Type III inhibitors

Type III inhibitors bind to sites distal to the ATP binding site and are often inactive in simple kinase enzyme assays. This apparent inactivity arises because these compounds can bind to the kinase and render it a poor substrate for an activating upstream kinase, thus disabling its activation. As a consequence, a cascade or cell-based assay may be required. 

Crataegus species (hawthorn, maybush, whitethorn) contain a variety of flavonoids, including rhamnosides, schaf-tosides, and spiraeosides. They have a positive inotropic effect on the heart by a mechanism different from that of cardiac glycosides, catecholamines, and the phosphodiesterase type III inhibitors (1) and are effective in mild heart failure (2). 

A less well-populated class of compounds, thought to have the potential to exhibit the highest levels of selectivity, modulate kinase activity in a non-ATP-competitive manner.10 Known as type III inhibitors, these avoid the highly... 

Inhibitors of the active form of the kinase are classified as Type I inhibitors, and inhibitors of inactive forms of the kinases are Type II inhibitors. Recently, there has been considerable interest in identifying additional binding sites remote from the active site but which can modulate kinase activity. Inhibitors binding to such allosteric or substrate sites would be considered type III inhibitors. 

Emerging data suggest that the issue of kinase inhibitor selectivity can be addressed by moving away from classic ATP-competitive (Type I) inhibitors and targeting the DFG-out pocket with type II and type III inhibitors (Figure 2.1c). 

Figure 2.2 FLiK constructs. Structurally flexible are labeled with a thiol reactive fluorophore. Conformational changes triggered by ligand binding alter the emission properties of the fluorophores. Activation loop labeled protein kinases for the detection and discrimination of type II and type III inhibitors. (Adapted with permission from Macmillan Publishers Ltd [6], copyright 2009.)...

Several prospective randomized trials have reported that cilostazol improves walking distance in patients with intermittent claudication by 40 to 50%, compared to placebo after 12 to 24 weeks of treatment (20,21). One of these placebo-controlled trials evaluated both pentoxifylline and cilostazol (17). Pentoxifylline demonstrated no benefit in either onset of claudication or absolute claudication distance as compared to placebo. Cilostazol, however, significantly improved both distances compared to placebo (17). A prevalent side effect of cilostazol is headache. Transient diarrhea, palpitations, and dizziness have also been reported. The FDA has issued a warning regarding the use of cilostazol in patients with congestive heart failure, because of the increased possibility of sudden cardiac death observed with other forms of diesterase type III inhibitors. Thus, it has become routine practice to assess cardiac function clinically and echocardiographically prior to initiating therapy with cilostazol for claudication, and periodically thereafter. As a result of the modest vasodilatation, heart rate may increase by a mean of 5.1 and 7.4 beats per... 

The inotropic effects of these agents are not mediated via direct stimulation of -adrenergic receptors or indirectly by release of catecholamines, but by selective inhibition of cardiac cAMP phosphodiesterase (PDE) type III [25,35-40]. Recently, it has been demonstrated that the imidazole core is primarily responsible for PDE isozyme specificity, whereas the dihydropyri-dazinone moiety is responsible for inhibitory potency the phenylene moiety obviously acts mainly as a spacer [26]. A five-point model for positive inotropic activity of PDE III inhibitors has been elaborated [41]. 

In the reduced state, cytochromes P-450 may also bind certain ligands to give particular difference spectra. The most well known is that which occurs when carbon monoxide binds giving an absorption maximum at 450 nm. A type III spectrum gives two peaks at 430 and 455 nm after binding of certain compounds such as ethyl isocyanide or methylenedioxyphenyl compounds to the reduced enzyme. The latter form stable complexes with the enzyme and are also inhibitors. 

Keratin IFs exhibit different motile behaviors compared with type III and IV IFs, and continue to move in the absence of MT in cells treated with MT inhibitors, such as nocodazole. It has been proposed that actin may be involved in their transport (Yoon et al., 2001). Time lapse imaging of keratin-GFP has revealed that keratin IF formation begins in the cell cortex in a region enriched in actin and required for both intact MT and actin, and that actin governs the organization and movement of keratin in Xenopus egg extracts (Weber and Bement, 2002). Less is known about specific mediators of these interactions, although the fact that myosin Va associates with NFs raises the interesting possibility that other such interactions may exist for keratins. 

