Cyclopropavir Research Articles

In vitro evaluation of current and novel antivirals in combination against human cytomegalovirus

Human cytomegalovirus (HCMV) can cause severe disease in patients with compromised or immature immune systems. Currently approved pharmacotherapies for the treatment of systemic HCMV infections [ganciclovir (GCV), cidofovir (CDV), foscarnet] are limited by a high incidence of adverse effects and/or the development of drug resistance. Given that many of these drugs have the same viral target (HCMV-encoded DNA polymerase), cross-resistance is relatively common. The primary means to combat drug resistance is combination pharmacotherapy using therapeutics with different molecular mechanisms of action with the expectation that those combinations result in an additive or synergistic enhancement of effect; combinations that result in antagonism can, in many cases, be detrimental to the outcome of the patient. We therefore tested select combinations of approved (GCV, CDV, letermovir (LMV)) and experimental (brincidofovir (BCV), cyclopropavir (CPV), maribavir (MBV), BDCRB) drugs with the hypothesis that combinations of drugs with different and distinct molecular mechanisms of action will produce an additive and/or synergistic enhancement of antiviral effect against HCMV in vitro. Using MacSynergy II (a statistical package that measures enhancement or lessening of effect relative to zero/additive), select drug combination studies demonstrated combination indices ranging from 160 to 372 with 95% confidence intervals greater than zero indicating that these combinations elicit a synergistic enhancement of effect against HCMV in vitro. These data suggest that administration of a viral DNA polymerase inhibitor, MBV, and/or a viral terminase inhibitor in combination has the potential to address the resistance/cross-resistance problems associated with currently available therapeutics.

Metabolism of Cyclopropavir and Ganciclovir in Human Cytomegalovirus-Infected Cells

Human cytomegalovirus (HCMV) is a widespread pathogen that can cause severe disease in immunologically immature and immunocompromised patients. The current standard of therapy for the treatment of HCMV infections is ganciclovir (GCV). However, high incidence rates of adverse effects are prevalent and limit the use of this drug. Cyclopropavir (CPV) is 10-fold more effective against HCMV in vitro than GCV (50% effective concentrations [EC50s]=0.46 and 4.1 μM, respectively) without any observed increase in cytotoxicity (S. Zhou, J. M. Breitenbach, K. Z. Borysko, J. C. Drach, E. R. Kern, E. Gullen, Y. C. Cheng, and J. Zemlicka, J. Med. Chem. 47:566-575, 2004, doi:10.1021/jm030316s). We have previously determined that the viral protein kinase pUL97 and endogenous cellular kinases are responsible for the conversion of CPV to a triphosphate (TP), the active compound responsible for inhibiting viral DNA synthesis and viral replication. However, this conversion has not been observed in HCMV-infected cells. To that end, we subjected HCMV-infected cells to equivalently effective concentrations (∼5 times the EC50) of either CPV or GCV and observed a time-dependent increase in triphosphate levels for both compounds (CPV-TP=121±11 pmol/10(6) cells; GCV-TP=43.7±0.4 pmol/10(6) cells). A longer half-life was observed for GCV-TP (48.2±5.7 h) than for CPV-TP (23.8±5.1 h). The area under the curve for CPV-TP produced from incubation with 2.5 μM CPV was 8,680±930 pmol·h/10(6) cells, approximately 2-fold greater than the area under the curve for GCV-TP of 4,520±420 pmol·h/10(6) cells produced from incubation with 25 μM GCV. We therefore conclude that the exposure of HCMV-infected cells to CPV-TP is greater than that of GCV-TP under these experimental conditions.

Resistance of Human Cytomegalovirus to Cyclopropavir Maps to a Base Pair Deletion in the Open Reading Frame of UL97

Human cytomegalovirus (HCMV) is a widespread pathogen in the human population, affecting many immunologically immature and immunocompromised patients, and can result in severe complications, such as interstitial pneumonia and mental retardation. Current chemotherapies for the treatment of HCMV infections include ganciclovir (GCV), foscarnet, and cidofovir. However, the high incidences of adverse effects (neutropenia and nephrotoxicity) limit the use of these drugs. Cyclopropavir (CPV), a guanosine nucleoside analog, is 10-fold more active against HCMV than GCV (50% effective concentrations [EC50s] = 0.46 and 4.1 μM, respectively). We hypothesize that the mechanism of action of CPV is similar to that of GCV: phosphorylation to a monophosphate by viral pUL97 protein kinase with further phosphorylation to a triphosphate by endogenous kinases, resulting in inhibition of viral DNA synthesis. To test this hypothesis, we isolated a CPV-resistant virus, sequenced its genome, and discovered that bp 498 of UL97 was deleted. This mutation caused a frameshift in UL97 resulting in a truncated protein that lacks a kinase domain. To determine if this base pair deletion was responsible for drug resistance, the mutation was engineered into the wild-type viral genome, which was then exposed to increasing concentrations of CPV. The results demonstrate that the engineered virus was approximately 72-fold more resistant to CPV (EC50 = 25.8 ± 3.1 μM) than the wild-type virus (EC50 = 0.36 ± 0.11 μM). We conclude, therefore, that this mutation is sufficient for drug resistance and that pUL97 is involved in the mechanism of action of CPV.

