A multi-dimensional, time-lapse, high content screening platform applied to schistosomiasis drug discovery.

Approximately 10% of the world's population is at risk of schistosomiasis, a disease of poverty caused by the Schistosoma parasite. To facilitate drug discovery for this complex flatworm, we developed an automated high-content screen to quantify the multidimensional responses of Schistosoma mansoni post-infective larvae (somules) to chemical insult. We describe an integrated platform to process worms at scale, collect time-lapsed, bright-field images, segment highly variable and touching worms, and then store, visualize, and query dynamic phenotypes. To demonstrate the methodology, we treated somules with seven drugs that generated diverse responses and evaluated 45 static and kinetic response descriptors relative to concentration and time. For compound screening, we used the Mahalanobis distance to compare multidimensional phenotypic effects induced by 1323 approved drugs. Overall, we characterize both known anti-schistosomals and identify new bioactives. Apart from facilitating drug discovery, the multidimensional quantification provided by this platform will allow mapping of chemistry to phenotype.

A Cell-Based Renilla Luminescence Reporter Plasmid Assay for High-Throughput Screening to Identify Novel FDA-Approved Drug Inhibitors of HPV-16 Infection.

Like cervical cancer, anal cancer is caused by human papillomavirus (HPV). HPV is the most common sexually transmitted agent and is found in the anal canal of almost all HIV-positive men who have sex with men (MSM). Rates of HPV anal cancer are disproportionately higher in this population. Although the nanovalent HPV vaccine is efficacious in protecting against oncogenic HPV types, a substantial proportion of MSM remains unvaccinated and anal HPV infection continues to be an important public health burden. Therefore, it is important to identify strategies to prevent HPV infection. We report on two promising and interlinked strategies: (1) the development of a cell-based Renilla luminescence reporter assay using HPV-16 pseudovirions that encapsidate SV40-driven Renilla luminescence reporter expression plasmid and (2) use of this assay for high-throughput screening (HTS) of FDA- and internationally approved drugs to identify those that could be repurposed to prevent HPV infection. We conducted a screen of 1906 drugs. The assay was valid with a Z' of 0.67 ± 0.04, percent coefficient of variance of 10.0, and signal-to-background noise window of 424.0 ± 8.0. Five drugs were chosen for further analyses based on selection parameters of ≥77.0% infection of HPV-16 pseudovirion-driven Renilla expression with <20.0% cytotoxicity. Of these, the antifungal pentamidine and a gamma-amino butyric acid receptor agonist securinine exhibited ≥90.0% infection with <10.0% cytotoxicity. This luminescent cell-based reporter expression plasmid assay for HTS is a valid method to identify FDA- and internationally approved drugs with the potential to be repurposed into prevention modalities for HPV infection.

Identification of Plasmodium dipeptidyl aminopeptidase allosteric inhibitors by high throughput screening.

Dipeptidyl aminopeptidases (DPAPs) are cysteine proteases that cleave dipeptides from the N-terminus of protein substrates and have been shown to play important roles in many pathologies including parasitic diseases such as malaria, toxoplasmosis and Chagas's disease. Inhibitors of the mammalian homologue cathepsin C have been used in clinical trials as potential drugs to treat chronic inflammatory disorders, thus proving that these enzymes are druggable. In Plasmodium species, DPAPs play important functions at different stages of parasite development, thus making them potential antimalarial targets. Most DPAP inhibitors developed to date are peptide-based or peptidomimetic competitive inhibitors. Here, we used a high throughput screening approach to identify novel inhibitor scaffolds that block the activity of Plasmodium falciparum DPAP1. Most of the hits identified in this screen also inhibit Plasmodium falciparum DPAP3, cathepsin C, and to a lesser extent other malarial clan CA proteases, indicating that these might be general DPAP inhibitors. Interestingly, our mechanism of inhibition studies indicate that most hits are allosteric inhibitors, which opens a completely new strategy to inhibit these enzymes, study their biological function, and potentially develop new inhibitors as starting points for drug development.

Site-Directed Fragment-Based Screening for the Discovery of Protein-Protein Interaction Stabilizers.

