David C. Tully

Targeting Plasmodium PI(4)K to eliminate malaria

Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.Summary Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here, we identify a lipid kinase, phosphatidylinositol 4-kinase (PI4K), as the target of imidazopyrazines, a novel antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens, P. falciparum and P. vivax, and inhibit liver stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI4K, altering the intracellular distribution of phosphatidylinositol 4-phosphate. Collectively, our data define PI4K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.

Inhibitors of cathepsin s

Cathepsin B is an abundant and ubiquitously expressed cysteine peptidase of the papain family. It is involved in many physiological processes, such as remodeling of the extracellular matrix (wound healing), apoptosis, and activation of thyroxine and renin. In addition to its physiological roles, cathepsin B is important in many pathological processes, such as inflammation, parasite infection and cancer, where it is highly up-regulated. In cancer patients, elevated cathepsin B activity correlates to poor therapy outcome. Therefore, it is not surprising that the use of cathepsin B inhibitors reduces both tumor cell motility and invasiveness in vitro. This review summarizes recent developments in cathepsin B inhibition. To date, numerous protein inhibitors of cathepsin B have been described, some of which are of endogenous origin and function as regulators of cathepsin B activity in the cell, such as the cystatins. In addition, some exogenous protein inhibitors of cathepsin B have been isolated from various natural sources, and the use of X-ray crystal structures of cathepsin B complexed with such protein inhibitors has resulted in the design and synthesis of many new small-molecular-weight compounds as inhibitors of cathepsin B. These synthetic compounds generally contain an electrophilic functionality that reacts with cathepsin B. In the present review, these inhibitors are divided according to their mechanisms of action, as reversible and irreversible, and then further subdivided into groups for their full descriptions.

Chemical activation of the mechanotransduction channel Piezo1

Piezo ion channels are activated by various types of mechanical stimuli and function as biological pressure sensors in both vertebrates and invertebrates. To date, mechanical stimuli are the only means to activate Piezo ion channels and whether other modes of activation exist is not known. In this study, we screened ∼3.25 million compounds using a cell-based fluorescence assay and identified a synthetic small molecule we termed Yoda1 that acts as an agonist for both human and mouse Piezo1. Functional studies in cells revealed that Yoda1 affects the sensitivity and the inactivation kinetics of mechanically induced responses. Characterization of Yoda1 in artificial droplet lipid bilayers showed that Yoda1 activates purified Piezo1 channels in the absence of other cellular components. Our studies demonstrate that Piezo1 is amenable to chemical activation and raise the possibility that endogenous Piezo1 agonists might exist. Yoda1 will serve as a key tool compound to study Piezo1 regulation and function. DOI: http://dx.doi.org/10.7554/eLife.07369.001

Imidazolopiperazines: hit to lead optimization of new antimalarial agents.

Starting from a hit series from a GNF compound library collection and based on a cell-based proliferation assay of Plasmodium falciparum, a novel imidazolopiperazine scaffold was optimized. SAR for this series of compounds is discussed, focusing on optimization of cellular potency against wild-type and drug resistant parasites and improvement of physiochemical and pharmacokinetic properties. The lead compounds in this series showed good potencies in vitro and decent oral exposure levels in vivo. In a Plasmodium berghei mouse infection model, one lead compound lowered the parasitemia level by 99.4% after administration of 100 mg/kg single oral dose and prolonged mice survival by an average of 17.0 days. The lead compounds were also well-tolerated in the preliminary in vitro toxicity studies and represents an interesting lead for drug development.

KAF156 Is an Antimalarial Clinical Candidate with Potential for Use in Prophylaxis, Treatment, and Prevention of Disease Transmission

ABSTRACT Renewed global efforts toward malaria eradication have highlighted the need for novel antimalarial agents with activity against multiple stages of the parasite life cycle. We have previously reported the discovery of a novel class of antimalarial compounds in the imidazolopiperazine series that have activity in the prevention and treatment of blood stage infection in a mouse model of malaria. Consistent with the previously reported activity profile of this series, the clinical candidate KAF156 shows blood schizonticidal activity with 50% inhibitory concentrations of 6 to 17.4 nM against P. falciparum drug-sensitive and drug-resistant strains, as well as potent therapeutic activity in a mouse models of malaria with 50, 90, and 99% effective doses of 0.6, 0.9, and 1.4 mg/kg, respectively. When administered prophylactically in a sporozoite challenge mouse model, KAF156 is completely protective as a single oral dose of 10 mg/kg. Finally, KAF156 displays potent Plasmodium transmission blocking activities both in vitro and in vivo. Collectively, our data suggest that KAF156, currently under evaluation in clinical trials, has the potential to treat, prevent, and block the transmission of malaria.