Synthesis and antiviral activities of methylenecyclopropane analogs with 6-alkoxy and 6-alkylthio substitutions that exhibit broad-spectrum antiviral activity against human herpesviruses.

Methylenecyclopropane nucleosides have been reported to be active against many of the human herpesviruses. The most active compound of this class is cyclopropavir (CPV), which exhibits good antiviral activity against human cytomegalovirus (HCMV), Epstein-Barr virus, both variants of human herpesvirus 6, and human herpesvirus 8. CPV has two hydroxymethyl groups on the methylenecyclopropane ring, but analogs with a single hydroxymethyl group, such as the prototypical (S)-synguanol, are also active and exhibit a broader spectrum of antiviral activity that also includes hepatitis B virus and human immunodeficiency virus. Here, a large set of monohydroxymethyl compounds with ether and thioether substituents at the 6 position of the purine was synthesized and evaluated for antiviral activity against a range of human herpesviruses. Some of these analogs had a broader spectrum of antiviral activity than CPV, in that they also inhibited the replication of herpes simplex viruses 1 and 2 and varicella-zoster virus. Interestingly, the antiviral activity of these compounds appeared to be dependent on the activity of the HCMV UL97 kinase but was relatively unaffected by the absence of thymidine kinase activity in HSV. These data taken together indicate that the mechanism of action of these analogs is distinct from that of CPV. They also suggest that they might be useful as broad-spectrum antiherpesvirus agents and may be effective in the treatment of resistant virus infections.

Cyclopropavir Susceptibility of Cytomegalovirus DNA Polymerase Mutants Selected after Antiviral Drug Exposure

Human cytomegalovirus (CMV) UL54 DNA polymerase (pol) mutants with known patterns of resistance to current antivirals ganciclovir (GCV), foscarnet (FOS), and cidofovir (CDV) were tested for cyclopropavir (CPV) susceptibility by a standardized reporter-based yield reduction assay. Exonuclease and A987G (region V) mutations at codons commonly associated with dual GCV-CDV resistance in clinical isolates paradoxically conferred increased CPV susceptibility. Various polymerase catalytic region mutations conferring FOS resistance with variable low-grade GCV and CDV cross-resistance also conferred CPV resistance, with 50% effective concentration (EC(50)) increases of 3- to 13-fold. CPV EC(50) values against several pol mutants were increased about 2-fold by adding UL97 mutation C592G. Propagation of a CMV exonuclease mutant under CPV selected for pol mutations less often than UL97 mutations. In 21 experiments, one instance each of mutations E756D and M844V, which were shown individually to confer 3- to 4-fold increases in CPV EC(50), was detected. Unlike GCV and CDV, exonuclease mutations are not a preferred mechanism of CPV resistance, but mutations in and near pol region III may confer CPV resistance by affecting its recognition as an incoming base for DNA polymerization.

Cyclopropavir inhibits the normal function of the human cytomegalovirus UL97 kinase.

Cyclopropavir (CPV) is active against human cytomegalovirus (CMV), as well as both variants of human herpesvirus 6 and human herpesvirus 8. The mechanism of action of CPV against CMV is similar to that of ganciclovir (GCV) in that it is phosphorylated initially by the CMV UL97 kinase, resulting in inhibition of viral DNA synthesis. Resistance to CPV maps to the UL97 kinase but is associated primarily with H520Q mutations and thus retains good antiviral activity against most GCV-resistant isolates. An examination of CMV-infected cultures treated with CPV revealed unusual cell morphology typically associated with the absence of UL97 kinase activity. A surrogate assay for UL97 kinase activity confirmed that CPV inhibited the activity of this enzyme and that its action was similar to the inhibition seen with maribavir (MBV) in this assay. Combination studies using real-time PCR indicated that, like MBV, CPV also antagonized the efficacy of GCV and were consistent with the observed inhibition of the UL97 kinase. Deep sequencing of CPV-resistant laboratory isolates identified a frameshift mutation in UL27, presumably to compensate for a loss of UL97 enzymatic activity. We conclude that the mechanism of action of CPV against CMV is complex and involves both the inhibition of DNA synthesis and the inhibition of the normal activity of the UL97 kinase.