Modulation of protein-protein interactions (PPIs) by small molecules has emerged as a valuable approach in drug discovery. Compared to direct inhibition, PPI stabilization is vastly underexplored but has strong advantages, including the ability to gain selectivity by targeting an interface formed only upon association of proteins. Here, we present the application of a site-directed screening technique based on disulfide trapping (tethering) to select for fragments that enhance the affinity between protein partners. We target the phosphorylation-dependent interaction between the hub protein 14-3-3σ and a peptide derived from Estrogen Receptor α (ERα), an important breast cancer target that is negatively regulated by 14-3-3σ. We identify orthosteric stabilizers that increase 14-3-3/ERα affinity up to 40-fold and propose the mechanism of stabilization based on X-ray crystal structures. These fragments already display partial selectivity toward ERα-like motifs over other representative 14-3-3 clients. This first of its kind study illustrates the potential of the tethering approach to overcome the hurdles in systematic PPI stabilizer discovery.

Functional Assessment of 2,177 U.S. and International Drugs Identifies the Quinoline Nitroxoline as a Potent Amoebicidal Agent against the Pathogen Balamuthia mandrillaris.

Balamuthia mandrillaris is a pathogenic free-living amoeba that causes a rare but almost always fatal infection of the central nervous system called granulomatous amoebic encephalitis (GAE). Two distinct forms of B. mandrillaris-a proliferative trophozoite form and a nonproliferative cyst form, which is highly resistant to harsh physical and chemical conditions-have been isolated from environmental samples worldwide and are both observed in infected tissue. Patients suffering from GAE are typically treated with aggressive and prolonged multidrug regimens that often include the antimicrobial agents miltefosine and pentamidine isethionate. However, survival rates remain low, and studies evaluating the susceptibility of B. mandrillaris to these compounds and other potential therapeutics are limited. To address the need for more-effective treatments, we screened 2,177 clinically approved compounds for in vitro activity against B. mandrillaris The quinoline antibiotic nitroxoline (8-hydroxy-5-nitroquinoline), which has safely been used in humans to treat urinary tract infections, was identified as a lead compound. We show that nitroxoline inhibits both trophozoites and cysts at low micromolar concentrations, which are within a pharmacologically relevant range. We compared the in vitro efficacy of nitroxoline to that of drugs currently used in the standard of care for GAE and found that nitroxoline is the most potent and selective inhibitor of B. mandrillaris tested. Furthermore, we demonstrate that nitroxoline prevents B. mandrillaris-mediated destruction of host cells in cultured fibroblast and primary brain explant models also at pharmacologically relevant concentrations. Taken together, our findings indicate that nitroxoline is a promising candidate for repurposing as a novel treatment of B. mandrillaris infections.IMPORTANCEBalamuthia mandrillaris is responsible for hundreds of reported cases of amoebic encephalitis, the majority of which have been fatal. Despite being an exceptionally deadly pathogen, B. mandrillaris is understudied, leaving many open questions regarding epidemiology, diagnosis, and treatment. Due to the lack of effective drugs to fight B. mandrillaris infections, mortality rates remain high even for patients receiving intensive care. This report addresses the need for new treatment options through a drug repurposing screen to identify novel B. mandrillaris inhibitors. The most promising candidate identified was the quinoline antibiotic nitroxoline, which has a long history of safe use in humans. We show that nitroxoline kills B. mandrillaris at pharmacologically relevant concentrations and exhibits greater potency and selectivity than drugs commonly used in the current standard of care. The findings that we present demonstrate the potential of nitroxoline to be an important new tool in the treatment of life-threatening B. mandrillaris infections.

Octopamine signaling in the metazoan pathogen Schistosoma mansoni: localization, small-molecule screening and opportunities for drug development.

