 

Two compounds other than the natural substrate SAM, l-VG and S -methyl-L-methionine (SMM), have been described so far as both substrates and inhibitors of ACS isozymes. l-VG was isolated 30 years ago from the fungus Rhodophyllus nidorosus It was shown to be a mechanism-based inhibitor of aspartate aminotransferase and kynurenine aminotransferase. First of all, l-VG is an alternative substrate of ACS in addition to being an inhibitor as described in the previous section. ... 

While requiring the availability of competitive inhibitors for each of the substrates, Fromm s use of competitive inhibitors to distinguish multisubstrate enzyme kinetic pathways represents the most powerful initial rate method. See Alternative Substrate Inhibition... 

Periodic acid oxidation has proved to be a very useful tool in enzymology since a wide variety of biochemicals contain hydroxyl groups on adjacent carbon atoms. For example, periodate-oxidized ATP (also called adenosine 5 -triphosphate 2, 3 -dialdehyde) has often been used as an alternative substrate or an irreversible inhibitor for a wide variety of ATP-utilizing enzymes. This compound, and many others, are now commercially available, even though they are readily synthesized e.g., periodic acid oxidized ADP, AMP, adenosine, P, P -di(adenosine-5 )pentaphosphate, P, P -di(adenosine-5 )tetraphos-phate, GTP, GDP, GMP, guanosine, CTP, CDP, CMP, etc. In the case of the nucleosides, commercial sources also can supply the dialcohol form of the nucleoside i.e., the nucleoside has first been oxidized with periodic acid and then reduced to the dialcohol with borohydride. 

If a noncompetitive or an uncompetitive inhibitor were present with the substrate at constant ratio, then graphical analysis would suggest that the phenomenon of substrate inhibition is present. If an investigator analyzed the apparent substrate inhibition via a Marmasse plot, wrong estimates of both the K a and K s values would be reported and the investigator would be mislead with respect to the kinetic mechanism. If partial inhibitors or alternative substrates are present in constant ratio, depending on the relative sizes of the Ymax and values,... 

ALTERNATIVE PRODUCT INHIBITION ABORTIVE COMPLEXES ALTERNATIVE SUBSTRATES COMPETITIVE INHIBITOR ABORTIVE COMPLEXES MAPPING SUBSTRATE INTERACTIONS USING KINETIC DATA MEMBRANE TRANSPORT ENERGY OF ACTIVATION Old... 

K. Osiecki-Newman, D. Fabbro, G. Legler, R. J. Desnick, and G. A. Grabowski, Human acid P-glucosidase Use of inhibitors, alternative substrates and amphiphiles to investigate the properties of the normal and Gaucher disease active sites, Biochim. Biophys. Acta, 915 (1987) 87-100. 

General aspects of enzymatic reactions cateuLyzed by kinases are briefly mentioned. Many alternate substrates, competitive inhibitors and affinity labels based either on the structure of ATP or on the structure of the non-ATP kinase substrates are described. Several examples are presented that should be of particular interest to the medicinal chemist. Finally, the design of an affinity label for creatine kinase is reviewed as an example of how such information can be used in the search for agents directed at an enzyme s active site. 

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