Giancarlo A. Biagini

Generation of quinolone antimalarials targeting the Plasmodium falciparum mitochondrial respiratory chain for the treatment and prophylaxis of malaria

There is an urgent need for new antimalarial drugs with novel mechanisms of action to deliver effective control and eradication programs. Parasite resistance to all existing antimalarial classes, including the artemisinins, has been reported during their clinical use. A failure to generate new antimalarials with novel mechanisms of action that circumvent the current resistance challenges will contribute to a resurgence in the disease which would represent a global health emergency. Here we present a unique generation of quinolone lead antimalarials with a dual mechanism of action against two respiratory enzymes, NADH:ubiquinone oxidoreductase (Plasmodium falciparum NDH2) and cytochrome bc1. Inhibitor specificity for the two enzymes can be controlled subtly by manipulation of the privileged quinolone core at the 2 or 3 position. Inhibitors display potent (nanomolar) activity against both parasite enzymes and against multidrug-resistant P. falciparum parasites as evidenced by rapid and selective depolarization of the parasite mitochondrial membrane potential, leading to a disruption of pyrimidine metabolism and parasite death. Several analogs also display activity against liver-stage parasites (Plasmodium cynomolgi) as well as transmission-blocking properties. Lead optimized molecules also display potent oral antimalarial activity in the Plasmodium berghei mouse malaria model associated with favorable pharmacokinetic features that are aligned with a single-dose treatment. The ease and low cost of synthesis of these inhibitors fulfill the target product profile for the generation of a potent, safe, and inexpensive drug with the potential for eventual clinical deployment in the control and eradication of falciparum malaria.

Functional Characterization and Target Validation of Alternative Complex I of Plasmodium falciparum Mitochondria

ABSTRACT This study reports on the first characterization of the alternative NADH:dehydrogenase (also known as alternative complex I or type II NADH:dehydrogenase) of the human malaria parasite Plasmodium falciparum, known as PfNDH2. PfNDH2 was shown to actively oxidize NADH in the presence of quinone electron acceptors CoQ1 and decylubiquinone with an apparent Km for NADH of approximately 17 and 5 μM, respectively. The inhibitory profile of PfNDH2 revealed that the enzyme activity was insensitive to rotenone, consistent with recent genomic data indicating the absence of the canonical NADH:dehydrogenase enzyme. PfNDH2 activity was sensitive to diphenylene iodonium chloride and diphenyl iodonium chloride, known inhibitors of alternative NADH:dehydrogenases. Spatiotemporal confocal imaging of parasite mitochondria revealed that loss of PfNDH2 function provoked a collapse of mitochondrial transmembrane potential (Ψm), leading to parasite death. As with other alternative NADH:dehydrogenases, PfNDH2 lacks transmembrane domains in its protein structure, and therefore, it is proposed that this enzyme is not directly involved in mitochondrial transmembrane proton pumping. Rather, the enzyme provides reducing equivalents for downstream proton-pumping enzyme complexes. As inhibition of PfNDH2 leads to a depolarization of mitochondrial Ψm, this enzyme is likely to be a critical component of the electron transport chain (ETC). This notion is further supported by proof-of-concept experiments revealing that targeting the ETCs Q-cycle by inhibition of both PfNDH2 and the bc1 complex is highly synergistic. The potential of targeting PfNDH2 as a chemotherapeutic strategy for drug development is discussed.

PfCRT and the trans-vacuolar proton electrochemical gradient: regulating the access of chloroquine to ferriprotoporphyrin IX.

It is accepted that resistance of Plasmodium falciparum to chloroquine (CQ) is caused primarily by mutations in the pfcrt gene. However, a consensus has not yet been reached on the mechanism by which resistance is achieved. CQ‐resistant (CQR) parasite lines accumulate less CQ than do CQ‐sensitive (CQS) parasites. The CQR phenotype is complex with a component of reduced energy‐dependent CQ uptake and an additional component that resembles energy‐dependent CQ efflux. Here we show that the required energy input is in the form of the proton electrochemical gradient across the digestive vacuole (DV) membrane. Collapsing the DV proton gradient (or starving the parasites of glucose) results in similar levels of CQ accumulation in CQS and CQR lines. Under these conditions the accumulation of CQ is stimulated in CQR parasite lines but is reduced in CQS lines. Energy deprivation has no effect on the rate of CQ efflux from CQR lines implying that mutant PfCRT does not function as an efflux pump or active carrier. Using pfcrt‐modified parasite lines we show that the entire CQ susceptibility phenotype is switched by the single K76T amino acid change in PfCRT. The efflux of CQ in CQR lines is not directly coupled to the energy supply, consistent with a model in which mutant PfCRT functions as a gated channel or pore, allowing charged CQ species to leak out of the DV.

