The cytochrome bc1 complex as an antipathogenic target.

The cytochrome bc1 complex is a key component of the mitochondrial respiratory chains of many eukaryotic microorganisms that are pathogenic for plants or humans, such as fungi responsible for crop diseases and Plasmodium falciparum, which causes human malaria. Cytochrome bc1 is an enzyme that contains two (ubi)quinone/quinol-binding sites, which can be exploited for the development of fungicidal and chemotherapeutic agents. Here, we review recent progress in determination of the structure and mechanism of action of cytochrome bc1 , and the associated development of antimicrobial agents (and associated resistance mechanisms) targeting its activity.

Treatment responses to Azithromycin and Ciprofloxacin in uncomplicated Salmonella Typhi infection: A comparison of Clinical and Microbiological Data from a Controlled Human Infection Model.

BACKGROUND: The treatment of enteric fever is complicated by the emergence of antimicrobial resistant Salmonella Typhi. Azithromycin is commonly used for first-line treatment of uncomplicated enteric fever, but the response to treatment may be sub-optimal in some patient groups when compared with fluoroquinolones. METHODS: We performed an analysis of responses to treatment with azithromycin (500mg once-daily, 14 days) or ciprofloxacin (500mg twice-daily, 14 days) in healthy UK volunteers (18-60 years) enrolled into two Salmonella controlled human infection studies. Study A was a single-centre, open-label, randomised trial. Participants were randomised 1:1 to receive open-label oral ciprofloxacin or azithromycin, stratified by vaccine group (Vi-polysaccharide, Vi-conjugate or control Men-ACWY vaccine). Study B was an observational challenge/re-challenge study, where participants were randomised to challenge with Salmonella Typhi or Salmonella Paratyphi A. Outcome measures included fever clearance time, blood-culture clearance time and a composite measure of prolonged treatment response (persistent fever ≥38.0°C for ≥72 hours, persistently positive S. Typhi blood cultures for ≥72 hours, or change in antibiotic treatment). Both trials are registered with ClinicalTrials.gov (NCT02324751 and NCT02192008). FINDINGS: In 81 participants diagnosed with S. Typhi in two studies, treatment with azithromycin was associated with prolonged bacteraemia (median 90.8 hours [95% CI: 65.9-93.8] vs. 20.1 hours [95% CI: 7.8-24.3], p<0.001) and prolonged fever clearance times <37.5°C (hazard ratio 2.4 [95%CI: 1.2-5.0]; p = 0.02). Results were consistent when studies were analysed independently and in a sub-group of participants with no history of vaccination or previous challenge. A prolonged treatment response was observed significantly more frequently in the azithromycin group (28/52 [54.9%]) compared with the ciprofloxacin group (1/29 [3.5%]; p<0.001). In participants treated with azithromycin, observed systemic plasma concentrations of azithromycin did not exceed the minimum inhibitory concentration (MIC), whilst predicted intracellular concentrations did exceed the MIC. In participants treated with ciprofloxacin, the observed systemic plasma concentrations and predicted intracellular concentrations of ciprofloxacin exceeded the MIC. INTERPRETATION: Azithromycin at a dose of 500mg daily is an effective treatment for fully sensitive strains of S. Typhi but is associated with delayed treatment response and prolonged bacteraemia when compared with ciprofloxacin within the context of a human challenge model. Whilst the cellular accumulation of azithromycin is predicted to be sufficient to treat intracellular S. Typhi, systemic exposure may be sub-optimal for the elimination of extracellular circulating S. Typhi. In an era of increasing antimicrobial resistance, further studies are required to define appropriate azithromycin dosing regimens for enteric fever and to assess novel treatment strategies, including combination therapies. TRIAL REGISTRATION: ClinicalTrials.gov (NCT02324751 and NCT02192008).

OptiMal-PK: an internet-based, user-friendly interface for the mathematical-based design of optimized anti-malarial treatment regimens.