Schistosomiasis is a tropical disease caused by a flatworm trematode parasite that infects over 200 million people worldwide. Treatment and control of the disease rely on just one drug, praziquantel. The possibility of drug resistance coupled with praziquantel's variable efficacy encourages the identification of new drugs and drug targets. Disruption of neuromuscular homeostasis in parasitic worms is a validated strategy for drug development. In schistosomes, however, much remains to be understood about the organization of the nervous system, its component neurotransmitters and potential for drug discovery. Using synapsin as a neuronal marker, we map the central and peripheral nervous systems in the Schistosoma mansoni adult and schistosomulum (post-infective larva). We discover the widespread presence of octopamine (OA), a tyrosine-derived and invertebrate-specific neurotransmitter involved in neuromuscular coordination. OA labeling facilitated the discovery of two pairs of ganglia in the brain of the adult schistosome, rather than the one pair thus far reported for this and other trematodes. In quantitative phenotypic assays, OA and the structurally related tyrosine-derived phenolamine and catecholamine neurotransmitters differentially modulated schistosomulum motility and length. Similarly, from a screen of 28 drug agonists and antagonists of tyrosine-derivative signaling, certain drugs that act on OA and dopamine receptors induced robust and sometimes complex concentration-dependent effects on schistosome motility and length; in some cases, these effects occurred at concentrations achievable in vivo The present data advance our knowledge of the organization of the nervous system in this globally important pathogen and identify a number of drugs that interfere with tyrosine-derivative signaling, one or more of which might provide the basis for a new chemotherapeutic approach to treat schistosomiasis.This article has an associated First Person interview with the first author of the paper.

A High-Content Larval Zebrafish Brain Imaging Method for Small Molecule Drug Discovery.

Drug discovery in whole-organisms such as zebrafish is a promising approach for identifying biologically-relevant lead compounds. However, high content imaging of zebrafish at cellular resolution is challenging due to the difficulty in orienting larvae en masse such that the cell type of interest is in clear view. We report the development of the multi-pose imaging method, which uses 96-well round bottom plates combined with a standard liquid handler to repose the larvae within each well multiple times, such that an image in a specific orientation can be acquired. We have validated this method in a chemo-genetic zebrafish model of dopaminergic neuron degeneration. For this purpose, we have developed an analysis pipeline that identifies the larval brain in each image and then quantifies neuronal health in CellProfiler. Our method achieves a SSMD* score of 6.96 (robust Z'-factor of 0.56) and is suitable for screening libraries up to 105 compounds in size.

Ceapins are a new class of unfolded protein response inhibitors, selectively targeting the ATF6α branch.

The membrane-bound transcription factor ATF6α plays a cytoprotective role in the unfolded protein response (UPR), required for cells to survive ER stress. Activation of ATF6α promotes cell survival in cancer models. We used cell-based screens to discover and develop Ceapins, a class of pyrazole amides, that block ATF6α signaling in response to ER stress. Ceapins sensitize cells to ER stress without impacting viability of unstressed cells. Ceapins are highly specific inhibitors of ATF6α signaling, not affecting signaling through the other branches of the UPR, or proteolytic processing of its close homolog ATF6ß or SREBP (a cholesterol-regulated transcription factor), both activated by the same proteases. Ceapins are first-in-class inhibitors that can be used to explore both the mechanism of activation of ATF6α and its role in pathological settings. The discovery of Ceapins now enables pharmacological modulation all three UPR branches either singly or in combination.

A gene-expression screen identifies a non-toxic sumoylation inhibitor that mimics SUMO-less human LRH-1 in liver.

SUMO-modification of nuclear proteins has profound effects on gene expression. However, non-toxic chemical tools that modulate sumoylation in cells are lacking. Here, to identify small molecule sumoylation inhibitors we developed a cell-based screen that focused on the well-sumoylated substrate, human Liver Receptor Homolog-1 (hLRH-1, NR5A2). Our primary gene-expression screen assayed two SUMO-sensitive transcripts, APOC3 and MUC1, that are upregulated by SUMO-less hLRH-1 or by siUBC9 knockdown, respectively. A polyphenol, tannic acid (TA) emerged as a potent sumoylation inhibitor in vitro (IC50 = 12.8 µM) and in cells. TA also increased hLRH-1 occupancy on SUMO-sensitive transcripts. Most significantly, when tested in humanized mouse primary hepatocytes, TA inhibits hLRH-1 sumoylation and induces SUMO-sensitive genes, thereby recapitulating the effects of expressing SUMO-less hLRH-1 in mouse liver. Our findings underscore the benefits of phenotypic screening for targeting post-translational modifications, and illustrate the potential utility of TA for probing the cellular consequences of sumoylation.