Inhibiting Plasmodium cytochrome bc1: a complex issue.

The cytochrome bc(1) complex is a key mitochondrial enzyme that catalyses transfer of electrons maintaining the membrane potential of mitochondria. Currently, atovaquone is the only drug in clinical use targeting the Plasmodium falciparum bc(1) complex. The rapid emergence of resistance to atovaquone resulted in a costly combination with proguanil (Malarone), limiting its widespread use in resource-poor disease-endemic areas. Cheaper alternatives that can overcome resistance are desperately required. Here we describe recent advances of bc(1)-targeted inhibitors that include hydroxynaphthoquinones (atovaquone analogues), pyridones (clodipol analogues), acridine related compounds (acridinediones and acridones) and quinolones. Significantly, many of these developmental compounds demonstrate little cross resistance with atovaquone-resistant parasite strains, and selected classes have excellent oral activity profiles in rodent models of malaria.

Acridinediones: Selective and Potent Inhibitors of the Malaria Parasite Mitochondrial bc1 Complex

The development of drug resistance to affordable drugs has contributed to a global increase in the number of deaths from malaria. This unacceptable situation has stimulated research for new drugs active against multidrug-resistant Plasmodium falciparum parasites. In this regard, we show here that deshydroxy-1-imino derivatives of acridine (i.e., dihydroacridinediones) are selective antimalarial drugs acting as potent (nanomolar Ki) inhibitors of parasite mitochondrial bc1 complex. Inhibition of the bc1 complex led to a collapse of the mitochondrial membrane potential, resulting in cell death (IC50 ∼15 nM). The selectivity of one of the dihydroacridinediones against the parasite enzyme was some 5000-fold higher than for the human bc1 complex, significantly higher (∼200 fold) than that observed with atovaquone, a licensed bc1-specific antimalarial drug. Experiments performed with yeast manifesting mutations in the bc1 complex reveal that binding is directed to the quinol oxidation site (Qo) of the bc1 complex. This is supported by favorable binding energies for in silico docking of dihydroacridinediones to P. falciparum bc1 Qo. Dihydroacridinediones represent an entirely new class of bc1 inhibitors and the potential of these compounds as novel antimalarial drugs is discussed.

A Medicinal Chemistry Perspective on 4-Aminoquinoline Antimalarial Drugs

A broad overview is presented describing the current knowledge and the ongoing research concerning the 4-aminoquinolines (4AQ) as chemotherapeutic antimalarial agents. Included are discussions of mechanism of action, structure activity relationships (SAR), chemistry, metabolism and toxicity and parasite resistance mechanisms. In discussions of SAR, particular emphasis has been given to activity versus chloroquine resistant strains of Plasmodium falciparum. Promising new lead compounds undergoing development are described and an overview of physicochemical properties of chloroquine and amodiaquine analogues is also included.

