Sequence homology, enzyme inhibitors

The standard model for the preclinical development of anti-osteoporosis therapies is the ovariectomized (OVX) rat. However, Cat K inhibitors developed specifically against the human enzyme are generally significantly less potent ( 2-orders of magnitude) against the rat and mouse enzymes than against human Cat K [9]. This loss of potency towards the rodent enzymes, which is consistent with their low sequence homology, therefore restricts the use of... 

The biogenic amines are the preferred substrates of MAO. The enzyme comes in two flavors, MAO-A and MAO-B, both of which, like FMO, rely on the redox properties of FAD for their oxidative machinery. The two isoforms share a sequence homology of approximately 70% (81) and are found in the outer mitochondrial membrane, but they differ in substrate selectivity and tissue distribution. In mammalian tissues MAO-A is located primarily in the placenta, gut, and liver, while MAO-B is predominant in the brain, liver, and platelets. MAO-A is selective for serotonin and norepinephrine and is selectively inhibited by the mechanism-based inhibitor clorgyline (82). MAO-B is selective for /1-phcncthylaminc and tryptamine, and it is selectively inhibited by the mechanism-based inhibitors, deprenyl and pargyline (82) (Fig. 4.32). Recently, both MAO-A (83) and MAO-B (84) were structurally characterized by x-ray crystallography. 

Remarkably, in this context, no significant sequence homology of amino acid residues could be detected. This successful structure elucidation should allow the design of non-natural specific inhibitors of this enzyme on the basis of structural information known from the bulgecins. 

Flavin-containing mitochondrial MAO-A and MAO-B catalyze the oxidative deamination of neurotransmitters, such as dopamine, serotonin, and norepinephrine in the central nervous system and peripheral tissues. The enzymes share 73% sequence homology and follow the same kinetic and chemical mechanism but have different substrate and inhibitor specificities. Chemical modification experiments provide evidence that a histidine residue is essential for the catalysis. There is also strong evidence that two cysteine residues are present in the active site of MAO. 

Today 11 members of the human PDE superfamily are known, all of which are class I phosphodiesterases and all of which are intracellular or membrane-bound enzymes. Several of the isoenzymes are encoded by more than one gene which, in combination with the presence of different splice variants, brings the number of different PDE proteins to well over 50. The different isoenzymes are characterized according to their substrate specificity, sequence homology, kinetic properties, and sensitivity to certain known PDE inhibitors. Table 9.1 shows these properties together with the predominant tissue expression of the various PDEs. 

COX-1 and COX-2 are of similar molecular mass (approximately 70 and 72 kDa respectively), with 65% amino acid sequence homology and near-identical catalytic sites. The most significant difference between the isoenzymes, which allows for selective inhibition, is the substitution of isoleucine at position 523 in COX-1 with valine in COX-2. The relatively smaller Val523 residue in COX-2 allows access to a hydrophobic side-pocket in the enzyme (which Ile523 sterically hinders). Drugs, such as the coxibs, bind to this alternative site and are considered to be selective inhibitors of COX-2. 

Finally, this inhibitor was cross-checked against five other phosphatases to determine the selectivity. Among those analyzed, TCPTP is closely related to PTPIB on the basis of sequence homology. Therefore, a difference in the affinity of the evaluated compounds for these two enzymes would be a remarkable achievement. The relative selectivities are snmmarized in Table 4, indicating that compound 18 is indeed able to distinguish between PTPIB and TCPTP. 

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