Selective Nonpeptidic Inhibitors of Herpes Simplex Virus Type 1 and Human Cytomegalovirus Proteases.

Abstract

Herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV) belong to the family Herpesviridae and cause severe opportunistic infections in immunocompromised individuals, including organ transplant recipients and AIDS patients. [1][2][3] Present clinical therapies commonly use viral DNA polymerase inhibitors such as acyclovir and ganciclovir, but this approach has been accompanied by the emergence of resistant mutants and various toxicities. 1)Therefore, treatment clinics need new antiviral drugs for combating herpesvirus infections.Herpesviruses, including HSV-1 and HCMV, encode a protease that is essential for the production of infectious virus particles. 4,5) The proofs obtained by site-directed mutagenesis 6,7) and chemical modification with diisopropyl-fluorophosphate [8][9][10] indicate that this protease is a serine protease. Recent results for the crystal structures of the HCMV, [11][12][13][14] varicella-zoster virus, 15) HSV-1 16) and HSV-2 16) proteases have shown that these proteases exhibit a novel fold and possess a unique catalytic triad in which the third member of the triad has a histidine substituted for an aspartic acid residue. Since their discovery, herpesvirus proteases have become a novel target of antiviral drugs. In this paper, we report the characterization of several potent inhibitors of HSV-1 and HCMV proteases obtained by random screening of a chemical compound library with the reverse-phase high-performance liquid chromatography (HPLC) assay system. MATERIALS AND METHODS Construction of HSV-1 Protease Expression VectorThe plasmid pRB4057 17) possessing the UL26 gene of HSV-1 was used as the template for polymerase chain reaction (PCR) amplification of the coding sequence for the protease catalytic domain (amino acids 1-247). Oligonucleotide primers were synthesized based on the published UL26 open reading frame.18) PCR was performed using Pwo DNA polymerase (Boehringer Mannheim) for 30 cycles of 2 min at 95°C, 2 min at 68°C and 2 min at 72°C. The resulting amplification product was digested with EcoRI and SmaI before ligation into corresponding sites of the pGEX-4T-1 (Pharmacia Biotech) plasmid. The resulting plasmid pGSTThHP was introduced into Escherichia coli BL21(DE3) pLysS to express HSV-1 protease fused in frame to glutathione S-transferase.Expression and Purification of HSV-1 Protease The bacterial cells harboring pGSTThHP were cultured in 1 l of 2ϫYT medium (1.6% Bacto tryptone, 1% Bacto yeast extract, 0.5% NaCl) containing 100 mg/ml ampicillin at 30°C to an OD 660 of 4, at which time isopropyl-1-thio-b-D-galactopyranoside was added to a final concentration of 0.1 mM, then growth was continued for 14 h. We then resuspended 11 g (wet weight) of the bacterial cell pellet in 55 ml of 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 1 mM EDTA, lysed the cells by sonication, and ultracentrifuged them at 180000ϫg for 30 min at 4°C. This supernatant was loaded on a glutathione Sepharose 4B column (Pharmacia Biotech), which was equilibrated with 50 mM Tris-HCl, pH 8.0, 150 mM NaCl,...

Modern Ethiotropic Chemotherapy of Herpesvirus Infections: Advances, New Trends and Perspectives. Alphaherpesviruses (Part Ii)

“…Несмотря на сходство структур, 1,4-дигидроксинафталин ингибирует протеазу ВПГ-1 по конкурентному механизму, тогда как 3 нафтохинона являются неконкурентными ингибиторами. Действие этих соединений селективно в отношении герпетических протеаз, они не проявляют существенной ингибирующей активности против сериновых протеаз млекопитающих (химотрипсин, трипсин, калликреин, плазмин, тромбин и фактор Xa) в концентрации 100 мкМ [43]. Таким образом, вышеописанные непептидные соединения могут представлять интерес для дальнейшего изучения их активности in vivo.…”

Protease inhibitors and their peptidomimetic derivatives as potential drugs

“…Through screening of a compound library, Matsumoto et al (2001) discovered 4 compounds, 1,4-dihydroxynaphthalene and 3 naphthoquinones, which effectively inhibit HSV-1 protease. Further study indicated that these compounds were potent and selective inhibitors of CMV protease, with extremely low IC 50 values.…”