Heme Binding Contributes to Antimalarial Activity of Bis-Quaternary Ammoniums

ABSTRACT Quaternary ammonium compounds have received recent attention due to their potent in vivo antimalarial activity based on their ability to inhibit de novo phosphatidylcholine synthesis. Here we show that in addition to this, heme binding significantly contributes to the antimalarial activity of these compounds. For the study, we used a recently synthesized bis-quaternary ammonium compound, T16 (1,12-dodecanemethylene bis[4-methyl-5-ethylthiazolium] diodide), which exhibits potent antimalarial activity (50% inhibitory concentration, ∼25 nM). Accumulation assays reveal that this compound is readily concentrated several hundredfold (cellular accumulation ratio, ∼500) into parasitized erythrocytes. Approximately 80% of the drug was shown to be distributed within the parasite, ∼50% of which was located in the parasite food vacuoles. T16 uptake was affected by anion substitution (permeation increasing in the order Cl− < Br− = NO3− < I− < SCN−) and was sensitive to furosemide—properties similar to substrates of the induced new permeability pathway in infected erythrocytes. Scatchard plot analysis of in situ T16 binding revealed high-affinity and low-affinity binding sites. The high-affinity binding site Kd was similar to that measured in vitro for T16 and ferriprotoporphyrin IX (FPIX) binding. Significantly, the capacity but not the Kd of the high-affinity binding site was decreased by reducing the concentration of parasite FPIX. Decreasing the parasite FPIX pool also caused a marked antagonism of T16 antimalarial activity. In addition, T16 was also observed to associate with parasite hemozoin. Binding of T16 to FPIX in the digestive food vacuole is shown to be critical for drug accumulation and antimalarial activity. These data provide additional new mechanisms of antimalarial activity for this promising new class of antimalarial compounds.

Identification, design and biological evaluation of heterocyclic quinolones targeting Plasmodium falciparum type II NADH:quinone oxidoreductase (PfNDH2).

A program was undertaken to identify hit compounds against NADH:ubiquinone oxidoreductase (PfNDH2), a dehydrogenase of the mitochondrial electron transport chain of the malaria parasite Plasmodium falciparum. PfNDH2 has only one known inhibitor, hydroxy-2-dodecyl-4-(1H)-quinolone (HDQ), and this was used along with a range of chemoinformatics methods in the rational selection of 17u2009000 compounds for high-throughput screening. Twelve distinct chemotypes were identified and briefly examined leading to the selection of the quinolone core as the key target for structure–activity relationship (SAR) development. Extensive structural exploration led to the selection of 2-bisaryl 3-methyl quinolones as a series for further biological evaluation. The lead compound within this series 7-chloro-3-methyl-2-(4-(4-(trifluoromethoxy)benzyl)phenyl)quinolin-4(1H)-one (CK-2-68) has antimalarial activity against the 3D7 strain of P. falciparum of 36 nM, is selective for PfNDH2 over other respiratory enzymes (inhibitory IC50 against PfNDH2 of 16 nM), and demonstrates low cytotoxicity and high metabolic stability in the presence of human liver microsomes. This lead compound and its phosphate pro-drug have potent in vivo antimalarial activity after oral administration, consistent with the target product profile of a drug for the treatment of uncomplicated malaria. Other quinolones presented (e.g., 6d, 6f, 14e) have the capacity to inhibit both PfNDH2 and P. falciparum cytochrome bc1, and studies to determine the potential advantage of this dual-targeting effect are in progress.

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.

Artemisinin activity-based probes identify multiple molecular targets within the asexual stage of the malaria parasites Plasmodium falciparum 3D7

Significance The mechanism of action of the artemisinin (ART) class of antimalarial drugs, the most important antimalarial drug class in use today, remains controversial, despite more than three decades of intensive research. We have developed an unbiased chemical proteomic approach using a suite of ART activity-based protein profiling probes to identify proteins within the malaria parasite that are alkylated by ART, including proteins involved in glycolysis, hemoglobin metabolism, and redox defense. The data point to a pleiotropic mechanism of drug action for this class and offer a strategy for investigating resistance mechanisms to ART-based drugs as well as mechanisms of action of other endoperoxide-based drugs. The artemisinin (ART)-based antimalarials have contributed significantly to reducing global malaria deaths over the past decade, but we still do not know how they kill parasites. To gain greater insight into the potential mechanisms of ART drug action, we developed a suite of ART activity-based protein profiling probes to identify parasite protein drug targets in situ. Probes were designed to retain biological activity and alkylate the molecular target(s) of Plasmodium falciparum 3D7 parasites in situ. Proteins tagged with the ART probe can then be isolated using click chemistry before identification by liquid chromatography–MS/MS. Using these probes, we define an ART proteome that shows alkylated targets in the glycolytic, hemoglobin degradation, antioxidant defense, and protein synthesis pathways, processes essential for parasite survival. This work reveals the pleiotropic nature of the biological functions targeted by this important class of antimalarial drugs.