Nfifi TCJK 26303

departure from other MEP inhibitors which contain a modified di- (or tri-) peptide backbone, with a critical secondary amide bond and a zinc-chelating ligand. Modification of the C-terminal carboxylic acid functionality of (8.65) to a tetrazole led to a highly potent, non-peptide MEP inhibitor CGS 26303 (8.66).

As with ACE inhibitors, these MEP inhibitors are not well absorbed orally, which limits their potential therapeutic usefulness. To improve pharmacokinetic profiles, the inhibitors have been further developed as prodrugs such as sinorphan (prodrug of (S)-thiorphan), SCH 34826 (a lipophilic ester of 8.59), UK 79 300 (an indanyl ester of (+)-isomer of (8.62)) and CGS 25462 and CGS 26393 (the aminomethyl phosphonate derivatives of 8.64 and 8.66 respectively).

A different approach to improving potential therapeutic efficacy in the development of non-addictive analgesics has been the realisation of combined inhibitors of more than one enzyme in a single inhibitor. Kelatorphan (8.67) inhibits MEP, aminopeptidase N and dipeptidylaminopeptidase, the enzymes involved in inactivation at different points of the enkephalin pentapeptides in the CNS. A variation of this approach has been the concept of covalently linking two different types of inhibitor in a 'prodrug'. An aminopeptidase N (APN) inhibitor and a MEP inhibitor have been linked by a thioester or a disulphide bond in order to increase the hydrophobicity, and so absorption, of each molecule (8.68). Hydrolysis or reduction, respectively, leading to the release of the two active inhibitors, occurs once the compound has passed the blood brain barrier.


(8.67); kelatorphan

Combined inhibitors such as the mercaptoalkyl derivatives alatrioprilat (8.69) and glycoprilat (8.70) display both MEP and ACE inhibitory activity and are being assesed for their therapeutic potential in the treatment of cardiovascular diseases. Aspartate proteases

HIV protease inhibitors

Two genetically distinct subtypes, HIV-1 and HIV-2, of human immunodeficiency virus (HIV) have been identified. Reverse transcriptase inhibitors such as AZT have had limited success because of emergence of viral resistance and drug toxicity. Blockade of the virally encoded protease, which is critical for viral replication, has become a major target in the search for an effective anti-viral agent and several inhibitors are now under development.

HIV-1 protease catalyzes the conversion of a polyprotein precursor (encoded by gag and pol genes) to mature proteins needed for the production of infectious HIV particle. A highly conserved triad, Asp-Thr(Ser)-Gly, in the viral enzyme which is also found in mammalian proteases belonging to the aspartic acid family, suggested a similar mechanistic class for HIV protease. This has now been confirmed by elucidation of the crystal structure of the native HIV protease and the HIV protease complexed with aspartyl protease inhibitors. There are however significant structural differences between the retroviral and classical aspartyl proteases such as renin. Mammalian and fungal aspartyl proteases generally comprise of 200 amino acids and consist of two homologous domains with the key catalytic triad occuring twice.

The structure of HIV protease has been identified by X-ray crystallographic methods as a homodimer comprising of two identically folded subunits (each comprising of 99 amino acids). Each subunit contributes one of the two conserved aspartates (Asp 25 and Asp 125) to the single hydrophobic active site cavity. It is believed that during hydrolysis, a water molecule attacks the carbonyl carbon of the peptide bond of the substrate while the carbonyl oxygen accepts the proton from one of the catalytic aspartic acid residues leading to the formation of a tetrahedral transition state. Catalytic studies have suggested that in the transition state, one of the aspartic acid residues exists in the neutral form whereas the other residue is negatively charged. However, the protonation state of the protease aspartic acid residues in the complex with its inhibitors remains controversial. After the formation of the transition state, two conformationally flexible flaps (one per subunit) close around the substrate.

HIV-protease cleaves the polyprotein precursor at eight different sites, of which Tyr-Pro and Phe-Pro residues (occuring as P1-P1' at three of the cleavage sites of HIV-1), are of particular interest in relation to the development of inhibitors. The amide bonds N-terminal to proline are not hydrolysed by mammalian aspartic proteases and therefore offer selectivity for the viral enzyme. Leu-Ala, Leu-Phe, Met-Met, and Phe-Leu are also found at HIV-1 cleavage sites. The amino-acid sequences flanking the cleavage have been divided into three classes.

Class 1: Phe-Pro or Tyr-Pro at P1-P1' Class 2: Phe-Leu at P1-P1' and Arg at P4 Class 3: Gln or Glu at P2'

Studies using oligopeptides have shown that seven residues spanning P4-P3' are required for specific and efficient hydrolysis of the P1-P1' amide bond and crystallographic data suggests multiple hydrogen bonding to the backbone of inhibitors spanning this site and close van der Waals contact for the P3-P3' side chains.

Incorporation of a transition-state mimic into substrate analogues has been one of the strategies used in the development of enzyme inhibitors. Substitution of the scissile amide bond with non hydrolyzable dipeptide isosteres in appropriate sequence context has also proved to be successful in the development of potent renin inhibitors. A number of such dipeptide isosteres (inserted into a heptapeptide template spanning P4-P3' and which mimic the tetrahedral intermediate of peptide hydrolysis) have been evaluated. Hydroxyethylene (8.71), dihydroxyethylene (8.72) and hydroxyethylamine

(8.73) isosteres provide the greatest intrinsic affinity for the enzyme. The order of affinity of other isosteres for HIV-1 protease has been established as difluoroketones

(8.74)=statine (8.75)> phosphinate (8.76)>reduced amide isostere (8.77). The principle structural features in most transition state analogues designed to inhibit HIV protease is the critical hydroxyl group shown by x-ray analysis to bind both aspartic acid groups.

Pepstatin A (Iva-Val-Val-Sta-Ala-Sta), a natural product, contains two residues of the amino-acid statine. It is a non-specific inhibitor of aspartic acid proteases and inhibits several retroviral proteases, including the hydrolysis of both polyprotein and oligopeptide

substrates by HIV-1 protease. The concentration of inhibitor required to inhibit HIV-1 protease are significantly higher than those required for mammalian or fungal aspartic proteases. The structure of H-261 (8.78) mimics the cleavage sequence of the renin substrate angiotensinogen (Leu-Val). It is also non-specific and inhibits both HIV-1 (Ki=5 nM) and HIV-2 (Ki=35 nM) protease. Analogues incorporating the cyclohexyalanine-Val hydroxyethylene isostere, U-81749 (8.79, Ki=70 nM) and the dihydroxyethylene isostere of cyclohexylalanine-Val, U-75875 (8.80, Ki<1 nM) both show potent antiviral activity in cell cultures.

(8.79); U-81749
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