Discovery of Novel Quinoline-Based Proteasome Inhibitors for Human African Trypanosomiasis (HAT).

Human African Trypanosomiasis (HAT) is a vector-borne disease caused by kinetoplastid parasites of the Trypanosoma genus. The disease proceeds in two stages, with a hemolymphatic blood stage and a meningo-encephalic brain stage. In the latter stage, the parasite causes irreversible damage to the brain leading to sleep cycle disruption and is fatal if untreated. An orally bioavailable treatment is highly desirable. In this study, we present a brain-penetrant, parasite-selective 20S proteasome inhibitor that was rapidly optimized from an HTS singleton hit to drug candidate compound 7 that showed cure in a stage II mouse efficacy model. Here, we describe hit expansion and lead optimization campaign guided by cryo-electron microscopy and an in silico model to predict the brain-to-plasma partition coefficient Kp as an important parameter to prioritize compounds for synthesis. The model combined with in vitro and in vivo experiments allowed us to advance compounds with favorable unbound brain-to-plasma ratios (Kp,uu) to cure a CNS disease such as HAT.

Targeting the trypanosome kinetochore with CLK1 protein kinase inhibitors.

The kinetochore is a macromolecular structure that assembles on the centromeres of chromosomes and provides the major attachment point for spindle microtubules during mitosis. In Trypanosoma brucei, the proteins that make up the kinetochore are highly divergent; the inner kinetochore comprises at least 20 distinct and essential proteins (KKT1-20) that include four protein kinases-CLK1 (also known as KKT10), CLK2 (also known as KKT19), KKT2 and KKT3. Here, we report the identification and characterization of the amidobenzimidazoles (AB) protein kinase inhibitors that show nanomolar potency against T. brucei bloodstream forms, Leishmania and Trypanosoma cruzi. We performed target deconvolution analysis using a selection of 29 T. brucei mutants that overexpress known essential protein kinases, and identified CLK1 as a primary target. Biochemical studies and the co-crystal structure of CLK1 in complex with AB1 show that the irreversible competitive inhibition of CLK1 is dependent on a Michael acceptor forming an irreversible bond with Cys 215 in the ATP-binding pocket, a residue that is not present in human CLK1, thereby providing selectivity. Chemical inhibition of CLK1 impairs inner kinetochore recruitment and compromises cell-cycle progression, leading to cell death. This research highlights a unique drug target for trypanosomatid parasitic protozoa and a new chemical tool for investigating the function of their divergent kinetochores.

Discovery and Characterization of Clinical Candidate LXE408 as a Kinetoplastid-Selective Proteasome Inhibitor for the Treatment of Leishmaniases.

Visceral leishmaniasis is responsible for up to 30,000 deaths every year. Current treatments have shortcomings that include toxicity and variable efficacy across endemic regions. Previously, we reported the discovery of GNF6702, a selective inhibitor of the kinetoplastid proteasome, which cleared parasites in murine models of leishmaniasis, Chagas disease, and human African trypanosomiasis. Here, we describe the discovery and characterization of LXE408, a structurally related kinetoplastid-selective proteasome inhibitor currently in Phase 1 human clinical trials. Furthermore, we present high-resolution cryo-EM structures of the Leishmania tarentolae proteasome in complex with LXE408, which provides a compelling explanation for the noncompetitive mode of binding of this novel class of inhibitors of the kinetoplastid proteasome.

Anti-Trypanosomal Proteasome Inhibitors Cure Hemolymphatic and Meningoencephalic Murine Infection Models of African Trypanosomiasis.

Current anti-trypanosomal therapies suffer from problems of longer treatment duration, toxicity and inadequate efficacy, hence there is a need for safer, more efficacious and 'easy to use' oral drugs. Previously, we reported the discovery of the triazolopyrimidine (TP) class as selective kinetoplastid proteasome inhibitors with in vivo efficacy in mouse models of leishmaniasis, Chagas Disease and African trypanosomiasis (HAT). For the treatment of HAT, development compounds need to have excellent penetration to the brain to cure the meningoencephalic stage of the disease. Here we describe detailed biological and pharmacological characterization of triazolopyrimidine compounds in HAT specific assays. The TP class of compounds showed single digit nanomolar potency against Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense strains. These compounds are trypanocidal with concentration-time dependent kill and achieved relapse-free cure in vitro. Two compounds, GNF6702 and a new analog NITD689, showed favorable in vivo pharmacokinetics and significant brain penetration, which enabled oral dosing. They also achieved complete cure in both hemolymphatic (blood) and meningoencephalic (brain) infection of human African trypanosomiasis mouse models. Mode of action studies on this series confirmed the 20S proteasome as the target in T. brucei. These proteasome inhibitors have the potential for further development into promising new treatment for human African trypanosomiasis.

