Human cytomegalovirus (HCMV) remains the leading viral cause of birth defects and life-threatening disease in transplant recipients. All approved antiviral drugs target the viral DNA polymerase and are associated with severe toxicity issues and the emergence of drug resistance. Attempts to discover improved anti-HCMV drugs led to the identification of the small-molecular-weight compound AIC246 (Letermovir). AIC246 exhibits outstanding anti-HCMV activity in vitro and in vivo and currently is undergoing a clinical phase IIb trial. The initial mode-of-action studies suggested that the drug acts late in the HCMV replication cycle via a mechanism distinct from that of polymerase inhibitors. Here, we extend our mode-of-action analyses and report that AIC246 blocks viral replication without inhibiting the synthesis of progeny HCMV DNA or viral proteins. The genotyping of mutant viruses that escaped AIC246 inhibition uncovered distinct point mutations in the UL56 subunit of the viral terminase complex. Marker transfer analyses confirmed that these mutations were sufficient to mediate AIC246 resistance. The mapping of drug resistance to open reading frame UL56 suggests that viral DNA processing and/or packaging is targeted by AIC246. In line with this, we demonstrate that AIC246 affects the formation of proper unit-length genomes from viral DNA concatemers and interferes with virion maturation. However, since AIC246-resistant viruses do not exhibit cross-resistance to previously published terminase inhibitors, our data suggest that AIC246 interferes with HCMV DNA cleavage/ packaging via a molecular mechanism that is distinct from that of other compound classes known to target the viral terminase.

Human cytomegalovirus (HCMV) remains a serious threat for immunocompromised individuals, including transplant recipients and newborns. To date, all drugs licensed for the treatment of HCMV infection and disease target the viral DNA polymerase. Although these drugs are effective, several drawbacks are associated with their use, including toxicity and emergence of drug resistance. Hence, new and improved antivirals with novel molecular targets are urgently needed. Here we report on the antiviral properties of AIC246, a representative of a novel class of low-molecular-weight compounds that is currently undergoing clinical phase II studies. The anti-HCMV activity of AIC246 was evaluated in vitro and in vivo using various cell culture assays and an engineered mouse xenograft model. In addition, antiviral properties of the drug were characterized in comparison to the current gold standard ganciclovir. We demonstrate that AIC246 exhibits excellent in vitro inhibitory activity against HCMV laboratory strains and clinical isolates, retains activity against ganciclovirresistant viruses, is well tolerated in different cell types (median selectivity index, 18,000), and exerts a potent in vivo efficacy in a mouse xenograft model. Moreover, we show that the antiviral block induced by AIC246 is reversible and the efficacy of the drug is not significantly affected by cell culture variations such as cell type or multiplicity of infection. Finally, initial mode-of-action analyses reveal that AIC246 targets a process in the viral replication cycle that occurs later than DNA synthesis. Thus, AIC246 acts via a mode of action that differs from that of polymerase inhibitors like ganciclovir.Human cytomegalovirus (HCMV) is a widespread opportunistic pathogen in immunocompromised individuals, including transplant recipients and tumor or AIDS patients, and remains the leading viral cause of birth defects (1,9,12,17,29). To date, a limited number of drugs are licensed for the systemic treatment of HCMV infection and disease: ganciclovir (GCV) (Cymevene; Roche), its oral prodrug valganciclovir (VGCV) (Valcyte; Roche), cidofovir (CDF) (Vistide; Gilead), and foscarnet (FOS) (Foscavir; Astra-Zeneca). In addition, valaciclovir (VACV) (Valtrex; GlaxoSmithKline), a drug that has been primarily developed for the treatment of herpes simplex virus (HSV) and varicella-zoster virus (VZV) infection, has gained marketing approval in certain countries for prophylaxis of HCMV infections in transplant patients. Although GCV, VGCV, CDF, and FOS are effective, several drawbacks are associated with the use of these drugs, including toxicity, poor oral bioavailability (except VGCV), and emergence of drug resistance (3,20). The active forms of GCV, CDF, and FOS share the same molecular target, the viral polymerase UL54. Consequently, drug-resistant strains of HCMV encoding UL54 mutations have been found for all three compounds, and the emergence of cross-resistant strains has been described in clinical settings. In addition, resistance to GCV is also associate...

