Amino acids protease inhibitors

Fig. 5.7. Structure analysis of a 56-amino-acid protease inhibitor peptide by 2D NMR spectroscopy. A. Onedimensional NMR spectrum of the peptide. B. A two-dimensional NMR spectrum of the same peptide. C. Set of possible structures obtained from 2D NMR spectra similar to the one shown in panel B. (Courtesy of Dr Sarah L.Heald, Miles Pharmaceutical Inc. For details see S.L.Heald et al. (1991) Biochemistry 30,10467 10477.)...

The simulations reported in the paper dealt with bovine pancreatic trypsin inhibitor (BPTI), a small (58 amino acid) protease inhibitor whose structure is stabilized by three disulfide crosslinks. The simulation began with a set of X-ray coordinates and zero velocities, and ran 100 steps of numerical integration of Newton s equations of motion, with each time step being about 1 fs. Since the X-ray coordinates did not correspond to a minimum of... 

An example of a pseudoirreversible inhibitor has been demonstrated for chymotrypsin (36). This enzyme is a serine protease, and its mechanism of catalysis may be outlined as follows, where or R2 preferentially is a hydrophobic amino acid residue. 

Figure 2.14 shows examples of both cases, an isolated ribbon and a p sheet. The isolated ribbon is illustrated by the structure of bovine trypsin inhibitor (Figure 2.14a), a small, very stable polypeptide of 58 amino acids that inhibits the activity of the digestive protease trypsin. The structure has been determined to 1.0 A resolution in the laboratory of Robert Huber in Munich, Germany, and the folding pathway of this protein is discussed in Chapter 6. Hairpin motifs as parts of a p sheet are exemplified by the structure of a snake venom, erabutoxin (Figure 2.14b), which binds to and inhibits... 

Uiastereoselecdve catalydc nitro-aldol reacdons of opdcally acdve iV-phthaloyl-c-phenyl-alanal with nitromethanein the presence of LLB proceed with high diastereoselecdvityfruirdryii = 99 11 as shown in Eq. 3.76. The product is converted via the Nef teacdon into f3S,35 -3-amino-3-hydroxy-4-phenylbutanoic acid, which is a subunit of the HIV-protease inhibitor... 

Davies and Reider (1996) have given some details of the HIV protease inhibitor CRDCIVAN (INDINAVIR) for which (lS,2R)-c -amino indanol is required. Indene is epoxidized enantioselectively, using the lacobsen strategy (SS-salen Mn catalyst, aqueous NaOH and PiNO), to (lS,2/ )-indene oxide in a two-phase system, in which the OH concentration is controlled. Indene oxide was subjected to the Ritter reaction with MeCN, in the presence of oleum, and subsequent hydrolysis and crystallization in the presence of tartaric acid gives the desired amino indanol. 

The amino acid residues of a protease substrate (or inhibitor) have been numerically designated P ... 

This approach has been mainly applied to peptide-based inhibitors of proteases, where the inhibitory molecule is a peptide with a transition state isostere appended to it, and the cognate substrate is simply a peptide of the same amino acid sequence, but lacking the isostere functionality. Examples where good correlations between the free energy of inhibitor binding and the free energy of kcJKM have been found, include peptide-trifluoromethyl ketone inhibitors of human leukocyte elastase (Stein et al., 1987) and peptide-phosphonamidate inhibitors of the metalloprotease ther-molysin (Bartlett and Marlowe, 1983). 

The human retrovirus HIV can be controlled using chemotherapy directed at the reverse transcriptase and aspartyl protease encoded by the viral genome as with other microbial pathogens, however, resistance to drug therapy becomes a major problem. Figure 7.3 shows a crystal structure (PDB 1HXW) of the HIV protease, where mutated amino acids (shown in cyan) lead to disrupted binding of the clinically effective inhibitor ritonavir [24]. 

Fig. 7.3 Binding of the HIV protease inhibitor ritonavir. Amino acids highlighted in cyan are mutated in resistant strains of the virus and tend to occur at the extremities of the inhibitor (see color plates, p. XXXIII).

From Sigma 3-aminoethylcarbazole (AEC) acrylamide/bis-acrylamide (30%) 37.5 1 amino acids alumina bentonite benzamidine bovine fiver tRNA bovine serum albumin (BSA) creatine phosphate (CP) diethyl pyrocarbonate (DEPC) dithiothreitol (DTT) Escherichia coli MRE600 tRNA pyrophosphatase (Ppase) Ca++ salt of folinic acid, (5-formyl THF) IIHPHS K salt of phospho-enol pyruvic acid, (PEP) creatine phospho kinase (CPK) protease inhibitor cocktail for fungal and yeast extracts phenylmethylsulfonyl fluoride (PMSF) spermidine trihydrochloride Tween 20. 

For example, a standard bond disconnection of a HIV protease inhibitor led to two key precursors AHPBA (3-amino 2-hydroxybutyric acid) and DMDFP (N-Boc 3,3-dimethyl 4,4-difluoroproline) (Scheme 5). 

The first protease inhibitor studied in human clinical trials was ciluprevir (BILN-2061), whose discovery was predicated on earlier studies that identified 54, a 6 amino acid N-terminal cleavage product of an NS5A/5B substrate, as a competitive inhibitor of NS3 [103,104], Although the development of ciluprevir was halted due to cardiotoxicity in animals, it demonstrated that a potent... 

Other potent peptide mimetic NS3 protease inhibitors have been reported that incorporate a serine trap on the C-terminal end of the peptide. Thus, the inhibitory activity of telaprevir (VX-950, 59), (7nM vs. NS3, 300 nM vs. the la replicon) is based on truncation of the polypeptide substrate, maximizing binding by alteration of amino acids at the scissile site, and capping both N- and C-terminal ends, the latter with a known dicarbonyl serine trap. This compound has exhibited impressive antiviral activity in animals, and showed a 4.4 log drop in viral load in genotype 1-infected patients in a Phase lb clinical trial [110]. Telaprevir is expected to enter Phase 3 clinical trials in 2007. Additional bicyclo-proline-based P2 tetrapeptides, represented by analog 60 (Kj = 22 nM), have been explored. Although the compounds are selective inhibitors of NS3, little or no cell-based replicon activity was reported, presumably due to poor cellular permeability [111-114], A diastereomer of telaprevir, has been reported to inhibit the replicon with an EC50 of 0.55 pM [115]. 

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