Other Irreversible Inhibitors

Two other irreversible inhibitors are penicillin G (4.18), a /3-lactam antibiotic, and orlistat (Xenical or Alii, 4.19), an antiobesity drug (Figure 4.19). Penicillin G inhibits cell wall synthesis in bacteria, while orlistat inhibits the breakdown of fats in the small intestine.16 Both drugs contain acid derivatives in a strained four-membered ring. Nucleophiles in the active sites of the inhibited enzymes attack the reactive carbonyl groups and open the strained ring in an energetically favorable, irreversible process. 

Incubation of AChE with onchidal resulted in the production of acetate, demonstrating that onchidal was a substrate for AChE, and approximately 3250 mol of onchidal was hydrolyzed/mol of enzyme irreversibly inhibited. Organophosphate and carbamate inhibitors of AChE have partition ratios (mol of toxin hydrolyzed/mol of enzyme irreversibly inhibited) that approach unity. Therefore, the relatively high partition ratio for onchidal suggests that the mechanism of inhibition utilized by onchidal may be distinctly different from other irreversible inhibitors (Walsh, 1984). The rate of hydrolysis of onchidal (Acat) was 325 min this value is relatively slow suggesting that onchidal is not a very good substrate. The ability of AChE to hydrolyze onchidal raised the question of whether inhibition of enzyme activity resulted from onchidal itself or from a product of the enzymatic hydrolysis of onchidal. Enzyme kinetics revealed that onchidal was unable to completely inhibit higher concentrations of AChE. From the experiments performed by Abramson et al. (1989), onchidal was in molar excess and was completely hydrolyzed. Thus,... 

The activity of P-lactamase inhibitors is often expressed as an IC q value, which is defined as the concentration of inhibitor that causes 50% inhibition of en2yme activity for a given set of conditions. IC q values, which vary widely according to substrate, time of incubation, and other factors, are presented herein solely to give an indication of potency and en2yme inhibitor specificity. Values that decrease with preincubation are indicative of irreversible inhibitors. 

Other Unusual P-Lactam Based Inhibitors. There are a number of other unusual p-lactams reported to have p-lactamase inhibition activity (191—194). In general these compounds are not very potent and are not irreversible inhibitors. Data are also very Umited. 

More than 50 endogenous and exogenous inhibitors of the calpains have been described as either transition-state reversible or irreversible inhibitors. The first transition-state inhibitors were the peptide aldehydes (e.g., leupeptin). Using this compound, new ones were synthesized that exhibited improved membrane permeability and calpain specificity (e.g., calpeptin). Other groups of inhibitors have since been discovered a-dicarbonyls (originally developed as serine protease inhibitors), nonpeptide quinolinecarboxamides,... 

Apart from these two Vertex compounds, only one other caspase inhibitor, BDN-6556, has been used in clinical trials. This compound belongs to the class of oxamyl dipeptides and was originally developed by Idun Pharmaceuticals (taken over by Pfizer). It is the only pan-caspase inhibitor that has been evaluated in humans. BDN-6556 displays inhibitory activity against all tested human caspases. It is also an irreversible, caspase-specific inhibitor that does not inhibit other major classes of proteases, or other enzymes or receptors. The therapeutic potential of BDN-6556 was first evaluated in several animal models of liver disease because numerous publications suggested that apoptosis contributes substantially to the development of some hepatic diseases, such as alcoholic hepatitis, hepatitis B and C (HBV, HCV), non-alcoholic steato-hepatitis (NASH), and ischemia/reperfusion injury associated with liver transplant. Accordingly, BDN-6556 was tested in a phase I study. The drug was safe and... 

In the first group of studies, involving kinetic inhibition studies, comparisons of the uilibrium (K ), phosphorylation (IC), and inhibition constant (K.) for the inhibition of electric eel and human erythrocyte AChE by ANTX-A(S) and DFP were done (Table II). From Table II it is seen that ANTX- A(S) has a higher affinity for human erythrocyte AChE (K =0.253 fiM) than electric eel AChE (K j=3.67 aM). AN DC-A(S) also shows greater affinity for AChE than DFP (K =300 fiM). And finally the bimolecular rate constant, Kj, which indicates the overall rate of reaction, shows AChE is more sensitive toward inhibition by ANTX-A(S) (Kj=1.36 pM- min- ) than DFP (K, = 0.033 /iM- min ). These studies add information to the comparative activity of ANTX-A(S) and other irreversible AChE inhibitors but do not show the site of inhibition. 

