Evaluation of the inhibitory effects of itraconazole on letermovir.

AIMS: Letermovir, a cytomegalovirus (CMV) DNA terminase complex inhibitor, is a substrate of ABCB1 (P-glycoprotein; P-gp), organic anion transporting polypeptide (OATP)1B1/3, UDP-glucuronosyltransferase (UGT)1A1, UGT1A3 and possibly ABCG2 (breast cancer resistance protein; BCRP). A study was conducted to evaluate the effects of itraconazole, a prototypic ABCB1/ABCG2 inhibitor, on letermovir pharmacokinetics (PK) and the effects of letermovir on itraconazole PK. METHODS: In an open-label, fixed-sequence study in 14 healthy participants, 200 mg oral itraconazole was administered once daily for 4 days. Following a 10-day washout, 480 mg oral letermovir was administered once daily for 14 days (Days 1-14) and then coadministered with 200 mg itraconazole once daily for 4 days (Days 15-18). Intensive PK sampling was performed for letermovir and itraconazole. PK and safety were evaluated. RESULTS: Letermovir geometric mean ratio (GMR; 90% confidence interval [CI]) for area under the concentration-time curve from time 0 to 24 h (AUC0-24 ) was 1.33 (1.17, 1.51) and for maximum concentration (Cmax ) was 1.21 (1.05, 1.39) following administration with/without itraconazole. Itraconazole GMR (90% CI) for AUC0-24 was 0.76 (0.71, 0.81) and for Cmax was 0.84 (0.76, 0.92) following administration with/without letermovir. Coadministration of letermovir with itraconazole was generally well tolerated. CONCLUSIONS: The increase in letermovir exposure with coadministration of itraconazole is likely predominantly due to inhibition of intestinal ABCB1 and potentially ABCG2 transport. The mechanism for the decrease in itraconazole exposure is unknown. The modest changes in letermovir and itraconazole PK are not considered clinically meaningful.

Safety and immunogenicity of intramuscular, single-dose V590 (rVSV-SARS-CoV-2 Vaccine) in healthy adults: Results from a phase 1 randomised, double-blind, placebo-controlled, dose-ranging trial.

BACKGROUND: Vaccines against COVID-19 are needed to overcome challenges associated with mitigating the global pandemic. We report the safety and immunogenicity of V590, a live recombinant vesicular stomatitis virus-based COVID-19 vaccine candidate. METHODS: In this placebo-controlled, double-blind, three-part phase 1 study, healthy adults were randomised to receive a single intramuscular dose of vaccine or placebo. In Part 1, younger (18-54 years) and, in Part 2, older (≥55 years) adults seronegative for SARS-CoV-2 nucleocapsid received one of four V590 dose levels (5.00 × 105; 2.40 × 106; 1.15 × 107; or 5.55 × 107 plaque-forming units [pfu]) or placebo. In Part 3, a single V590 dose level (5.55 × 107 pfu) or placebo was administered to younger SARS-CoV-2 seropositive adults. Primary endpoints included adverse events (AEs) and for Parts 1 and 2 anti-SARS-CoV-2 serum neutralising antibody responses measured by 50% plaque reduction neutralisation (PRNT50) assay at Day 28. Registration NCT04569786 [P001-02]. FINDINGS: 232 participants were randomised and 219 completed the study. In seronegative participants, anti-SARS-CoV-2 spike-specific antibody responses to V590 were low and comparable to placebo across the lower dose levels. At the highest dose level (5.55 × 107 pfu), anti-SARS-CoV-2 spike-specific PRNT50 was 2.3-fold higher than placebo. The most frequently reported AEs were injection-site pain (38.4%), headache (15.1%) and fatigue (13.4%). INTERPRETATION: V590 was generally well-tolerated. However, Day 28 anti-SARS-Cov-2 spike-specific antibody responses in seronegative participants following a single intramuscular administration of V590 were not sufficient to warrant continued development. FUNDING: The study was funded by Merck Sharp & Dohme LLC., a subsidiary of Merck & Co., Inc., Rahway, NJ, USA.

Acute and Chronic Effects of Rifampin on Letermovir Suggest Transporter Inhibition and Induction Contribute to Letermovir Pharmacokinetics.

