Covalent inhibitors

As an alternative to peptidic inhibitors, which display electrostatic interactions with the active site, covalent inhibitors have also been described recently. Such peptides bear a functional group that can react reversibly with the catalytic serine of the protease. These include aldehydes, a-ketoacid derivates, lactams and boronates. 

Table 2.5 PEPTIDIC KETO-AMIDES AS COVALENT INHIBITORS...

The most convenient way of categorizing the classes of cathepsin inhibitors is based on the nature of the electrophilic warhead that interacts with the sulfhydryl group of the active site cysteine residue. Since a large portion of the binding energy of a cysteine protease inhibitor comes from the covalent interaction with this thiol, the properties of the resulting molecules are largely derived from the electrophile. In broad terms, these inhibitors can be broken down into ketone and nitrile-based reversible covalent inhibitors, or the more recent non-covalent inhibitors based on an aminoaniline template. 

The past 3 years have seen tremendous advances in both the design of Cat K inhibitors and in our understanding of the effect of Cat K inhibition on bone remodeling. The structural diversity of Cat K inhibitors has expanded considerably from simple peptidomimetics to non-peptidic derivatives and even non-covalent inhibitors. The potency, selectivity and pharmacokinetic properties of key compounds are very attractive and seem well-suited to further development. The disclosure of clinical validation of the effect of Cat K inhibition on bone mineral density, plus the provocative data suggesting a decoupling of bone resorption and bone formation provides a compelling framework for further development of Cat K inhibitors for the treatment of osteoporosis. 

A. Gilbert, K. Young Applications of GPC spin colunm/ESI-MS for identifying non-covalent inhibitors of RGS4 protein, a potential CNS target, in Proceedings of the 52nd ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, 2003. 

Curcumin represents a covalent inhibitor of CBP. Beyond, it showed anti-angiogenic effects and is currently in phase II/III of clinical trials for cancer therapy, as well as in phase II for Alzheimer s disease and psoriasis [21, 22[. Garcinol, a potent inhibitor of PCAF and p300 (IC50 = 5 and 7 J-M, respectively) was found to induce apoptosis and alter global gene expression in HeLa cells. It was identified to repress chromatin transcription. N one of these inhibitors had effects on H D AC activity or on histone-free DNA transcription [23]. 

Gehlhaar, D. K., Bouzida, D., and Rejto, P. A. (1999) Reduced dimensionality in ligand-protein structure prediction covalent inhibitors of serine proteases and design of site-directed combinatorial libraries. ACS Symposium Series 719, 292-311. 

B. P. Rempel and S. G. Withers, Covalent inhibitors of glycosidases and their applications in... 

The probes of this type were shown to selectively label at least 75% of human kinases in crude cell lysates, thus demonstrating their selectivity and promiscuity for kinases [101]. As a follow up, the labeled kinases were subjected to proteolytic digestion, and the biotinylated peptides purified on avidin beads and analyzed by LC-MS/MS. This analysis demonstrated that the site of probe labeling was indeed the conserved active-site lysine as predicted. In contrast to the promiscuity demonstrated by the acyl phosphate probes, several selective covalent inhibitors of protein kinases have been used as ABPP probes. Wortmannin is a natural product derived from the fungus Penicillium funiculosum. It is a potent and specific covalent inhibitor of phosphoinositide 3-kinase (PI3K) and the PI3K-related kinase (PIKK) families [102, 103]. The use of natural products in relation to ABPP is covered by Breinbauer et al. [104]. 

Al-Shawi M.K., Urbatsch I.L., Senior A.E. (1994) Covalent inhibitors of P-glycoprotein ATPase activity, J Biol Chem 269(12) 8986—8992. 

E. Elhanany, A. Ordentlich, O. Dgany, D. Kaplan, Y. Segall, R. Barak, B. Velan and A. Shafferman, Resolving pathways of interaction of covalent inhibitors with the active site of acetylcholinesterases MALDI-TOF/MS analysis of various nerve agent phosphyl adducts, Chem. Res. Toxicol., 14, 912-918 (2001). 

Irreversible (covalent) inhibitors. This case is less common than reversible inhibition or activation. Example Phenoxybenzamine, an antagonist at a-adreneigic receptors. 

While diclofenac and most other cyclooxygenase inhibitors act competitively (i.e., non-covalently), acetylsalicylic acid causes covalent modification of serine 530. Its effect may therefore last longer than that of a non-covalent inhibitor. Interestingly, the half-life of acetylsalicylic acid is rather short - about 15 minutes most of the drug is just hydrolysed to acetic acid and salicylic acid. However, salicylic acid itself still acts as a (competitive) inhibitor of Cox. Also, the covalent modification of Cox achieved early on will persist after elimination of acetylsalicylic acid, so that the clinical effect of this drug will outlast its elimination. 

Inhibition constants for covalent inhibitors are given as inact Kj. 

Specific small molecules or ions can inhibit even nonallosteric enzymes. In irreversible inhibition, the inhibitor is covalently linked to the enzyme or bound so tightly that its dissociation from the enzyme is very slow. Covalent inhibitors provide a means of mapping the enzyme s active site. In contrast, reversible inhibition is characterized by a rapid equilibrium between enzyme and inhibitor. A competitive inhibitor prevents the substrate from binding to the active site. It reduces the reaction velocity by diminishing the proportion of enzyme molecules that are bound to substrate. In noncompetitive inhibition, the inhibitor decreases the turnover number. Competitive inhibition can be distinguished from noncompetitive inhibition by determining whether the inhibition can be overcome by raising the substrate concentration. 

Fig. 5.6. Secondary structure of the /3 subunit of E. coli F,. The primary structure of the protein was deduced from the DNA sequence [11]. The binding residues and numbers of binding sites for covalent inhibitors are also shown.

Covalent inhibitors were synthesized by Withers and coworkers.148 149 The use of 3F-sialyl fluoride 136 as donor substrate allowed identification of the covalent mechanism for TcTS. Fluoride 136 completely inactivates the enzyme, but only at concentrations as high as 20 mM, and separation of excess inactivator caused recovery of the activity.148 Since the 3D structure of TcTS showed that the donor site is more spacious and hydrophobic than in a human sialidase, derivatives at C-9 of the fluoride 136 were synthesized by a chemoenzymatic method and tested as inhibitors (Fig. 10).149 Inhibition was analyzed with trifluoromethylumbelliferyl sialic acid for measuring hydrolase activity, however, as the site is the same for... 

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