Engineering yeast endosymbionts as a step toward the evolution of mitochondria.

It has been hypothesized that mitochondria evolved from a bacterial ancestor that initially became established in an archaeal host cell as an endosymbiont. Here we model this first stage of mitochondrial evolution by engineering endosymbiosis between Escherichia coli and Saccharomyces cerevisiae An ADP/ATP translocase-expressing E. coli provided ATP to a respiration-deficient cox2 yeast mutant and enabled growth of a yeast-E. coli chimera on a nonfermentable carbon source. In a reciprocal fashion, yeast provided thiamin to an endosymbiotic E. coli thiamin auxotroph. Expression of several SNARE-like proteins in E. coli was also required, likely to block lysosomal degradation of intracellular bacteria. This chimeric system was stable for more than 40 doublings, and GFP-expressing E. coli endosymbionts could be observed in the yeast by fluorescence microscopy and X-ray tomography. This readily manipulated system should allow experimental delineation of host-endosymbiont adaptations that occurred during evolution of the current, highly reduced mitochondrial genome.

Gene Expression Profiling of Endoplasmic Reticulum Stress in Hepatitis C Virus-Containing Cells Treated with an Inhibitor of Protein Disulfide Isomerases.

Protein disulfide isomerases (PDIs) catalyze disulfide bond formation between protein cysteine residues during protein folding in the endoplasmic reticulum (ER) lumen and are essential for maintaining ER homoeostasis. The life cycle of the hepatitis C virus (HCV) is closely associated with the ER. Synthesis and maturation of HCV proteins occur in the ER membrane and are mediated by multiple host cell factors that include also PDI. Here, we present a study investigating the effect of PDI inhibition on Huh7 human hepatoma cells harboring an HCV subgenomic replicon using the abscisic acid-derived PDI inhibitor origamicin. Transcriptional profiling shows that origamicin changed the expression levels of genes involved in the oxidative and ER stress responses and the unfolded protein response, as indicated by the upregulation of antioxidant enzymes and chaperone proteins, the downregulation of cell-cycle proteins, and induction of apoptosis-associated genes. Our data suggest that origamicin negatively impacts HCV replication by causing an imbalance in cellular homoeostasis and induction of stress responses. These insights suggest that inhibition of PDIs by low-molecular-weight inhibitors could be a promising approach to the discovery of novel antiviral compounds.

Proteasome inhibition for treatment of leishmaniasis, Chagas disease and sleeping sickness.

Chagas disease, leishmaniasis and sleeping sickness affect 20 million people worldwide and lead to more than 50,000 deaths annually. The diseases are caused by infection with the kinetoplastid parasites Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp., respectively. These parasites have similar biology and genomic sequence, suggesting that all three diseases could be cured with drugs that modulate the activity of a conserved parasite target. However, no such molecular targets or broad spectrum drugs have been identified to date. Here we describe a selective inhibitor of the kinetoplastid proteasome (GNF6702) with unprecedented in vivo efficacy, which cleared parasites from mice in all three models of infection. GNF6702 inhibits the kinetoplastid proteasome through a non-competitive mechanism, does not inhibit the mammalian proteasome or growth of mammalian cells, and is well-tolerated in mice. Our data provide genetic and chemical validation of the parasite proteasome as a promising therapeutic target for treatment of kinetoplastid infections, and underscore the possibility of developing a single class of drugs for these neglected diseases.

Antitrypanosomal Treatment with Benznidazole Is Superior to Posaconazole Regimens in Mouse Models of Chagas Disease.

