Antimalarial 4(1H)-pyridones bind to the Qi site of cytochrome bc1.
ABSTRACT: 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.
Project description:The cytochrome bc1 complex (cyt bc1) is the third component of the mitochondrial electron transport chain and is the target of several potent antimalarial compounds, including the naphthoquinone atovaquone (ATV) and the 4(1H)-quinolone ELQ-300. Mechanistically, cyt bc1 facilitates the transfer of electrons from ubiquinol to cytochrome c and contains both oxidative (Qo) and reductive (Qi) catalytic sites that are amenable to small-molecule inhibition. Although many antimalarial compounds, including ATV, effectively target the Qo site, it has been challenging to design selective Qi site inhibitors with the ability to circumvent clinical ATV resistance, and little is known about how chemical structure contributes to site selectivity within cyt bc1. Here, we used the proposed Qi site inhibitor ELQ-300 to generate a drug-resistant Plasmodium falciparum clone containing an I22L mutation at the Qi region of cyt b. Using this D1 clone and the Y268S Qo mutant strain, P. falciparum Tm90-C2B, we created a structure-activity map of Qi versus Qo site selectivity for a series of endochin-like 4(1H)-quinolones (ELQs). We found that Qi site inhibition was associated with compounds containing 6-position halogens or aryl 3-position side chains, while Qo site inhibition was favored by 5,7-dihalogen groups or 7-position substituents. In addition to identifying ELQ-300 as a preferential Qi site inhibitor, our data suggest that the 4(1H)-quinolone scaffold is compatible with binding to either site of cyt bc1 and that minor chemical changes can influence Qo or Qi site inhibition by the ELQs.
Project description:Cytochrome bc1 inhibitors have been broadly studied as human and veterinary medicines and agricultural fungicides. For the most part, cytochrome bc1 inhibitors compete with ubiquinol at the ubiquinol oxidation (Qo) site or with ubiquinone at the quinone reduction (Qi) site. 4(1 H)-Quinolones with 3-position substituents may inhibit either site based on quinolone ring substituents. 4(1 H)-Quinolones that inhibit the Qi site are highly effective against toxoplasmosis, malaria, and babesiosis and do not inhibit human cytochrome bc1. We tested a series of 4(1 H)-Quinolones against wild-type and drug resistant strains of Toxoplasma gondii and Plasmodium falciparum. These experiments identified very potent compounds that inhibit T. gondii proliferation at picomolar concentrations. The most potent compounds target the Qo site, and for these compounds, an alkyl side chain confers potency against T. gondii greater than that of bulkier side chains. Our experiments also show that substituents on the quinolone ring influenced selectivity between T. gondii and P. falciparum and between Qo and Qi site-mediated activity. Comparison of the parasite cytochrome b sequences identified amino acids that are associated with drug resistance in P. falciparum that exist naturally in wild-type T. gondii. These underlying differences may influence drug susceptibility. Finally, a Qo site active 4(1 H)-quinolone-3-diarylether tested in a murine model of toxoplasmosis was superior to atovaquone, resulting in survival from Type I strain T. gondii infection. These experiments identify highly effective compounds for toxoplasmosis and provide valuable insight into the structure-activity relationship of cytochrome bc1 inhibitors.
Project description:Cytochrome bc1, a dimeric multi-subunit electron-transport protein embedded in the inner mitochondrial membrane, is a major drug target for the treatment and prevention of malaria and toxoplasmosis. Structural studies of cytochrome bc1 from mammalian homologues co-crystallized with lead compounds have underpinned structure-based drug design to develop compounds with higher potency and selectivity. However, owing to the limited amount of cytochrome bc1 that may be available from parasites, all efforts have been focused on homologous cytochrome bc1 complexes from mammalian species, which has resulted in the failure of some drug candidates owing to toxicity in the host. Crystallographic studies of the native parasite proteins are not feasible owing to limited availability of the proteins. Here, it is demonstrated that cytochrome bc1 is highly amenable to single-particle cryo-EM (which uses significantly less protein) by solving the apo and two inhibitor-bound structures to ∼4.1 Å resolution, revealing clear inhibitor density at the binding site. Therefore, cryo-EM is proposed as a viable alternative method for structure-based drug discovery using both host and parasite enzymes.
Project description:A previous phenotypic screen by GSK identified 2-(quinolin-4-yloxy)acetamides as potent growth inhibitors of Mycobacterium tuberculosis (Mtb). We report the results of a preliminary structure-activity relationship (SAR) study of the compound class which has yielded more potent inhibitors. An Mtb cytochrome bd oxidase deletion mutant (cydKO) was found to be hypersensitive to most members of the compound library, while strains carrying single-nucleotide polymorphisms of the qcrB gene, which encodes a subunit of the menaquinol cytochrome c oxidoreductase (bc1) complex, were resistant to the library. These results identify that the 2-(quinolin-4-yloxy)acetamide class of Mtb growth inhibitors can be added to the growing number of scaffolds that target the M. tuberculosis bc1 complex.