Rifampin has acute inhibitory and chronic inductive effects that can cause complex drug-drug interactions. Rifampin inhibits transporters including organic-anion-transporting polypeptide (OATP)1B and P-glycoprotein (P-gp), and induces enzymes and transporters including cytochrome P450 3A, UDP-glucuronosyltransferase (UGT)1A, and P-gp. This study aimed to separate inhibitory and inductive effects of rifampin on letermovir disposition and elimination (indicated for cytomegalovirus prophylaxis in hematopoietic stem cell transplant recipients). Letermovir is a substrate of UGT1A1/3, P-gp, and OATP1B, with its clearance primarily mediated by OATP1B. Letermovir (single-dose) administered with rifampin (single-dose) resulted in increased letermovir exposure through transporter inhibition. Chronic coadministration with rifampin (inhibition plus potential OATP1B induction) resulted in modestly decreased letermovir exposure vs. letermovir alone. Letermovir administered 24 hours after the last rifampin dose (potential OATP1B induction) resulted in markedly decreased letermovir exposure. These data suggest rifampin may induce transporters that clear letermovir; the modestly reduced letermovir exposure with chronic rifampin coadministration likely reflects the net effect of inhibition and induction. OATP1B endogenous biomarkers coproporphyrin (CP) I and glycochenodeoxycholic acid-sulfate (GCDCA-S) were also analyzed; their exposures increased after single-dose rifampin plus letermovir, consistent with OATP1B inhibition and prior reports of inhibition by rifampin alone. CP I and GCDCA-S exposures were substantially reduced with letermovir administered 24 hours after the last dose of rifampin vs. letermovir plus chronic rifampin coadministration. This study suggests that OATP1B induction may contribute to reduced letermovir exposure after chronic rifampin administration, although given the complexity of letermovir disposition alternative mechanisms are not fully excluded.

Dual Plasmepsin-Targeting Antimalarial Agents Disrupt Multiple Stages of the Malaria Parasite Life Cycle.

Artemisin combination therapy (ACT) is the main treatment option for malaria, which is caused by the intracellular parasite Plasmodium. However, increased resistance to ACT highlights the importance of finding new drugs. Recently, the aspartic proteases Plasmepsin IX and X (PMIX and PMX) were identified as promising drug targets. In this study, we describe dual inhibitors of PMIX and PMX, including WM382, that block multiple stages of the Plasmodium life cycle. We demonstrate that PMX is a master modulator of merozoite invasion and direct maturation of proteins required for invasion, parasite development, and egress. Oral administration of WM382 cured mice of P. berghei and prevented blood infection from the liver. In addition, WM382 was efficacious against P. falciparum asexual infection in humanized mice and prevented transmission to mosquitoes. Selection of resistant P. falciparum in vitro was not achievable. Together, these show that dual PMIX and PMX inhibitors are promising candidates for malaria treatment and prevention.

The Malaria Parasite Cyclin H Homolog PfCyc1 Is Required for Efficient Cytokinesis in Blood-Stage Plasmodium falciparum.

All well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs), and these protein kinase complexes are viable drug targets. The regulatory control of the Plasmodium falciparum cell division cycle remains poorly understood, and the roles of the various CDKs and cyclins remain unclear. The P. falciparum genome contains multiple CDKs, but surprisingly, it does not contain any sequence-identifiable G1-, S-, or M-phase cyclins. We demonstrate that P. falciparum Cyc1 (PfCyc1) complements a G1 cyclin-depleted Saccharomyces cerevisiae strain and confirm that other identified malaria parasite cyclins do not complement this strain. PfCyc1, which has the highest sequence similarity to the conserved cyclin H, cannot complement a temperature-sensitive yeast cyclin H mutant. Coimmunoprecipitation of PfCyc1 from P. falciparum parasites identifies PfMAT1 and PfMRK as specific interaction partners and does not identify PfPK5 or other CDKs. We then generate an endogenous conditional allele of PfCyc1 in blood-stage P. falciparum using a destabilization domain (DD) approach and find that PfCyc1 is essential for blood-stage proliferation. PfCyc1 knockdown does not impede nuclear division, but it prevents proper cytokinesis. Thus, we demonstrate that PfCyc1 has a functional divergence from bioinformatic predictions, suggesting that the malaria parasite cell division cycle has evolved to use evolutionarily conserved proteins in functionally novel ways.IMPORTANCE Human infection by the eukaryotic parasite Plasmodium falciparum causes malaria. Most well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs) to promote essential cell division processes. Remarkably, there are no identifiable cyclins that are predicted to control the cell cycle in the malaria parasite genome. Thus, our knowledge regarding the basic mechanisms of the malaria parasite cell cycle remains unsatisfactory. We demonstrate that P. falciparum Cyc1 (PfCyc1), a transcriptional cyclin homolog, complements a cell cycle cyclin-deficient yeast strain but not a transcriptional cyclin-deficient strain. We show that PfCyc1 forms a complex in the parasite with PfMRK and the P. falciparum MAT1 homolog. PfCyc1 is essential and nonredundant in blood-stage P. falciparum PfCyc1 knockdown causes a stage-specific arrest after nuclear division, demonstrating morphologically aberrant cytokinesis. This work demonstrates a conserved PfCyc1/PfMAT1/PfMRK complex in malaria and suggests that it functions as a schizont stage-specific regulator of the P. falciparum life cycle.