Two CYP51 inhibitors, posaconazole and the ravuconazole prodrug E1224, were recently tested in clinical trials for efficacy in indeterminate Chagas disease. The results from these studies show that both drugs cleared parasites from the blood of infected patients at the end of the treatment but that parasitemia rebounded over the following months. In the current study, we sought to identify a dosing regimen of posaconazole that could permanently clear Trypanosoma cruzi from mice with experimental Chagas disease. Infected mice were treated with posaconazole or benznidazole, an established Chagas disease drug, and parasitological cure was defined as an absence of parasitemia recrudescence after immunosuppression. Twenty-day therapy with benznidazole (10 to 100 mg/kg of body weight/day) resulted in a dose-dependent increase in antiparasitic activity, and the 100-mg/kg regimen effected parasitological cure in all treated mice. In contrast, all mice remained infected after a 25-day treatment with posaconazole at all tested doses (10 to 100 mg/kg/day). Further extension of posaconazole therapy to 40 days resulted in only a marginal improvement of treatment outcome. We also observed similar differences in antiparasitic activity between benznidazole and posaconazole in acute T. cruzi heart infections. While benznidazole induced rapid, dose-dependent reductions in heart parasite burdens, the antiparasitic activity of posaconazole plateaued at low doses (3 to 10 mg/kg/day) despite increasing drug exposure in plasma. These observations are in good agreement with the outcomes of recent phase 2 trials with posaconazole and suggest that the efficacy models combined with the pharmacokinetic analysis employed here will be useful in predicting clinical outcomes of new drug candidates.

Utilizing Chemical Genomics to Identify Cytochrome b as a Novel Drug Target for Chagas Disease.

Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc1) in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, QN and QP. The L197F mutation is located in the QN site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another QN site inhibitor, but not to strobilurin or myxothiazol, which target the QP site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC50) of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease.

Lead identification to clinical candidate selection: drugs for Chagas disease.

Chagas disease affects 8 million people worldwide and remains a main cause of death due to heart failure in Latin America. The number of cases in the United States is now estimated to be 300,000, but there are currently no Food and Drug Administration (FDA)-approved drugs available for patients with Chagas disease. To fill this gap, we have established a public-private partnership between the University of California, San Francisco and the Genomics Institute of the Novartis Research Foundation (GNF) with the goal of delivering clinical candidates to treat Chagas disease. The discovery phase, based on the screening of more than 160,000 compounds from the GNF Academic Collaboration Library, led to the identification of new anti-Chagas scaffolds. Part of the screening campaign used and compared two screening methods, including a colorimetric-based assay using Trypanosoma cruzi expressing ß-galactosidase and an image-based, high-content screening (HCS) assay using the CA-I/72 strain of T. cruzi. Comparing molecules tested in both assays, we found that ergosterol biosynthesis inhibitors had greater potency in the colorimetric assay than in the HCS assay. Both assays were used to inform structure-activity relationships for antiparasitic efficacy and pharmacokinetics. A new anti-T. cruzi scaffold derived from xanthine was identified, and we describe its development as lead series.

Substituted 2-phenylimidazopyridines: a new class of drug leads for human African trypanosomiasis.

A phenotypic screen of a compound library for antiparasitic activity on Trypanosoma brucei, the causative agent of human African trypanosomiasis, led to the identification of substituted 2-(3-aminophenyl)oxazolopyridines as a starting point for hit-to-lead medicinal chemistry. A total of 110 analogues were prepared, which led to the identification of 64, a substituted 2-(3-aminophenyl)imidazopyridine. This compound showed antiparasitic activity in vitro with an EC50 of 2 nM and displayed reasonable druglike properties when tested in a number of in vitro assays. The compound was orally bioavailable and displayed good plasma and brain exposure in mice. Compound 64 cured mice infected with Trypanosoma brucei when dosed orally down to 2.5 mg/kg. Given its potent antiparasitic properties and its ease of synthesis, compound 64 represents a new lead for the development of drugs to treat human African trypanosomiasis.

Targeting the ERAD pathway via inhibition of signal peptide peptidase for antiparasitic therapeutic design.

Early secretory and endoplasmic reticulum (ER)-localized proteins that are terminally misfolded or misassembled are degraded by a ubiquitin- and proteasome-mediated process known as ER-associated degradation (ERAD). Protozoan pathogens, including the causative agents of malaria, toxoplasmosis, trypanosomiasis, and leishmaniasis, contain a minimal ERAD network relative to higher eukaryotic cells, and, because of this, we observe that the malaria parasite Plasmodium falciparum is highly sensitive to the inhibition of components of this protein quality control system. Inhibitors that specifically target a putative protease component of ERAD, signal peptide peptidase (SPP), have high selectivity and potency for P. falciparum. By using a variety of methodologies, we validate that SPP inhibitors target P. falciparum SPP in parasites, disrupt the protein's ability to facilitate degradation of unstable proteins, and inhibit its proteolytic activity. These compounds also show low nanomolar activity against liver-stage malaria parasites and are also equipotent against a panel of pathogenic protozoan parasites. Collectively, these data suggest ER quality control as a vulnerability of protozoan parasites, and that SPP inhibition may represent a suitable transmission blocking antimalarial strategy and potential pan-protozoan drug target.