Summary The Clp protease complex in Mycobacterium tuberculosis is unusual in its composition, functional importance, and activation mechanism. While most bacterial species contain a single ClpP protein that is dispensable for normal growth, mycobacteria have two ClpPs, ClpP1 and ClpP2, which are essential for viability and together form the ClpP1P2 tetradecamer. Acyldepsipeptide antibiotics of the ADEP class inhibit the growth of Gram-positive firmicutes by activating ClpP and causing unregulated protein degradation. Here we show that, in contrast, mycobacteria are killed by ADEP through inhibition of ClpP function. Although ADEPs can stimulate purified M. tuberculosis ClpP1P2 to degrade larger peptides and unstructured proteins, this effect is weaker than for ClpP from other bacteria and depends on the presence of an additional activating factor (e.g. the dipeptide benzyloxycarbonyl-leucyl-leucine in vitro) to form the active ClpP1P2 tetradecamer. The cell division protein FtsZ, which is a particularly sensitive target for ADEP-activated ClpP in firmicutes, is not degraded in mycobacteria. Depletion of the ClpP1P2 level in a conditional Mycobacterium bovis BCG mutant enhanced killing by ADEP unlike in other bacteria. In summary, ADEPs kill mycobacteria by preventing interaction of ClpP1P2 with the regulatory ATPases, ClpX or ClpC1, thus inhibiting essential ATP-dependent protein degradation.

Pritelivir reduced the rates of genital HSV shedding and days with lesions in a dose-dependent manner in otherwise healthy men and women with genital herpes. (Funded by AiCuris; ClinicalTrials.gov number, NCT01047540.).

Letermovir is a novel antiviral compound currently in clinical development for the prevention of human cytomegalovirus (HCMV) infections. In contrast to all currently approved anti-HCMV drugs that target the viral DNA polymerase, letermovir acts via a distinct mode of action involving the viral terminase subunit pUL56. To extend our understanding of potential letermovir resistance mechanisms, we used marker transfer to characterize mutations identified in letermovir-resistant HCMV variants that were selected in cell culture. Human cytomegalovirus (HCMV) disease continues to be a serious and life-threatening condition in immunocompromised patients such as transplant recipients. Currently approved anti-HCMV drugs, including (val)ganciclovir, cidofovir, and foscarnet, are associated with profound toxicity as well as drug resistance that restricts their long-term clinical benefit. All these substances ultimately target the viral DNA polymerase (pUL54), though ganciclovir requires prior activation by the viral pUL97 kinase. Accordingly, mutations in open reading frame (ORF) UL97 are associated with (val)ganciclovir resistance whereas mutations in ORF UL54 can confer cross-resistance to all approved anti-HCMV drugs, an increasingly frequent scenario representing a serious threat for specific patient populations (1-3).Letermovir (AIC246) is a new anti-HCMV drug with a distinct but well-characterized mechanism of action targeting the viral terminase subunit pUL56 (4, 5). The drug has proven to be well tolerated in numerous (pre)clinical studies and has demonstrated clinical efficacy in a recent phase IIb trial, meeting all primary endpoints as a prophylactic drug (6).In line with its distinct mode of action, in vitro studies have shown that letermovir is active against HCMV strains resistant to current anti-HCMV drugs (4, 5, 7). In vivo confirmation of resistance-breaking potential was demonstrated in a patient infected with a multidrug-resistant HCMV strain (8). So far, no letermovir-resistant HCMV strains have been reported from clinical trials. Nonetheless, as letermovir is a direct-acting antiviral, the emergence of resistance is ultimately to be expected.HCMV resistance testing previously required virus cultivation and phenotypic characterization of virus isolates; this is slow, labor-intensive, and nonstandardized and often has a poor success rate (3, 9). Current standard clinical practice uses genotyping and virtual phenotyping based, e.g., on mutation databases (10). Once a database is established, this genetic approach is faster, more sensitive, and more cost-effective and provides detailed and quantitative resistance information to the physician (3). However, genetic monitoring of drug resistance requires detailed knowledge of potential resistance mutations in order to develop virtual phenotyping profiles for screening databases (6).To date, 2 letermovir resistance mutations leading to amino acid substitutions have been identified in vitro, both within the coding region for the pUL56 subunit of the viral termi...