An APC/C-Cdh1 Biosensor Reveals the Dynamics of Cdh1 Inactivation at the G1/S Transition.

B-type cyclin-dependent kinase activity must be turned off for mitotic exit and G1 stabilization. B-type cyclin degradation is mediated by the anaphase-promoting complex/cyclosome (APC/C); during and after mitotic exit, APC/C is dependent on Cdh1. Cdh1 is in turn phosphorylated and inactivated by cyclin-CDK at the Start transition of the new cell cycle. We developed a biosensor to assess the cell cycle dynamics of APC/C-Cdh1. Nuclear exit of the G1 transcriptional repressor Whi5 is a known marker of Start; APC/C-Cdh1 is inactivated 12 min after Whi5 nuclear exit with little measurable cell-to-cell timing variability. Multiple phosphorylation sites on Cdh1 act in a redundant manner to repress its activity. Reducing the number of phosphorylation sites on Cdh1 can to some extent be tolerated for cell viability, but it increases variability in timing of APC/C-Cdh1 inactivation. Mutants with minimal subsets of phosphorylation sites required for viability exhibit striking stochasticity in multiple responses including budding, nuclear division, and APC/C-Cdh1 activity itself. Multiple cyclin-CDK complexes, as well as the stoichiometric inhibitor Acm1, contribute to APC/C-Cdh1 inactivation; this redundant control is likely to promote rapid and reliable APC/C-Cdh1 inactivation immediately following the Start transition.

An essential malaria protein defines the architecture of blood-stage and transmission-stage parasites.

Blood-stage replication of the human malaria parasite Plasmodium falciparum occurs via schizogony, wherein daughter parasites are formed by a specialized cytokinesis known as segmentation. Here we identify a parasite protein, which we name P. falciparum Merozoite Organizing Protein (PfMOP), as essential for cytokinesis of blood-stage parasites. We show that, following PfMOP knockdown, parasites undergo incomplete segmentation resulting in a residual agglomerate of partially divided cells. While organelles develop normally, the structural scaffold of daughter parasites, the inner membrane complex (IMC), fails to form in this agglomerate causing flawed segmentation. In PfMOP-deficient gametocytes, the IMC formation defect causes maturation arrest with aberrant morphology and death. Our results provide insight into the mechanisms of replication and maturation of malaria parasites.

Regulated degradation of the APC coactivator Cdc20.

