Transition-state inhibitors

The substrate in an enzyme catalysed reaction is converted to the product through a series of transition state structures (Appendix 7). Although these transition state structures are transient, they bind to the active site of the enzyme and therefore must have structures that are compatible with the structure of the 

SELECTED EXAMPLES OF DRUG ACTION AT SOME COMMON TARGET AREAS 

Related to this phenomenon is the ability of some inhibitors to induce more conformational change in the enzyme than the substrate can. Thus the kinetics of the binding of allopurinol 9,51) [which is an anti-metabolite of hypoxanthine (9.52)] to xanthine oxidase demonstrate that binding is slow, but tight, and a steady state is not encountered (Cha, Agarwal and Parks, 1975). The authors think that this behaviour indicates a large conformational change. 

Let us now turn to those enzyme inhibitors which are designed to resemble, not the substrate, but the transition state of that substrate. Interest in this derives from the widely held belief that the active site of a free enzyme is more nearly complementary to the transition state of the substrate than to the free substrate (Pauling, 1948). The aim of such work is to produce inhibitors that are much more tightly held. 

It is usually not known what the transition state of a substrate is when it is on 

Amide (9.66) lIlH, Tetrahedral intermediate (9.67) Carboxylic acid (9.66) 

Pentostatin (deoxycoformycin) 4.18) and its ribose analogue, coformycin, strongly inhibit adenosine deaminase, the enzyme that converts adenosine 9.72) to inosine 9.74). presumably through the intermediate 9.73). This reaction is similar to the hydrolysis of an amidine to an amide, and it needs to be prevented when medicating patients with adenine-formulated drugs such as vidarabine 4.16). It can be seen that the structure of pentostatin resembles the 

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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,... 

P-site ligands inhibit adenylyl cyclases by a noncompetitive, dead-end- (post-transition-state) mechanism (cf. Fig. 6). Typically this is observed when reactions are conducted with Mn2+ or Mg2+ on forskolin- or hormone-activated adenylyl cyclases. However, under- some circumstances, uncompetitive inhibition has been noted. This is typically observed with enzyme that has been stably activated with GTPyS, with Mg2+ as cation. That this is the mechanism of P-site inhibition was most clearly demonstrated with expressed chimeric adenylyl cyclase studied by the reverse reaction. Under these conditions, inhibition by 2 -d-3 -AMP was competitive with cAMP. That is, the P-site is not a site per se, but rather an enzyme configuration and these ligands bind to the post-transition-state configuration from which product has left, but before the enzyme cycles to accept new substrate. Consequently, as post-transition-state inhibitors, P-site ligands are remarkably potent and specific inhibitors of adenylyl cyclases and have been used in many studies of tissue and cell function to suppress cAMP formation. 

The kinetically deduced existence of two classes of substrate sites may also account for the molar ratio between ATP analogs and inhibitors on the one hand and phosphoenzyme on the other hand. This ratio has been reported to be 2 1 for the ATP analogs adenylyl imido diphosphate (AMP-PNP) [135] and 2, 3 -0-(2,4,6-trinitrophenylcyclohexadienylidine)-ATP (TNP-ATP) [97], and also 2 1 for the ATP-site directed fluorescent inhibitors eosin [99] and FITC [49,50] and the transition-state inhibitor vanadate [126]. 

The first substrate analogue inhibitors of FAAH were reported in 1994. The anandamide analogues prepared represented three elasses of putative transition-state inhibitors a-trifluoromethyl ketones, a-ketoesters and a-ketoamides [62], In the initial sereening studies, it was found that the trifluoromethyl ketone eompounds tested were effeetive inhibitors of AEA hydrolysis. A selected set of a-keto esters also inhibited hydrolysis, while a-keto amides were ineffective. In particular, arachidonyl trifluoromethyl ketone (32), gave almost 100% inhibition of anandamide hydrolysis. A detailed investigation of the structural requirements for FAAH inhibition with a-trifluoromethyl ketones has been carried out by Roger and co-workers [63]. 

The review articles by Schramm (1998, 2003) provide a number of examples of the successful application of this protocol to the design of enzyme-specific transition state-like inhibitors. Among these, the transition state inhibitors of human purine nucleoside phosphorylase (PNP) are particularly interesting from a medicinal chemistry perspective, as examples of these compounds have entered human clinical trials for the treatment of T-cell cancers and autoimmune disorders. 

A number of methods can assist in identifying and characterizing enol intermediates (as well as eneamine and carbanion intermediates) in enzyme-catalyzed reactions. These include (1) proton isotope exchange (2) oxidation of the intermediate (3) coupled elimination (4) spectrophotometric methods (5) use of transition-state inhibitors (6) use of suicide inhibitors (7) isolation of the enol and (8) destructive analysis. 

ENTROPY TRAP MODEL MOLECULAR SIMILARITY TRANSITION-STATE INHIBITORS pCa pD... 

Bachand B, Tarazi M, St-Denis Y, Edmunds JJ, Winocour PD, Leblond L, Siddiqui MA (2001) Potent and selective bicyclic lactam inhibitors of thrombin. Part 4 transition state inhibitors. Bioorg Med Chem Lett ll(3) 287-290... 

M. Degano, S. C. Almo, J. C. Sacchettini, and V. L. Schramm, Trypanosomal nucleoside hydrolase. A novel mechanism from the structure with a transition-state inhibitor, Biochemistry, 37 (1998) 6277-6285. 

Transition state inhibitors. Suppose that a chemical reaction of a compound S takes place with rate constant /cx through transition state T. Let the equilibrium constant for formation of T be Kj-. Assume that an enzyme E can combine either with S with dissociation constant Kds or with the compound in its transition state structure T with dissociation constant Kdj (Eq. 9-83). 

The anion formed by removal of the 3-H is analogous to the enolate anion of Eq. 13-6 and has a strong structural similarity to the readily formed anions of organic nitro compounds. The nitronate anions may, perhaps, be regarded as transition state inhibitors. 

Ban et al 7 Courtesy of T. A. Steitz. The peptidyltransferase center is marked by the green image of the transition state inhibitor shown in Fig. 29-13. (F) Model of three tRNAs bound to a ribosome from Thermus thermophilus in the A (a mi noacyl), P (pepti-dyl), and E (exit) sites. These are based on 0.75-nm X-ray data and a number of difference electron density maps. The 3-CCA end of the A-site tRNA is not modeled hut is... 

SCHEME 4.12 Coformycin (4.11), a transition state inhibitor of AMP deaminase... 

Competitive, reversible inhibitors are the most common type of inhibitor developed for pharmaceutical use. If the substrate of an enzyme is known, then a competitive inhibitor will likely somewhat resemble the substrate. The search for an inhibitor will typically start with molecules of similar structure to the substrate. Because enzymes theoretically bind most strongly to a transition state, competitive inhibitors are often designed to resemble a transition state or a high energy intermediate along the reaction coordinate. These types of drugs are called transition state analogues or transition state inhibitors. 

Pepstatin, another representative of the group of transition state inhibitors and a very potent inhibitor of pepsin, is a modified pentapeptide (McConnell... 

Miles RW, Tyler PC, Furneaux RH, Bagdassarian CK, Schramm VL (1998) One-third-the-sites transition state inhibitors for purine nucleoside phosphorylase. Biochemistry 37 8615-8621... 

Furneaux RH, Limberg G, Tyler PC, Schramm VL (1997) Synthesis of transition state inhibitors for N-riboside hydrolases and transferases. Tetrahedron 53 2915-2930... 

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