As discussed in previous sections, the stepwise deiodination of T4 is mediated by at least three different enzymes. Deiodination of the outer ring of T4 and reverse T3 is mediated by the type I and II enzymes while deiodination of the inner ring of T4 and T3 is catalysed by the type I and III enzymes. The contribution of the different enzymes to the peripheral production and clearance of T3 and rT3 can be estimated using PTU as a specific inhibitor of the type I deiodinase (for potential pitfalls of this approach, see Section 3.3). Thyroid hormone has a positive effect on the type I and type III enzymes but down-regulates the type II deiodinase. 

In addition to the obvious deactivating role of deiodinases, there has been recent evidence that a relationship exists between regulation of deiodination of thyroid hormones in target cells and the intracellular effects of T4 and T3 on pituitary and hypothalamus function. In the rat pituitary, and probably the human, type-II deiodinase-catalyzed conversion of T4 to T3 is a prerequisite for inhibition of TRH release. rT3, produced from T4 by type-III deiodinase, is a potent inhibitor of type-II deiodinase. In a postulated regulatory circuit, rT3 formed from T4 by type-III deiodinase in surrounding CNS (Central Nervous System) tissue enters the pituitary and inhibits type-II enzyme. The resulting decrease in T3 concentration, in turn, causes an increase in TSH secretion49. 

Cyclic derivatives of type III include cyclic Mannich bases, such as dihydroben-zoxazines 497, employed as detergents for lubricating oils, - and cyclic urcides 498, precursors of crosslinking agents for fabrics, as well as other cyclic derivatives prepared by conversion of Mannich bases. Macromolecular derivatives of type IV are relatively small in size and have branched (star-shaped) structures they are of considerable importance as, for example, corrosion inhibitors 499, plastics stabilizers 500, - pre-polymers for epoxy-based electrophoretic paints, and polyols in polyurethane synthesis. ... 

Type III Topographical mimetics Structure-based design Non-peptide protease inhibitors... 

Type III mimetics represent the Farmer definition ofpeptidomimetics in that they possess novel templates, which appear unrelated to the original peptides but contain the essential groups, positioned on a novel non-peptide scaffold to serve as topographical mimetics. Several type III peptidomimetic protease inhibitors have been characterized where direct... 

Recently, the importance of generating inhibitors that target receptor conformational ensembles has been pointed out (10). This method goes beyond the current docking of knovm structures to known active site con-formers and can lead to type III and GRAB peptidomimetics. 

Workers at DuPont used a pharmacophore model and database search to develop the first type III mimetic inhibitor of HIV protease, DuP450 (87)(Fig. 15.38). This evolved from a 3D pharnacophorethat retained two key interactions replacement of the flap-bound water and a hydroxyl transition-state isostere (155). Molecular modeling led to a cyclohexanone as a better spacer between these groups, and finally the seven-membered cyclic urea (87) was created (Fig. 15.38). The development of these inhibitors illustrates the importance of conformational analysis in the design of constrained analogs. 

Thrombin inhibitors (92) and (93) illustrate a novel type III peptidomimetic. Most protease inhibitors bind in an extended jS-strand conformation that is stabilized by multiple enzyme ligand hydrogen bonds. 

Other type III peptidomimetic inhibitors of thrombin have been developed from screening leads (166, 167) such as inhibitor (94) (Fig. 15.40). SAR led to the design of (95)Inhibitor (96) was derived from docking studies with the 5-amidino indole nucleus, followed by addition of a lipophilic side-chain to interact with the important Sg subsite of thrombin. The crystal structures of both (95) and (96) in the active site of thrombin shows that the aromatic core, binds in the site as expected, but... 

Another type III peptidomimetic inhibitor was derived from the crystal structure of a bicyclic [3.1.3] inhibitor (170) complexed to thrombin (97) (Fig. 15.41). The X-ray structure revealed that one of the carbonyls was oriented towards the hydrophobic P-pocket (S2). The desolvation necessary to place a carbonyl in a hydrophobic pocket is unfavorable and various alkyl groups were used as possible replacements. This led to the potent (iCj = 13 nM) and selective (>760 for thrombin over tiypsin) inhibitor (98). 

New approaches to design inhibitors of Factor Xa as potential anticoagulants have been reviewed (173), and important type III mimetics have been described (Fig. 15.42). All inhibitors contain amidine or basic groups that bind in the enzyme s site none of the inhibitors contains a classical electrophilic center of the type employed in TSA inhibitors (174-180). 

Other type III peptidomimetic inhibitors of this enzyme have also been reported. Inhibitor (112) (Fig. 15.47) was developed by replacing the A1A2 dipeptidyl sequence with a benzodiazepine scaffold (209). Later, SAR modifications of the benzodiazepine nucleus that included a hydrophilic 7-cyano group and a 4-sulfonyl group provided the potent, orally available and in vivo active (113) (210). 

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