BACKGROUND: The search for highly effective anti-malarial therapies has gathered pace and recent years have seen a number of promising single and combined therapies reach the late stages of development. A key drug development challenge is the need for early assessment of the clinical utility of new drug leads as it is often unclear for developers whether efforts should be focused on efficacy or metabolic stability/exposure or indeed whether the continuation of iterative QSAR (quantitative structure-activity and relationships) cycles of medicinal chemistry and biological testing will translate to improved clinical efficacy. Pharmacokinetic and pharmacodynamic (PK/PD)-based measurements available from in vitro studies can be used for such clinical predictions. However, these predictions often require bespoke mathematical PK/PD modelling expertise and are normally performed after candidate development and, therefore, not during the pre-clinical development phase when such decisions need to be made. METHODS: An internet-based tool has been developed using STELLA(®) software. The tool simulates multiple differential equations that describe anti-malarial PK/PD relationships where the user can easily input PK/PD parameters. The tool utilizes a simple stop-light system to indicate the efficacy of each combination of parameters. This tool, called OptiMal-PK, additionally allows for the investigation of the effect of drug combinations with known or custom compounds. RESULTS: The results of simulations obtained from OptiMal-PK were compared to a previously published and validated mathematical model on which this tool is based. The tool has also been used to simulate the PK/PD relationship for a number of existing anti-malarial drugs in single or combined treatment. Simulations were predictive of the published clinical parasitological clearance activities for these existing therapies. CONCLUSIONS: OptiMal-PK is designed to be implemented by medicinal chemists and pharmacologists during the pre-clinical anti-malarial drug development phase to explore the impact of different PK/PD parameters upon the predicted clinical activity of any new compound. It can help investigators to identify which pharmacological features of a compound are most important to the clinical performance of a new chemical entity and how partner drugs could potentially improve the activity of existing therapies.

Antimalarial 4(1H)-pyridones bind to the Qi site of cytochrome bc1.

Cytochrome bc1 is a proven drug target in the prevention and treatment of malaria. The rise in drug-resistant strains of Plasmodium falciparum, the organism responsible for malaria, has generated a global effort in designing new classes of drugs. Much of the design/redesign work on overcoming this resistance has been focused on compounds that are presumed to bind the Q(o) site (one of two potential binding sites within cytochrome bc1 using the known crystal structure of this large membrane-bound macromolecular complex via in silico modeling. Cocrystallization of the cytochrome bc1 complex with the 4(1H)-pyridone class of inhibitors, GSK932121 and GW844520, that have been shown to be potent antimalarial agents in vivo, revealed that these inhibitors do not bind at the Q(o) site but bind at the Q(i )site. The discovery that these compounds bind at the Q(i) site may provide a molecular explanation for the cardiotoxicity and eventual failure of GSK932121 in phase-1 clinical trial and highlight the need for direct experimental observation of a compound bound to a target site before chemical optimization and development for clinical trials. The binding of the 4(1H)-pyridone class of inhibitors to Q(i) also explains the ability of this class to overcome parasite Q(o)-based atovaquone resistance and provides critical structural information for future design of new selective compounds with improved safety profiles.

Synthesis and antimalarial activities of a diverse set of triazole-containing furamidine analogues.

Four different series of triazole diamidines have been prepared by the Pinner method from the corresponding triazole dinitriles. Copper-catalyzed "click chemistry" was used for the synthesis of 1,4- and 4,5-substituted triazoles, aryl magnesium acetylide reagents for the 1,5-substituted triazoles, with a thermal dipolar addition reaction employed for the 2,4-substituted triazoles. In vitro antimalarial activity against two different PfCRT-modified parasite lines (Science 2002, 298, 210-213) of Plasmodium falciparum and inhibition of hemozoin formation were determined for each compound. Several diamidines with potent nanomolar antimalarial activities were identified, and selected molecules were resynthesized as their diamidoxime triazole prodrugs. One of these prodrugs, OB216, proved to be highly potent in vivo with an ED50 value of 5 mg kg(-1) (po) and an observed 100 % cure rate (CD100) of just 10 mg kg(-1) by oral (po) administration in mice infected with P. vinckei.

Candidate selection and preclinical evaluation of N-tert-butyl isoquine (GSK369796), an affordable and effective 4-aminoquinoline antimalarial for the 21st century.

N-tert-Butyl isoquine (4) (GSK369796) is a 4-aminoquinoline drug candidate selected and developed as part of a public-private partnership between academics at Liverpool, MMV, and GSK pharmaceuticals. This molecule was rationally designed based on chemical, toxicological, pharmacokinetic, and pharmacodynamic considerations and was selected based on excellent activity against Plasmodium falciparum in vitro and rodent malaria parasites in vivo. The optimized chemistry delivered this novel synthetic quinoline in a two-step procedure from cheap and readily available starting materials. The molecule has a full industry standard preclinical development program allowing first into humans to proceed. Employing chloroquine (1) and amodiaquine (2) as comparator molecules in the preclinical plan, the first preclinical dossier of pharmacokinetic, toxicity, and safety pharmacology has also been established for the 4-aminoquinoline antimalarial class. These studies have revealed preclinical liabilities that have never translated into the human experience. This has resulted in the availability of critical information to other drug development teams interested in developing antimalarials within this class.

