115 Protease Inhibitors

Inhibitors of well known metallo-, sulfhydi yl and serine proteases block viral proteolysis in infected cells. The use of iodoacetamide (lAM) to stabilize poliovirus and foot-and-mouth virus precursors has been described (36, 52). The preciirsors were subsequently cleaved in vitro to yield normal-sized products (36)1 and polio RHA replicase activity was stabilized in cell-free extracts of lAM-treated cells (55) H-ethyl maleimide inactivates an EMC virus-specific protease in reticulocyte lysates (20). These results could signify that a sulfhydryl-type protease, e.g., cathepsin B, is required for some of the cleavage reactions. However, when such compounds are added to cells, numerous proteins react, and there is substantial inhibition of protein synthesis. Energy metabolism in such cells is depressed, and ATP-reqiiiring proteolytic reactions blocked. 

Inhibition of HeLa Cell Protein Synthesis by Bhinovirus Type 1A 

The protease/esterase inhibitor diisopropyl phosphofluoridate (LFP) was shown to block the cleavage of poliovirus polyprotein (7) This implicates a protease with a serine-containing active site. 

The most interesting class of protease inhibitors in viral studies has been the chloromethyl ketone derivatives of amino acids (54j 55) These were designed as affinity labels of serine-type proteases, and react irreversibly with histidine and serine residues in the active sites of proteases. There is a basis for selectivity of the chloromethyl ketones phenylalanyl and lysyl derivatives were synthesized, which had specificity for chymotrypsin and trypsin, respectively (54) This specificity led to studies on inhibition of poliovirus protein cleavage, with positive results (25, 26). 

In summary, chemical inhibitors of cleavage are available, but one must be foimd which is absolutely directed against either a viral substrate or a virus-specific protease. Additional experimentation will be required, if eventually an inhibitor of proteolysis is to develop into a clinically useful antiviral. 

Another kind of drug is targeted at an enzyme called protease which is present in HIV-1 virus. The virus needs to produce some proteins and enzymes besides its RNA in order to replicate itself. Two genes called gag and pol (on the RNA) produces a multiprotein (let us call it gag-pol protein ) that contains two proteins fused together. The enzyme HIV-1 protease splits this fused protein into the individual active structural proteins. If this enzyme is made inactive, the essential structural proteins cannot be produced, and hence the virus becomes noninfectious. This suggests that a drug may be created that inhibits this enzyme. 

Protease (also called proteinase) is an enzyme that splits the bond called peptide bond that connects amino acids in a protein. The reaction is a hydrolysis that means splitting of a bond by adding water and is expressed as follows  

There are several types of proteases. HIV protease is an example of aspartic acid proteases. The structure of HIV protease as determined by X-ray crystallography is shown in Fig. 21.11. How the peptide bond is cleaved is shown schematically in Fig. 7.3. The set of two aspartic acid residues shown catalyzes the addition of water molecule to gag-pol protein (a). The result is the formation of an intermediate shown as (b) in the figure, (b) is known to decompose into two separate entities as shown in (c). This completes the hydrolysis. The region in the gag-pol protein where this splitting occurs has an amino acid sequence of -Leu-Asn-Phe-Pro-lle- (Asn=aparagine, He=isoleucine, Leu=leucine, Phe=phenyl alanine, Pro=proline). 

then how would you make the enzyme inactive Compound (A) or (B) would bind to the enzyme with certain strength. If another compound binds to the same spot of the enzyme with a greater strength and yet cannot be decomposed, it will block the binding of the proper compound (A) or (B). And hence, (A) would not be able to bind and undergo hydrolysis. This is the idea of inhibition of an enzyme. 

A number of drug companies have tried to develop AIDS drugs based on this principle. These drugs are hence called HIV protease inhibitors. Four HIV protease inhibitors have been approved for use in treating AIDS. They are saquinavir (trade name=Inverase developed by Hoffman-La Roche), ritonavir (Norvir by Abbots Lab), indinavir sulfate (Crixivan by Merck), and nellinavir mesylate (Viracept by Agouron Pharmaceuticals). All of these compounds try to mimic the structure of intermediate (B), but they are not decomposable. 