Present status and advances in pharmacotherapy for cytomegalovirus retinitis associated with acquired immunodeficiency syndrome

Cytomegalovirus retinitis has been the most common opportunistic infection and leading cause of visual loss in AIDS patients. There are five drugs approved by the Food and Drug Administration in the USA for the treatment of cytomegalovirus retinitis: ganciclovir, foscarnet/phosphonoformic acid, valganciclovir, cidofovir and formivirsen. Progress has been made in the treatment of cytomegalovirus retinitis. Nowadays, maribavir, monoclonal antibody MSL-109, cyclopropavir and BAY 38-4766 are entering clinical trials. This review summarizes present status and advances in pharmacotherapy for cytomegalovirus retinitis in AIDS.

Phosphorylation of antiviral and endogenous nucleotides to di- and triphosphates by guanosine monophosphate kinase

Many fraudulent nucleosides including the antivirals acyclovir (ACV) and ganciclovir (GCV) must be metabolized to triphosphates to be active. Cyclopropavir (CPV) is a newer, related guanosine nucleoside analog that is active against human cytomegalovirus (HCMV) in vitro and in vivo. We have previously demonstrated that CPV is phosphorylated to its monophosphate (CPV-MP) by the HCMV pUL97 kinase. Consequently, like other nucleoside analogs phosphorylated by viral kinases, CPV most likely must be converted to a triphosphate (CPV-TP) in order to elicit antiviral activity. Once formed by pUL97, we hypothesized that guanosine monophosphate kinase (GMPK) is the enzyme responsible for the conversion of CPV-MP to CPV-DP. Incubation of CPV-MP with GMPK resulted in the formation of CPV-DP and, surprisingly, CPV-TP. When CPV-DP was incubated with GMPK, a time-dependent increase in CPV-TP occurred corresponding to a decrease in CPV-DP thereby demonstrating that CPV-DP is a substrate for GMPK. Substrate specificity experiments revealed that GMP, dGMP, GDP, and dGDP are substrates for GMPK. In contrast, GMPK recognized only acyclovir and ganciclovir monophosphates as substrates, not their diphosphates. Kinetic studies demonstrated that CPV-DP has a K M value of 45 ± 15 μM. We were, however, unable to determine the K M value for CPV-MP directly, but a mathematical model of experimental data gave a theoretical K M value for CPV-MP of 332 ± 60 μM. We conclude that unlike many other antivirals, cyclopropavir can be converted to its active triphosphate by a single cellular enzyme once the monophosphate is formed by a virally encoded kinase.

Stereoselective Phosphorylation of Cyclopropavir by pUL97 and Competitive Inhibition by Maribavir

Human cytomegalovirus (HCMV) is a widespread pathogen that can cause severe disease in immunologically immature and immunocompromised individuals. Cyclopropavir (CPV) is a guanine nucleoside analog active against human and murine cytomegaloviruses in cell culture and efficacious in mice by oral administration. Previous studies established that the mechanism of action of CPV involves inhibition of viral DNA synthesis. Based upon this action and the structural similarity of CPV to ganciclovir (GCV), we hypothesized that CPV must be phosphorylated to a triphosphate to inhibit HCMV DNA synthesis and that pUL97 is the enzyme responsible for the initial phosphorylation of CPV to a monophosphate (CPV-MP). We found that purified pUL97 phosphorylated CPV 45-fold more extensively than GCV, a known pUL97 substrate and the current standard of treatment for HCMV infections. Kinetic studies with CPV as the substrate for pUL97 demonstrated a Km of 1,750+/-210 microM. Introduction of 1.0 or 10 nM maribavir, a known pUL97 inhibitor, and subsequent Lineweaver-Burk analysis demonstrated competitive inhibition of CPV phosphorylation, with a Ki of 3.0+/-0.3 nM. Incubation of CPV with pUL97 combined with GMP kinase [known to preferentially phosphorylate the (+)-enantiomer of CPV-MP] established that pUL97 stereoselectively phosphorylates CPV to its (+)-monophosphate. These results elucidate the mechanism of CPV phosphorylation and help explain its selective antiviral action.

Stereospecific Phosphorylation of Cyclopropavir by pUL97 and Inhibition by Maribavir

Stereospecific Phosphorylation of Cyclopropavir by pUL97 and Inhibition by Maribavir