Selective and specific inhibition of the plasmodium falciparum lysyl-tRNA synthetase by the fungal secondary metabolite cladosporin.

With renewed calls for malaria eradication, next-generation antimalarials need be active against drug-resistant parasites and efficacious against both liver- and blood-stage infections. We screened a natural product library to identify inhibitors of Plasmodium falciparum blood- and liver-stage proliferation. Cladosporin, a fungal secondary metabolite whose target and mechanism of action are not known for any species, was identified as having potent, nanomolar, antiparasitic activity against both blood and liver stages. Using postgenomic methods, including a yeast deletion strains collection, we show that cladosporin specifically inhibits protein synthesis by directly targeting P. falciparum cytosolic lysyl-tRNA synthetase. Further, cladosporin is >100-fold more potent against parasite lysyl-tRNA synthetase relative to the human enzyme, which is conferred by the identity of two amino acids within the enzyme active site. Our data indicate that lysyl-tRNA synthetase is an attractive, druggable, antimalarial target that can be selectively inhibited.

A high-throughput screen to identify inhibitors of ATP homeostasis in non-replicating Mycobacterium tuberculosis.

Growing evidence suggests that the presence of a subpopulation of hypoxic non-replicating, phenotypically drug-tolerant mycobacteria is responsible for the prolonged duration of tuberculosis treatment. The discovery of new antitubercular agents active against this subpopulation may help in developing new strategies to shorten the time of tuberculosis therapy. Recently, the maintenance of a low level of bacterial respiration was shown to be a point of metabolic vulnerability in Mycobacterium tuberculosis. Here, we describe the development of a hypoxic model to identify compounds targeting mycobacterial respiratory functions and ATP homeostasis in whole mycobacteria. The model was adapted to 1,536-well plate format and successfully used to screen over 600,000 compounds. Approximately 800 compounds were confirmed to reduce intracellular ATP levels in a dose-dependent manner in Mycobacterium bovis BCG. One hundred and forty non-cytotoxic compounds with activity against hypoxic non-replicating M. tuberculosis were further validated. The resulting collection of compounds that disrupt ATP homeostasis in M. tuberculosis represents a valuable resource to decipher the biology of persistent mycobacteria.

A single mutation in the first transmembrane domain of yeast COX2 enables its allotopic expression.

During the course of evolution, a massive reduction of the mitochondrial genome content occurred that was associated with transfer of a large number of genes to the nucleus. To further characterize factors that control the mitochondrial gene transfer/retention process, we have investigated the barriers to transfer of yeast COX2, a mitochondrial gene coding for a subunit of cytochrome c oxidase complex. Nuclear-recoded Saccharomyces cerevisiae COX2 fused at the amino terminus to various alternative mitochondrial targeting sequences (MTS) fails to complement the growth defect of a yeast strain with an inactivated mitochondrial COX2 gene, even though it is expressed in cells. Through random mutagenesis of one such hybrid MTS-COX2, we identified a single mutation in the first Cox2 transmembrane domain (W56 --> R) that (i) results in the cellular expression of a Cox2 variant with a molecular mass indicative of MTS cleavage, which (ii) supports growth of a cox2 mutant on a nonfermentable carbon source, and that (iii) partially restores cytochrome c oxidase-specific respiration by the mutant mitochondria. COX2(W56R) can be allotopically expressed with an MTS derived from S. cerevisiae OXA1 or Neurospora crassa SU9, both coding for hydrophobic mitochondrial proteins, but not with an MTS derived from the hydrophilic protein Cox4. In contrast to some other previously transferred genes, allotopic COX2 expression is not enabled or enhanced by a 3'-UTR that localizes mRNA translation to the mitochondria, such as yeast ATP2(3)('-UTR). Application of in vitro evolution strategies to other mitochondrial genes might ultimately lead to yeast entirely lacking the mitochondrial genome, but still possessing functional respiratory capacity.

Identification of human kinases involved in hepatitis C virus replication by small interference RNA library screening.

