HCMV protease

In order to identify novel lead compounds with antiviral effects, methanol and aqueous extracts of some medicinal plants in the Zingiberaceae family were screened for inhibition of proteases from human immunodeficiency virus type 1 (HIV-1), hepatitis C virus (HCV) and human cytomegalovirus (HCMV). By bioassay-guided fractionation, eight fiavones were isolated from the black rhizomes of Kaempferia parviflora Wall, ex Baker. The most effective inhibitors, 5-hydroxy-7-methoxyfiavone and 5,7-dimethoxyflavone, inhibited HIV-1 protease, with an inhibitory concentration 50 (IC50) values of 19 0,M. Moreover, 5-hydroxy-3,7-dimethoxyflavone inhibited HCV protease and HCMV protease, with IC50 values of 190 and 250 pM, respectively. 

HCMV, a p-herpesvirus, is an opportunistic pathogen in immunocompromised individuals such as AIDS patients and organ transplant recipients [411]. Thus HCMV protease has become a viable target for antiviral chemotherapy [412, 413]. 

Several monobactams incorporating a benzyl side-chain at the C-4 carbon atom, such as compounds of Fig. 48, have been reported to be selective inhibitors of HCMV protease [367]. 

Gerona-Navarro and coworkers in 2004 have reported the synthesis and the evaluation of a series of new 2-azetidinones (Fig. 50), derived from phenylalanine [281], which were designed on the basis of the structure of the reported (3-lactam inhibitors [367] and the residues implicated in the active site of the HCMV protease [417]. These compounds have been evaluated against HCMV in human embryonic lung cells [418], and the results compared to those obtained for the reference compounds, which were the model (3-lactam la of Fig. 49, the viral DNA polymerase inhibitors DHPG (ganciclovir), and HPMPC (cidofovir). 

Human cytomegalovirus (HCMV) disease is the most common life-threatening opportunistic viral infection in the inmunocompromised. HCMV protease, a serine protease, plays a critical role in capsid assembly and viral maturation and is an attractive target for antiviral chemotherapy. Slater et al investigated the interaction of various 1,4-naphthoquinones derivatives with HCMV protease. They identified potent irreversible naphthoquinones inhibitors of HCMV protease which covalently modify... 

The protein superfamily of proteases [78, 79], however, is an ideal framework for a directed privileged structure-based masterkey concept. It has already been reported that the 5,5-trans-fused lactam moiety was systematically optimized and explored as a serine protease-directed scaffold by GlaxoSmithKline and has delivered progressible lead compounds for various members of that target class [3], such as thrombin [80, 81], elastase [82, 83], HCMV protease [84, 85], and the hepatitis C virus-encoded NS3-4A protease [86, 87]. Here, the initially identified scaffold was engineered toward the serine protease-wide commonality in substrate binding and processing [3],... 

The synthesis and biological activity of pyrimido[l,2-i>]-l,2,4,5-tetrazin-6-ones as HCMV protease inhibitors has been reported <03BMCL3483>. Novel 6-azapteridines from bifunctional... 

The first step in identification of herpesvirus inhibitors that act via this mechanism is the evaluation of compounds for their ability to inhibit the protease enzyme. A number of assay formats have been used to examine the effect of herpesvirus protease inhibitors. This chapter describes four of these assays, and their relative merits and problems are discussed. In particular, the conditions for HCMV protease are described, with reference to modifications for other herpesvirus proteases where possible in the notes. A source of purified recombinant enzyme will be assumed, because a number of studies have described the use of Escherichia coli to express active herpesvirus proteases from a number of viruses, along with suitable purification methods (4-6). 

Determine the concentration of enzyme required to achieve 30-50% substrate cleavage. This typically corresponds to a final concentration of approx 0.5 pM for HCMV protease. 

Determine the concentration of enzyme required to achieve 50-70% substrate cleavage. This can be calculated from the formula % cleavage = 100 x (SCcpm-samplecpm)/(SCcpm) assuming that the substrate is fully cleavable. A suitable enzyme concentration typically corresponds to a final concentration of approx 0.5 pM for HCMV protease. 

DMSO Compounds and peptides are commonly dissolved in DMSO as the most universal solvent. HCMV protease is tolerant of DMSO at up to 10% (v/v) with only slight loss in activity. Higher levels of DMSO may be used, and may be compensated for by reducing the level of glycerol in the assay buffer to 10%. 

Glycerol Glycerol has been shown to increase the activity of HCMV protease severalfold. Glycerol concentrations in the range of 10-50% are useful, but samples become hard to pipet and mix at higher concentrations. 

Table 1.22 Peptidyl methyl and perfluoroalkyl ketone inhibitors of HCMV protease...

V-Containine. Protein and Peptide Compounds Briodionen (318) Streptomyces sp. WC76599 Human CMV HCMV Protease [228]... 

X. P. Lin, L. Wenwei, Z. Xiaomin, Synthesis 2002, 1017-1026. Synthesis of a peptidomimetic HCMV protease inhibitor library. 

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