BACKGROUND: Cdc20 is a highly conserved activator of the anaphase-promoting complex (APC), promoting cell-cycle-regulated ubiquitination and proteolysis of a number of critical cell-cycle-regulatory targets including securin and mitotic cyclins. APC-Cdc20 activity is tightly regulated, and this regulation is likely important for accurate cell cycle control. One significant component of Cdc20 regulation is thought to be Cdc20 proteolysis. However, published literature suggests different mechanisms and requirements for Cdc20 proteolysis. The degree to which Cdc20 proteolysis is cell-cycle regulated, the dependence of Cdc20 proteolysis on Cdc20 destruction boxes (recognition sequences for APC-mediated ubiqutination, either by Cdc20 or by the related Cdh1 APC activator), and the need for APC itself for Cdc20 proteolysis all have been disputed to varying extents. In animals, Cdc20 proteolysis is thought to be mediated by Cdh1, contributing an intrinsic order of APC activation by Cdc20 and then by Cdh1. One report suggests a Cdh1 requirement for Cdc20 proteolysis in budding yeast; this idea has not been tested further. RESULTS: We characterized Cdc20 proteolysis using Cdc20 expressed from its endogenous locus; previous studies generally employed strongly overexpressed Cdc20, which can cause significant artifacts. We analyzed Cdc20 proteolysis with or without mutations in previously identified destruction box sequences, using varying methods of cell cycle synchronization, and in the presence or absence of Cdh1. Cdc20 instability is only partially dependent on destruction boxes. A much stronger dependence on Cdh1 for Cdc20 proteolysis was observed, but Cdh1-independent proteolysis was also clearly observed. Cdc20 proteolysis independent of both destruction boxes and Cdh1 was especially detectable around the G1/S transition; Cdh1-dependent proteolysis was most notable in late mitosis and G1. CONCLUSIONS: Cdc20 proteolysis is under complex control, with different systems operating at different points in the cell cycle. This complexity is likely to explain apparent conflicts in previously published literature on this subject. A major mode of control of Cdc20 proteolysis occurs in late mitosis/early G1 and is Cdh1-dependent, as in animal cells; this mode may contribute to the known sequential activation of the APC by Cdc20 followed by Cdh1. An independent mode of Cdc20 proteolysis, independent of destruction boxes and Cdh1, occurs at G1/S; we do not know the mechanism or function of this mode of proteolysis, but speculate that it may contribute to sharpening and restricting activation of APC-Cdc20 to early mitosis.

Requirements and reasons for effective inhibition of the anaphase promoting complex activator CDH1.

Anaphase promoting complex (APC)-Cdh1 targets multiple mitotic proteins for degradation upon exit from mitosis into G1; inhibitory phosphorylation of Cdh1 by cyclin-dependent kinase (CDK) and Polo kinase has been proposed to prevent the premature degradation of substrates in the ensuing cell cycle. Here, we demonstrate essentiality of CDK phosphorylation of Cdh1 in Saccharomyces cerevisiae by exact endogenous gene replacement of CDH1 with CDK-unphosphorylatable CDH1-m11; in contrast, neither Cdh1 polo kinase sites nor polo interaction motifs are required. CDH1-m11 cells arrest in the first cycle with replicated DNA and sustained polarized growth; most cells have monopolar spindles. Blocking proteolysis of the Cin8 kinesin in CDH1-m11 cells does not promote spindle pole body (SPB) separation. In contrast, expression of undegradable mitotic cyclin results in both SPB separation and the restoration of isotropic growth. A minority of CDH1-m11 cells arrest with short bipolar spindles that fail to progress to anaphase; this can be accounted for by a failure to accumulate Cdc20 and consequent failure to cleave cohesin. Bipolar spindle assembly in CDH1-m11 cells is strikingly sensitive to gene dosage of the stoichiometric Cdh1 inhibitor ACM1. Thus, different spindle-regulatory pathways have distinct sensitivities to Cdh1, and ACM1 may buffer essential CDK phosphorylation of Cdh1.

A chromatin immunoprecipitation screen reveals protein kinase Cbeta as a direct RUNX1 target gene.

RUNX1 (also known as AML1) is a DNA-binding transcription factor that functions as a tumor suppressor and developmental determinant in hematopoietic cells. Target promoters have been identified primarily through the use of differential expression strategies and candidate gene approaches but not biochemical screens. Using a chromatin immunoprecipitation screen, we identified protein kinase Cbeta as a direct RUNX1 target gene and demonstrate that endogenous RUNX1 binds the chromatinized protein kinase Cbeta promoter of U937 cells. A phylogenetically conserved RUNX1-binding site within the PKCbeta promoter binds RUNX1 in electrophoretic mobility shift analyses and confers RUNX1 responsiveness on a heterologous promoter. Changes in RUNX1 activity affect endogenous protein kinase Cbeta expression, and a dominant-negative form of RUNX1 protects U937 cells from apoptotic stimuli previously shown to be dependent on protein kinase Cbeta. This protection can be reversed by the ectopic expression of protein kinase Cbeta. Together these findings demonstrate that protein kinase Cbeta is a direct, downstream target of RUNX1 and links RUNX1 to a myeloid apoptotic pathway.