Imidazolopiperazines:Lead Optimization of the Second-Generation Antimalarial Agents

On the basis of the initial success of optimization of a novel series of imidazolopiperazines, a second generation of compounds involving changes in the core piperazine ring was synthesized to improve antimalarial properties. These changes were carried out to further improve the potency and metabolic stability of the compounds by leveraging the outcome of a set of in vitro metabolic identification studies. The optimized 8,8-dimethyl imidazolopiperazine analogues exhibited improved potency, in vitro metabolic stability profile and, as a result, enhanced oral exposure in vivo in mice. The optimized compounds were found to be more efficacious than the current antimalarials in a malaria mouse model. They exhibit moderate oral exposure in rat pharmacokinetic studies to achieve sufficient multiples of the oral exposure at the efficacious dose in toxicology studies.

Camostat attenuates airway epithelial sodium channel function in vivo through the inhibition of a channel-activating protease

Inhibition of airway epithelial sodium channel (ENaC) function enhances mucociliary clearance (MCC). ENaC is positively regulated by channel-activating proteases (CAPs), and CAP inhibitors are therefore predicted to be beneficial in diseases associated with impaired MCC. The aims of the present study were to 1) identify low-molecular-weight inhibitors of airway CAPs and 2) to establish whether such CAP inhibitors would translate into a negative regulation of ENaC function in vivo, with a consequent enhancement of MCC. To this end, camostat, a trypsin-like protease inhibitor, provided a potent (IC50 ∼50 nM) and prolonged attenuation of ENaC function in human airway epithelial cell models that was reversible upon the addition of excess trypsin. In primary human bronchial epithelial cells, a potency order of placental bikunin > camostat > 4-guanidinobenzoic acid 4-carboxymethyl-phenyl ester > aprotinin >> soybean trypsin inhibitor = α1-antitrypsin, was largely consistent with that observed for inhibition of prostasin, a molecular candidate for the airway CAP. In vivo, topical airway administration of camostat induced a potent and prolonged attenuation of ENaC activity in the guinea pig trachea (ED50 = 3 μg/kg). When administered by aerosol inhalation in conscious sheep, camostat enhanced MCC out to at least 5 h after inhaled dosing. In summary, camostat attenuates ENaC function and enhances MCC, providing an opportunity for this approach toward the negative regulation of ENaC function to be tested therapeutically.

Active site conformational changes of prostasin provide a new mechanism of protease regulation by divalent cations.

Prostasin or human channel‐activating protease 1 has been reported to play a critical role in the regulation of extracellular sodium ion transport via its activation of the epithelial cell sodium channel. Here, the structure of the extracellular portion of the membrane associated serine protease has been solved to high resolution in complex with a nonselective d‐FFR chloromethyl ketone inhibitor, in an apo form, in a form where the apo crystal has been soaked with the covalent inhibitor camostat and in complex with the protein inhibitor aprotinin. It was also crystallized in the presence of the divalent cation Ca+2. Comparison of the structures with each other and with other members of the trypsin‐like serine protease family reveals unique structural features of prostasin and a large degree of conformational variation within specificity determining loops. Of particular interest is the S1 subsite loop which opens and closes in response to basic residues or divalent ions, directly binding Ca+2 cations. This induced fit active site provides a new possible mode of regulation of trypsin‐like proteases adapted in particular to extracellular regions with variable ionic concentrations such as the outer membrane layer of the epithelial cell.

Discovery, Optimization, and Biological Evaluation of 5-(2-(Trifluoromethyl)phenyl)indazoles as a Novel Class of Transient Receptor Potential A1 (TRPA1) Antagonists

A high throughput screening campaign identified 5-(2-chlorophenyl)indazole compound 4 as an antagonist of the transient receptor potential A1 (TRPA1) ion channel with IC50 = 1.23 μM. Hit to lead medicinal chemistry optimization established the SAR around the indazole ring system, demonstrating that a trifluoromethyl group at the 2-position of the phenyl ring in combination with various substituents at the 6-position of the indazole ring greatly contributed to improvements in vitro activity. Further lead optimization resulted in the identification of compound 31, a potent and selective antagonist of TRPA1 in vitro (IC50 = 0.015 μM), which has moderate oral bioavailability in rodents and demonstrates robust activity in vivo in several rodent models of inflammatory pain.

Lead optimization of imidazopyrazines: a new class of antimalarial with activity on Plasmodium liver stages.

Imidazopyridine 1 was identified from a phenotypic screen against P. falciparum (Pf) blood stages and subsequently optimized for activity on liver-stage schizonts of the rodent parasite P. yoelii (Py) as well as hypnozoites of the simian parasite P. cynomolgi (Pc). We applied these various assays to the cell-based lead optimization of the imidazopyrazines, exemplified by 3 (KAI407), and show that optimized compounds within the series with improved pharmacokinetic properties achieve causal prophylactic activity in vivo and may have the potential to target the dormant stages of P. vivax malaria.