Open-label comparative clinical study of chlorproguanil-dapsone fixed dose combination (Lapdap) alone or with three different doses of artesunate for uncomplicated Plasmodium falciparum malaria.

UNLABELLED: The objective of this study was to determine the appropriate dose of artesunate for use in a fixed dose combination therapy with chlorproguanil-dapsone (CPG-DDS) for the treatment of uncomplicated falciparum malaria. METHODS: Open-label clinical trial comparing CPG-DDS alone or with artesunate 4, 2, or 1 mg/kg at medical centers in Blantyre, Malawi and Farafenni, The Gambia. The trial was conducted between June 2002 and February 2005, including 116 adults (median age 27 years) and 107 children (median age 38 months) with acute uncomplicated Plasmodium falciparum malaria. Subjects were randomized into 4 groups to receive CPG-DDS alone or plus 4, 2 or 1 mg/kg of artesunate once daily for 3 days. Assessments took place on Days 0-3 in hospital and follow-up on Days 7 and 14 as out-patients. Efficacy was evaluated in the Day 3 per-protocol (PP) population using mean time to reduce baseline parasitemia by 90% (PC90). A number of secondary outcomes were also included. Appropriate artesunate dose was determined using a pre-defined decision matrix based on primary and secondary outcomes. Treatment emergent adverse events were recorded from clinical assessments and blood parameters. Safety was evaluated in the intent to treat (ITT) population. RESULTS: In the Day 3 PP population for the adult group (N = 85), mean time to PC90 was 19.1 h in the CPG-DDS group, significantly longer than for the +artesunate 1 mg/kg (12.5 h; treatment difference -6.6 h [95%CI -11.8, -1.5]), 2 mg/kg (10.7 h; -8.4 h [95%CI -13.6, -3.2]) and 4 mg/kg (10.3 h; -8.7 h [95%CI -14.1, -3.2]) groups. For children in the Day 3 PP population (N = 92), mean time to PC90 was 21.1 h in the CPG-DDS group, similar to the +artesunate 1 mg/kg group (17.7 h; -3.3 h [95%CI -8.6, 2.0]), though the +artesunate 2 mg/kg and 4 mg/kg groups had significantly shorter mean times to PC90 versus CPG-DDS; 14.4 h (treatment difference -6.4 h [95%CI -11.7, -1.0]) and 12.8 h (-7.4 h [95%CI -12.9, -1.8]), respectively. An analysis of mean time to PC90 for the Day 14 PP and ITT populations was consistent with the primary analysis. Treatment emergent, drug-related adverse events were experienced in 35.3% (41/116) of adults and 70.1% (75/107) of children; mostly hematological and gastroenterological. The nature and incidence of adverse events was similar between the groups. No dose-related changes in laboratory parameters were observed. Nine serious adverse events due to any cause occurred in five subjects including two cases of hemolysis believed to be associated with drug treatment (one adult, one child). One adult died of anaphylactic shock, not associated with investigational therapy. CONCLUSIONS: CPG-DDS plus artesunate demonstrated advantages over CPG-DDS alone for the primary efficacy endpoint (mean time to PC90) except in children for the 1 mg/kg artesunate dose. Based on a pre-defined decision matrix, the primary endpoint in the child group supported an artesunate dose of 4 mg/kg. Secondary endpoints also supported a 4 mg/kg artesunate dose to take forward into the remainder of the development program. TRIAL REGISTRATION: ClinicalTrials.gov NCT00519467.

Resisting resistance: dealing with the irrepressible problem of malaria.

The burgeoning problem of malaria in the developing world and the relentless march of drug resistance demand that we continue to seek new chemotherapeutic strategies. Given the enormous expense of developing and marketing new chemical entities, we often rely on an increased understanding of the pharmacology of older drugs and judicious use of drug combinations. Development is being driven primarily by public-private partnerships from academic investigations. Two such agents are the antifolate combination Lapdap, already licensed and soon to be combined with artesunate, and isoquine, a novel isoquinoline, about to enter clinical trials. Other drug combinations designed to minimize the spread of resistance are in the pipeline. Such developments are crucial as it becomes clear that existing drugs, even those used in combinations, may have limited lifetimes.