Antiviral Efficacy and Clinical Use. These drugs inhibit an enzyme known as HIV protease. This enzyme is needed to manufacture specific HIV proteins, in- 

Protease inhibitors are often incorporated into the comprehensive treatment of people with HIV infection. Use of these drugs in combination with other anti-HIV agents is discussed in more detail later in this chapter (see HIV and the Treatment of AIDS ). In addition, a specific protease inhibitor can be combined with a low dose of ritonavir—a process known as protease-inhibitor boosting. 34,69 Ritonavir inhibits the hepatic breakdown of the other (primary) protease drug, thereby enabling the primary drug to exert better therapeutic effects at a lower dose.34 

Mechanism of Action. Protease inhibitors bind to the HIV protease and prevent this enzyme from acting on HIV substrates.32 This effect negates the ability of the protease enzyme to cleave polypeptide precursors from larger, polypeptide chains.32 If these precursors are not available for the manufacture of HIV proteins, the virus cannot fully develop.102 Treatment with protease inhibitors therefore results in the manufacture of incomplete and noninfectious fragments of HIV rather than the mature virus.32 

Thermolysin (TLN) is a well-characterized zinc endopeptidase that hydrolyzes peptide bonds linking two hydrophobic residues. Several FEP studies have focused on TLN, since high resolution X-ray structures for TLN and a 

The initial FEP calculations by Kollman and coworkers on TLN focused on tripeptide analogs in which the peptide bond was replaced by P(02)X to form a phosphoramidate (X = NH), a phosphonate (X = O), and a phosphinic acid (X = CH2). The FEP calculations were carried out using AMBER and the TLN-phosphoramidate X-ray structure. The for the 

Rhizopus pepsin belongs to the aspartyl protease family, whose members are distributed in microorganisms, plants, and animals. This protease class cleaves peptide bonds on the N-terminal side of Phe or Tyr. 

In an FEP study, Rao and Singh used AMBER 3.3 to determine the relative binding free enei of pepstatin and its derivatives to Rhizopus pepsin. A united-atom and an all-atom representation were used for the residues of the enzyme and for the inhibitors, respectively. The pepstatin-Rhizopus pepsin complex was modeled starting from the high resolution crystal structure, where the inhibitor was in the reduced form. All pepstatin derivatives studied had modifications made on statine. The AAGlfia mutation of 

Trypsin is a digestive enzyme that hydrolyzes peptides on the C-terminal side of basic amino acid residues. Trypsin is one of the most well-characterized enzymes with a crystal structure first solved in 1972.i ° 

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Lebon et al., 1996] Lebon, F., Vinals, C., Feytmans, E., and Durant, F. Computational drug design of new HIV-1 protease inhibitors. Arch. Phys. Biochem. 104 (1996) B44. 

Structure-based Design Methods to Design HiV-1 Protease Inhibitors... 

Flow chart showing the design of novel orally active HIV-1 protease inhibitor. (Figure adapted from Lam P K ]adhav, C E Eyermann, C N Hodge, Y Ru, L T Bacheler, ] L Meek, M ] Otto, M M Rayner, Y N V /ong, ang, P C Weber, D A Jackson, T R Sharpe and S Erickson-Viitanen 1994. Rational Design of Potent, able. Nonpeptide Cyclic Ureas as HIV Protease Inhibitors. Science 263 380-384.)... 

Extraction of hGH from pituitary glands has been accompHshed by a variety of procedures with or without protease inhibitors. One of the first commercial procedures used glacial acetic acid at 70°C to extract the hGH from the glands (39). 

Sequences have been determined for plasminogen and bovine Factor XII, and they are not homologous with the other serine proteases. The amino-terminal sequence of Factor XII is homologous, however, with the active site of several naturally occurring protease inhibitors (11). 

Permeation enhancers are used to improve absorption through the gastric mucosa. Eor example, oral dehvery of insulin (mol wt = 6000) has been reported from a water-in-oH- emulsion containing lecithin, nonesterified fatty acids, cholesterol [57-88-5], and the protease inhibitor aprotinin [9087-70-1] (23). 