Project description:Single-dose therapies for malaria have been proposed as a way to reduce the cost and increase the effectiveness of antimalarial treatment. However, no compound to date has shown single-dose activity against both the blood-stage Plasmodium parasites that cause disease and the liver-stage parasites that initiate malaria infection. Here, we describe a subset of cytochrome bc1 (cyt bc1) inhibitors, including the novel 4(1H)-quinolone ELQ-400, with single-dose activity against liver, blood, and transmission-stage parasites in mouse models of malaria. Although cyt bc1 inhibitors are generally classified as slow-onset antimalarials, we found that a single dose of ELQ-400 rapidly induced stasis in blood-stage parasites, which was associated with a rapid reduction in parasitemia in vivo. ELQ-400 also exhibited a low propensity for drug resistance and was active against atovaquone-resistant P. falciparum strains with point mutations in cyt bc1. Ultimately, ELQ-400 shows that cyt bc1 inhibitors can function as single-dose, blood-stage antimalarials and is the first compound to provide combined treatment, prophylaxis, and transmission blocking activity for malaria after a single oral administration. This remarkable multi-stage efficacy suggests that metabolic therapies, including cyt bc1 inhibitors, may be valuable additions to the collection of single-dose antimalarials in current development.
Project description:Toxoplasma gondii is an apicomplexan parasite that causes fatal and debilitating brain and eye disease. Endochinlike quinolones (ELQs) are preclinical compounds that are efficacious against apicomplexan-caused diseases, including toxoplasmosis, malaria, and babesiosis. Of the ELQs, ELQ-316 has demonstrated the greatest efficacy against acute and chronic experimental toxoplasmosis. Although genetic analyses in other organisms have highlighted the importance of the cytochrome bc1 complex Qi site for ELQ sensitivity, the mechanism of action of ELQs against T. gondii and the specific mechanism of ELQ-316 remain unknown. Here, we describe the selection and genetic characterization of T. gondii clones resistant to ELQ-316. A T. gondii strain selected under ELQ-316 drug pressure was found to possess a Thr222-Pro amino acid substitution that confers 49-fold resistance to ELQ-316 and 19-fold resistance to antimycin, a well-characterized Qi site inhibitor. These findings provide further evidence for ELQ Qi site inhibition in T. gondii and greater insight into the interactions of Qi site inhibitors with the apicomplexan cytochrome bc1 complex.
Project description:The high incidence of fungal pathogens has become a global issue for crop protection. A promising strategy to control fungal plant infections is based on the use of nature-inspired compounds. The cytochrome bc1 complex is an essential component of the cellular respiratory chain and is one of the most important fungicidal targets. Natural products have played a crucial role in the discovery of cytochrome bc1 inhibitors, as proven by the development of strobilurins, one of the most important classes of crop-protection agents, over the past two decades. In this review, we summarize advances in the exploration of natural product scaffolds for the design and development of new bc1 complex inhibitors. Particular emphasis is given to molecular modeling-based approaches and structure-activity relationship (SAR) studies performed to improve the stability and increase the potency of natural precursors. The collected results highlight the versatility of natural compounds and provide an insight into the potential development of nature-inspired derivatives as antifungal agents.
Project description:Complex III or the cytochrome (cyt) bc1 complex constitutes an integral part of the respiratory chain of most aerobic organisms and of the photosynthetic apparatus of anoxygenic purple bacteria. The function of cyt bc1 is to couple the reaction of electron transfer from ubiquinol to cytochrome c to proton pumping across the membrane. Mechanistically, the electron transfer reaction requires docking of its Rieske iron-sulfur protein (ISP) subunit to the quinol oxidation site (QP) of the complex. Formation of an H-bond between the ISP and the bound substrate was proposed to mediate the docking. Here we show that the binding of oxazolidinedione-type inhibitors famoxadone, jg144, and fenamidone induces docking of the ISP to the QP site in the absence of the H-bond formation both in mitochondrial and bacterial cyt bc1 complexes, demonstrating that ISP docking is independent of the proposed direct ISP-inhibitor interaction. The binding of oxazolidinedione-type inhibitors to cyt bc1 of different species reveals a toxophore that appears to interact optimally with residues in the QP site. The effect of modifications or additions to the toxophore on the binding to cyt bc1 from different species could not be predicted from structure-based sequence alignments, as demonstrated by the altered binding mode of famoxadone to bacterial cyt bc1.
Project description:Available treatments for Chagas' disease and visceral leishmaniasis are inadequate, and there is a pressing need for new therapeutics. Drug discovery efforts for both diseases principally rely upon phenotypic screening. However, the optimization of phenotypically active compounds is hindered by a lack of information regarding their molecular target(s). To combat this issue we initiate target deconvolution studies at an early stage. Here, we describe comprehensive genetic and biochemical studies to determine the targets of three unrelated phenotypically active compounds. All three structurally diverse compounds target the Qi active-site of cytochrome b, part of the cytochrome bc1 complex of the electron transport chain. Our studies go on to identify the Qi site as a promiscuous drug target in Leishmania donovani and Trypanosoma cruzi with a propensity to rapidly mutate. Strategies to rapidly identify compounds acting via this mechanism are discussed to ensure that drug discovery portfolios are not overwhelmed with inhibitors of a single target.
Project description:The rapid advancement of a series of noncovalent inhibitors of T790M mutants of EGFR is discussed. The optimization of pyridone 1, a nonselective high-throughput screening hit, to potent molecules with high levels of selectivity over wtEGFR and the broader kinome is described herein.