Potency and Stereoselectivity of Cyclopropavir Triphosphate Action on Human Cytomegalovirus DNA Polymerase

Abstract

C yclopropavir (CPV), a methylenecyclopropane analogue (1) of 2=-deoxyguanosine, is currently undergoing phase I clinical safety studies as a potential drug against human cytomegalovirus (HCMV) (2-4), a common opportunistic pathogen responsible for diseases in multiple organ systems, particularly in immunocompromised patients and newborns (5). Previous mechanism of action studies have revealed that CPV, like ganciclovir (GCV), the first-line therapy for HCMV infection, is initially phosphorylated by the virus-encoded UL97 kinase (6), while conversion of CPV monophosphate (CPV-MP) to CPV triphosphate (CPV-TP) can be performed by cellular kinases (7,8). Accordingly, certain HCMV UL97 mutations confer CPV resistance (9, 10). CPV inhibits viral DNA replication (3), and accumulation of CPV-TP has been detected during viral infection (11). As certain mutations affecting the catalytic subunit (UL54, Pol) of the HCMV DNA polymerase also confer CPV resistance, it has been hypothesized that CPV-TP targets HCMV Pol (12) and terminates viral DNA synthesis. However, whether and how CPV-TP acts to inhibit HCMV Pol have not been determined. In particular, whether CPV-TP inhibits competitively, whether it is a substrate for incorporation into DNA, and whether it causes chain termination are not known.Interestingly, CPV is substantially more potent than GCV against HCMV replication in cell culture (2, 3). Some of this increased potency may be due to greater accumulation in infected cells of CPV-TP than GCV-TP at equivalently effective concentrations, which is consistent with more extensive phosphorylation of CPV than GCV by UL97 (6, 11). However, CPV is also more potent than GCV against HCMV lacking UL97 (3, 9, 13). We hypothesized that this could be due to more potent inhibition of HCMV Pol by CPV-TP than by GCV-TP.The initial phosphorylation and the interaction of the triphosphate with viral polymerase are the most crucial steps in the determination of enantioselectivity of antiviral nucleoside analogues (14). Previous studies showed that the (ϩ) enantiomers are the preferred substrates during enzymatic conversion of CPV to CPVdiphosphate by UL97 and GMP kinase (6, 7). Although there is precedent for stereoselective inhibition of a herpesvirus polymerase by GCV-TP (15), whether CPV-TP is also stereoselective in its activity against HCMV Pol has not been tested.To investigate these questions, we synthesized CPV-TP in both enantiomeric forms and investigated their actions on HCMV Pol. MATERIALS AND METHODSChemicals and reagents. All solvents and reagents, unless otherwise indicated, were analysis-grade commercial products and were used as received. CPV-TP enantiomers were synthesized according to the routes presented in Fig. 1, as described below. CPV-TP enantiomers are soluble in water at a concentration of 5 mM, and their purity was confirmed using high-pressure liquid chromatography. Chemical syntheses. (i) (Z)-9-{[2-(Acetoxymethyl)-2-(hydroxymethyl)cyclopropylidene]methyl}-N2 -isobutyrylguanine phosphate (compound 3). A mixt...

Phase Ib Trial To Evaluate the Safety and Pharmacokinetics of Multiple Ascending Doses of Filociclovir (MBX-400, Cyclopropavir) in Healthy Volunteers

“…However, PK profiles of the unphosphorylated form of the drug in plasma may underestimate in vivo efficacy, as the active triphosphate form of filociclovir in CMV-infected cells (not measureable in uninfected individuals) is expected to decay less rapidly between doses than concentrations of filociclovir in plasma. Compared to that of ganciclovir, filociclovir has a 2-fold lower in vitro IC 50 (11,16,17), a 2-fold higher peak triphosphate level in infected cells (18), and an ϳ10-fold higher affinity of the nucleotide triphosphate form to CMV polymerase (6).…”

“…Additional phosphorylation steps are then catalyzed by cellular kinases to the di-and triphosphate forms. The triphosphate form inhibits viral DNA polymerase (expression of both phosphoproteins ppUL44 and pp28, essential for viral assembly and replication), which blocks viral replication (6)(7)(8)(9). Since uninfected cells do not contain pUL97, they are not expected to phosphorylate filociclovir.…”

Ganciclovir‐Resistant Cytomegalovirus Infection in Abdominal Solid Organ Transplant Recipients: Case Series and Review of the Literature

“…Like GCV, it requires UL97 ‐dependent phosphorylation for activation and subsequently inhibits CMV DNA polymerase . In vitro, it is shown to be more potent than GCV and retains activity against some GR‐CMV strains, theoretically due to stereoselectivity of the UL97 kinase . Preclinical studies in animals demonstrated anti‐CMV activity in vivo, including oral administration .…”

“…76 In vitro, it is shown to be more potent than GCV and retains activity against some GR-CMV strains, theoretically due to stereoselectivity of the UL97 kinase. 77 Preclinical studies in animals demonstrated anti-CMV activity in vivo, including oral administration. 78 However, its mechanism of action appears to be more similar to that of maribavir in that it does not directly inhibit DNA polymerase.…”

Synthesis of New Imidazopyridine Nucleoside Derivatives Designed as Maribavir Analogues

“…The development of Brincidofovir for therapy of HCMV has been halted because of increased gastrointestinal toxicity of the oral formulation in adult hematopoietic cell transplant recipients. The non-nucleoside guanosine analogue Cyclopropavir, which shares the same mechanism of action with GCV, has proven to be more potent in HCMV inhibition in vitro [ 12 ] and Phase I trials have been recently completed. Furthermore, the search for novel molecular targets within the viral life cycle has led to the fast track approval of the terminase inhibitor Letermovir in late 2017, for the prophylaxis of HCMV infection and disease in adult HCMV-seropositive recipients of an allogeneic human stem cell transplant (HSCT) [ 13 ].…”