Phenylmethanesulfonyl fluoride (PMSF) [329-98-6] M 174.2, m 90-91 , 92-93 . Purified by recrystn from ""CgHe, pet ether or CHCl3-pet ether. [Davies and Dick J Chem Soc 483 1932 cf Tullock and Coffman J Org Chem 23 2016 I 960.] It is a general protease inhibitor (specific for trypsin and chymotrypsin) and is a good substitute for diisopropylphosphoro floridate [Fahrney and Gould 7 Am Chem Soc 85 997 1963]. 

SG Deeks, M Smith, M Holdniy, JO Kahn. HIV-1 protease inhibitors A review for clinicians. J Am Med Assoc 277 145-153, 1997. 

Test by pooling fractions to detect cofactor Add protease inhibitors... 

The most recent advance in treating HIV infections has been to simultaneously attack the virus on a second front using a protease inhibitor. Recall from Section 27.10 that proteases are enzymes that catalyze the hydrolysis of proteins at specific points. When HIV uses a cell s DNA to synthesize its own proteins, the initial product is a long polypeptide that contains several different proteins joined together. To be useful, the individual proteins must be separated from the aggregate by protease-catalyzed hydrolysis of peptide bonds. Protease inhibitors prevent this hydrolysis and, in combination with reverse transcriptase inhibitors, slow the reproduction of HIV. Dramatic reductions in the viral load in HIV-infected patients have been achieved with this approach. 

Protease inhibitor (Section 28.13) A substance that interferes with enzyme-catalyzed hydrolysis of peptide bonds. 

Protease Inhibitors Give Life to AIDS Patients... 

Candidate protease inhibitor drugs must be relatively specific for the HIV-1 protease. Many other aspartic proteases exist in the human body and are essential to a variety of body functions, including digestion of food and processing of hormones. An ideal drug thus must strongly inhibit the HIV-1 protease, must be delivered effectively to the lymphocytes where the protease must be blocked, and should not adversely affect the activities of the essential human aspartic proteases. 

A final but important consideration is viral mutation. Certain mutant HIV strains are resistant to one or more of the protease inhibitors, and even for patients who respond initially to protease inhibitors it is possible that mutant viral forms may eventually thrive in the infected individual. The search for new and more effective protease inhibitors is ongoing. 

Chen, Z., Li, Y, Schock, H. B., et al., 1995. Three-dimensional structure of a mutant HIV-1 protease displaying cros.s-resi.stance to all protease inhibitors in clinical txiAs. Journal of Biological Chemistry 270 21433-21436. 

A Scheme for the preparation of a series of eyelie urea HIV protease inhibitors eontaining alkynyl-tethered heteroeyeles in the P2 region ineludes hydrogenation with LiAlH4 in THF in 60-80% yields (96BMCL797) (Sehemes 83 and 84). 

In the rosary pea Abrus precatorius L. Trigollenine as well as its gallic acid ester Precatorine (209) is found (71P195) (Scheme 69). 1-Carboxymethyl-nicotinic acid (210) was isolated as a colorless solid from the marine sponge Anthosigmella cf. raromicrosclera as a cysteine protease inhibitor (98JNP671). This compound was first synthesized in 1991. The sodium... 

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... 

The Henry reactions of A, ALdibenzyl-L-phenylalaninal with nitroalkanes using 1.2 equiv of tetrabutylammonium fluoride (TBAF) as the catalyst proceed in ahighly stereoselective manner, as shown in Eqs. 3.82 and 3.83. This reaction provides rapid and stereoselective access to important molecules containing 1,3-diamino-2-hydroxypropyl segments, which are cenhal structural subunit of the HIV protease inhibitor amprenavir (in Scheme 3.21). 

Today, 3D databases, which provide the means for storing and searching for 3D information of compounds, are proven to be useful tools in drug discovery programs. This is well exemplified with the recent discovery of novel nonpeptide HIV-1 protease inhibitors using pharmacophore searches of the National Cancer Institute 3D structural database [13-15]. 

P-Blockers, benzodiazepines, NSAIDs, barbiturates NSAIDs, protease inhibitors, P-blockers, benzodiazepines Antimalarials, NSAIDs, P-blockers, bronchodilators Phosphine oxides, NSAIDs, anticonvulsants Bronchodilators, P-blockers... 

Efficient coupling between a chiral 3-phenylpropionamide enolate and (S)-glyci-dyl tosylate was achieved in a practical route to the HIV-1 protease inhibitor L-735-524 [68b]. 

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