Suicide or irreversible inhibitors of GST Pl-1 include agents that bind covalently to glutathione, thereby forming thioether adducts that are stabilized at the active site of the enzyme. These agents include activated aromatic systems (2, 3), epoxides (4, 5), esters (6), and Michael acceptors such as ethacrynic acid (7), cycloalkenones (8, 9), and haloenol lactones (10-13), among others [3,48,54-57],... 

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. 

Vigabatrin is a new antiepileptic for use in epilepsy unresponsive to other therapy. It is an irreversible inhibitor of GABA-transaminase, the enzyme responsible for inactivation of the neurotransmitter GABA and it has shown efficacy against partial and secondarily generalized seizures. The principal unwanted effects are psychiatric disorders, including depression and psychosis, in a small number of patients. 

Tranylcypromine ( rans-2-phenylcyclopropylamine, TCP, 8a) has close structural similarity to amphetamine (2-amino-1-phenylpropane) and is known as a nonhydrazine, nonselective, and irreversible inhibitor of both MAO A and B. It is also a potent reversible inhibitor of CAOs [36,37], Tranylcypromine has an important clinical use for treatment of certain depressive illnesses, particularly of nonendo-genous and atypical depressions and depressions associated with anxiety, agitation, phobias, and anergia [38-40], In combination with lithium, it is also applied for treatment of refractory depression [41], Recent reports also discussed MAO inhibitors as useful agents against neurodegenerative disorders such as Parkinson s or Alzheimer s diseases [42], Despite impressive clinical successes, clinical use of tranylcypromine and other MAO inhibitors is limited by various problems, including the cheese effect discussed in Section 1,... 

A detailed study of the inhibition of MAOs by fluorophenyl cyclopropyl amines shows that the presence of fluorine has very important effects on this inhibition. While some of the regioisomers are inhibitors of the CAO (copper-containing amine oxidase), some other ones, such as 2-fluoro-l-arylcylopro-pyl amines, are excellent selective and irreversible inhibitors of MAO A. In this latter case, the nonfluorinated parent compound is a poor inhibitor of MAO B (Figure 7.55). ° " ... 

Another important characteristic of M AOIs is the production of reversible versus irreversible enzyme inhibition. An irreversible inhibitor permanently disables the enzyme. This means that MAO must be resynthesized, in the absence of the drug, before the activity of the enzyme can be reestablished. Resynthesis of the enzyme may take up to 2 weeks. For this reason, an interval of 10-14 days is required after discontinuing irreversible inhibitors and before instituting treatment with other antidepressants or permitting the use of contraindicated drugs or the consumption of contraindicated foods. On the other hand, a reversible inhibitor can move away from the active site of the enzyme, making the enzyme available to metabo-hze other substances. The reversibility and selectivity of the currently available MAOIs are summarized in Table 2-4. 

Irreversible Inhibitors of EGFR and Other ErbB Proteins... 

Iressa (gefitinib, 2) is a reversible inhibitor, thus mechanistically it is possible to excert less toxicity in comparison to similar irreversible inhibitors, which bond to the protein covalently. Gefitinib (2) is also quite selective. It is at least 100-fold selective against other tyrosine kinases such as erbB-2, KDR, c-flt or serine/threonine kinases such as PKC, MEK-1, and ERK-2. 

Peptidyl fluoromethyl ketones are widely used as fairly potent inhibitors for a variety of proteases, including serine, cysteine, and aspartyl proteases. Unlike other halomethyl ketones (Section 15.1.3), fluoromethyl ketones are reversible transition-state mimics. The electron-withdrawing fluorine(s) next to the carbonyl group enhances the electrophilicity of the a-fluoroalkyl ketone functionality, thereby making the carbonyl more susceptible to nucleophilic attack. a-Fluoroalkyl ketones are good mimics of peptide bonds due to the small size of the fluorine and the stability of C F bonds. There are three general classes of peptidyl fluoromethyl ketones fluoromethyl ketones (irreversible inhibitors of cysteine proteases), difluoromethyl ketones (reversible inhibitors of both serine and aspartyl proteases), and trifluoromethyl/perfluoroalkyl ketones, which typically exist in hydrated forms and are excellent inhibitors of both serine and cysteine proteasesJ1 ... 

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