The propagation of the hepatitis C virus (HCV) is a complex process that requires both host and viral proteins. To facilitate identification of host cell factors that are required for HCV replication, we screened a panel of small interference RNAs that preferentially target human protein kinases using an HCV replicon expressing the firefly luciferase gene as a genetic reporter. Small interference RNAs specific for three human kinases, Csk, Jak1, and Vrk1, were identified that reproducibly reduce viral RNA and viral protein levels in HCV replicon-bearing cells. Treatment of replicon cells with a small molecule inhibitor of Csk also resulted in a significant reduction in HCV RNA and proteins, further supporting a role for Csk in HCV replication. The effects of siRNAs targeting eight kinases known to be negatively regulated by Csk were then examined; knock down of one of these kinases, Fyn, resulted in up-regulation of the HCV replicon, suggesting that Csk mediates its effect on HCV replication through Fyn. This conclusion was further corroborated by demonstration that replicon cells treated with Csk inhibitor contained lower levels of the phosphorylated form of Fyn than control cells.

siRNA in human cells selectively localizes to target RNA sites.

Recent observations of RNA interference (RNAi) in the nuclei of human cells raise key questions about the extent to which nuclear and cytoplasmic RNAi pathways are shared. By directly visualizing the localization of small interfering RNA (siRNA) in live human cells, we show here that siRNA either selectively localizes in the cytoplasm or translocates into the nucleus, depending on where the silencing target RNA resides. Two siRNAs that target the small nuclear 7SK and U6 RNAs localize into the nucleus as duplexes. In contrast, an siRNA targeting the cytoplasmic hepatitis C virus replicon RNA dissociates, and only antisense strand distributes in the cytoplasm of the cells harboring the target RNA, whereas sense strand gets degraded. At the same time, both strands of the latter siRNA are distributed throughout the cytoplasm and nucleus in cells lacking the silencing target RNA. These results suggest the existence of a mechanism by which the RNAi machinery orchestrates a target-determined localization of the siRNA and the corresponding RNAi activity, and also provide evidence for formation of nuclear-programmed active RNA induced silencing complexes directly in the nucleus.

A genome-wide overexpression screen in yeast for small-molecule target identification.

We describe a multicopy gene suppression screen of drug sensitivity in Saccharomyces cerevisiae that facilitates the identification of cellular targets of small molecules. An array of yeast transformants harboring a multicopy yeast genomic library was screened for resistance to growth inhibitors. Comparison of array growth patterns for several such inhibitors allowed the differentiation of general and molecule-specific genetic suppressors. Specific resistance to phenylaminopyrimidine (1), an inhibitor identified from a kinase-directed library, was associated with the overexpression of Pkc1 and a subset of downstream kinases. Components of two other pathways (pheromone response/filamentous growth and Pho85 kinase) that genetically interact with the PKC1 MAPK signaling cascade were also identified. Consistent with the suppression screen, inhibitor 1 bound to Pkc1 in yeast cell lysate and inhibited its activity in vitro. These results demonstrate the utility of this approach for the rapid deconvolution of small-molecule targets.

Ubiquitin signals protein trafficking via interaction with a novel ubiquitin binding domain in the membrane fusion regulator, Vps9p.

The conserved vacuolar protein-sorting (Vps) pathway controls the trafficking of proteins to the vacuole/lysosome. Both the internalization of ubiquitylated cargo from the plasma membrane and its sorting at the late endosome via the Vps pathway depend on ubiquitin (Ub) binding motifs present in trafficking regulators. Here we report that Ub controls yet a third step in the Vps pathway. Vps9p, which promotes endosomal and Golgi-derived vesicle fusion, binds directly to Ub via a Cue1p-homologous (CUE) domain. The CUE domain is structurally related to the Ub-associated (UBA) domain. In an assay for vacuolar delivery of a transmembrane receptor fused to Ub, a Ub mutation impairing interaction with Vps9p led to a cytoplasmic block in receptor trafficking. This block resembled that of a receptor fused to wild-type Ub but expressed in a vps9-null background. Strikingly, this trafficking defect caused by a mutant Ub was rescued by deletion of the Vps9p CUE domain, indicating that lack of the CUE domain renders Vps9p independent of Ub for activation in vivo. We thus provide evidence for biochemical and genetic interactions between Ub and a novel Ub binding domain in Vps9p. Ub plays a positive role, whereas the CUE domain plays both positive and negative roles in Vps9p function in trafficking.