Novel Chemical Class of pUL97 Protein Kinase-Specific Inhibitors with Strong Anticytomegaloviral Activity

Abstract

Human cytomegalovirus (HCMV) is a major human pathogen frequently associated with life-threatening disease in immunosuppressed patients and newborns. The HCMV UL97-encoded protein kinase (pUL97) represents an important determinant of viral replication. Recent studies demonstrated that pUL97-specific kinase inhibitors are powerful tools for the control of HCMV replication. We present evidence that three related quinazoline compounds are potent inhibitors of the pUL97 kinase activity and block in vitro substrate phosphorylation, with 50% inhibitory concentrations (IC 50 s) between 30 and 170 nM. Replication of HCMV in primary human fibroblasts was suppressed with a high efficiency. The IC 50 s of these three quinazoline compounds (2.4 ؎ 0.4, 3.4 ؎ 0.6, and 3.9 ؎ 1.1 M, respectively) were in the range of the IC 50 of ganciclovir (1.2 ؎ 0.2 M), as determined by the HCMV green fluorescent protein-based antiviral assay. Importantly, the quinazolines were demonstrated to have strong inhibitory effects against clinical HCMV isolates, including ganciclovir-and cidofovir-resistant virus variants. Moreover, in contrast to ganciclovir, the formation of resistance to the quinazolines was not observed. The mechanisms of action of these compounds were confirmed by kinetic analyses with infected cells. Quinazolines specifically inhibited viral early-late protein synthesis but had no effects at other stages of the replication cycle, such as viral entry, consistent with a blockage of the pUL97 function. In contrast to epithelial growth factor receptor inhibitors, quinazolines affected HCMV replication even when they were added hours after virus adsorption. Thus, our findings indicate that quinazolines are highly efficient inhibitors of HCMV replication in vitro by targeting pUL97 protein kinase activity.

Cytomegaloviral protein kinase pUL97 interacts with the nuclear mRNA export factor pUL69 to modulate its intranuclear localization and activity

“…This finding prompted us to analyse additional protein kinase inhibitors, particularly those inhibiting the viral protein kinase pUL97. Two inhibitors of pUL97 kinase activity, Ax7396 and Gö6976, belonging to two different chemical classes, quinazoline and indolocarbazole, both of which inhibit pUL97 kinase activity as well as HCMV replication in vitro (Herget et al, 2004;Marschall et al, 2001Marschall et al, , 2002Schleiss et al, 2008), also induced intranuclear microspeckled accumulation of pUL69 similarly to roscovitine (Fig. 1a,v,vi and viii).…”

“…of 1) and analysed 72 h p.i. During HCMV replication, the following protein kinase inhibitors were used: indolocarbazoles (2 mM), Gö6976 (active against HCMV pUL97 and PKC) and Gö7874 (active only against PKC; Marschall et al, 2001Marschall et al, , 2002; quinazoline (10 mM), Ax7396 [active against HCMV pUL97 and epidermal growth factor receptor (EGFR); Herget et al, 2004]; tyrosine kinase inhibitors (2 mM), AG490 (tyrphostin; active against Janus kinases), AG1478 and PD153035 (active against EGFR); CDK inhibitors (10 mM), Rosco (roscovitine, active against CDKs 1, 2, 7 and 9) and Flavo (flavopiridol, active against CDKs 1, 2, 4 and 9). In vitro kinase assay (IVKA).…”

“…Previous studies demonstrated that deletion of the ORF UL97 from the viral genome or pharmacological inhibition of pUL97 kinase activity drastically reduced virus replication in cell culture (Biron et al, 2002;Herget et al, 2004;Marschall et al, 2002;Prichard et al, 1999). pUL97 phosphorylates the viral DNA polymerase processivity factor pUL44; therefore it may be important for viral DNA replication (Marschall et al, 2003).…”

Synthesis and Antimicrobial Evaluation of Some Tricyclic Substituted benzo[h]quinazolines, benzo[h]quinolines and naphthaleno[d]thiazoles

“…Quinazoline nucleus is one of fused heterocyclic ring systems that possesses a wide array of biological activities. These pharmacological activities include DNA binding 4 , antitumor [5][6][7] , benzodiazepine and GABA receptor ligand activity 8 , antiviral 9,10 , antibacterial 11 , antituberculosis 12,13 and cellular phosphorylation and tyrosine kinase inhibition 14 . Many marketed drugs comprise quinazoline nucleus in their structures such as; , treatment of lupus 25 , treatment of neurodegenerative diseases 26 , antitubercular 27,28 , antiprotozoal 29,30 and anticancer 31,32 .…”

One-pot reductive cyclization to antitumor quinazoline precursors

“…[5][6][7] Among other pharmacological activities, quinazoline derivatives show remarkable antimicrobial properties against microorganisms associated with death in patients carrying immunocompromised diseases. 8 We recently embarked on a program to synthesize anilinoquinazoline analogs as experimental cancer therapeutics. The preparation of The syntheses of quinazolinone derivatives has been accomplished by cyclocondensation of formamide with anthranilic acid derivatives (Scheme 1, route a) or the less common FriedelCrafts annulation of aminoaryl substrates (Scheme 1, route b).…”