Tackling reproducibility in academic preclinical drug discovery.

The reproducibility of biomedical research on novel drug targets has become suspect. Here, we highlight how drug discovery centres embedded in academic institutions, but with a translational imperative, can help address this reproducibility crisis.

A Fragment-Based Ligand Screen Against Part of a Large Protein Machine: The ND1 Domains of the AAA+ ATPase p97/VCP.

The ubiquitous AAA+ ATPase p97 functions as a dynamic molecular machine driving several cellular processes. It is essential in regulating protein homeostasis, and it represents a potential drug target for cancer, particularly when there is a greater reliance on the endoplasmic reticulum-associated protein degradation pathway and ubiquitin-proteasome pathway to degrade an overabundance of secreted proteins. Here, we report a case study for using fragment-based ligand design approaches against this large and dynamic hexamer, which has multiple potential binding sites for small molecules. A screen of a fragment library was conducted by surface plasmon resonance (SPR) and followed up by nuclear magnetic resonance (NMR), two complementary biophysical techniques. Virtual screening was also carried out to examine possible binding sites for the experimental hits and evaluate the potential utility of fragment docking for this target. Out of this effort, 13 fragments were discovered that showed reversible binding with affinities between 140 µM and 1 mM, binding stoichiometries of 1:1 or 2:1, and good ligand efficiencies. Structural data for fragment-protein interactions were obtained with residue-specific [U-(2)H] (13)CH3-methyl-labeling NMR strategies, and these data were compared to poses from docking. The combination of virtual screening, SPR, and NMR enabled us to find and validate a number of interesting fragment hits and allowed us to gain an understanding of the structural nature of fragment binding.

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.

Screening for noise in gene expression identifies drug synergies.

Stochastic fluctuations are inherent to gene expression and can drive cell-fate specification. We used such fluctuations to modulate reactivation of HIV from latency-a quiescent state that is a major barrier to an HIV cure. By screening a diverse library of bioactive small molecules, we identified more than 80 compounds that modulated HIV gene-expression fluctuations (i.e., "noise"), without changing mean expression. These noise-modulating compounds would be neglected in conventional screens, and yet, they synergized with conventional transcriptional activators. Noise enhancers reactivated latent cells significantly better than existing best-in-class reactivation drug combinations (and with reduced off-target cytotoxicity), whereas noise suppressors stabilized latency. Noise-modulating chemicals may provide novel probes for the physiological consequences of noise and an unexplored axis for drug discovery, allowing enhanced control over diverse cell-fate decisions.

UCSF Small Molecule Discovery Center: innovation, collaboration and chemical biology in the Bay Area.

The Small Molecule Discovery Center (SMDC) at the University of California, San Francisco, works collaboratively with the scientific community to solve challenging problems in chemical biology and drug discovery. The SMDC includes a high throughput screening facility, medicinal chemistry, and research labs focused on fundamental problems in biochemistry and targeted drug delivery. Here, we outline our HTS program and provide examples of chemical tools developed through SMDC collaborations. We have an active research program in developing quantitative cell-based screens for primary cells and whole organisms; here, we describe whole-organism screens to find drugs against parasites that cause neglected tropical diseases. We are also very interested in target-based approaches for so-called "undruggable", protein classes and fragment-based lead discovery. This expertise has led to several pharmaceutical collaborations; additionally, the SMDC works with start-up companies to enable their early-stage research. The SMDC, located in the biotech-focused Mission Bay neighborhood in San Francisco, is a hub for innovative small-molecule discovery research at UCSF.

A high-throughput functional screen identifies small molecule regulators of temperature- and mechano-sensitive K2P channels.

K2P (KCNK) potassium channels generate "leak" potassium currents that strongly influence cellular excitability and contribute to pain, somatosensation, anesthesia, and mood. Despite their physiological importance, K2Ps lack specific pharmacology. Addressing this issue has been complicated by the challenges that the leak nature of K2P currents poses for electrophysiology-based high-throughput screening strategies. Here, we present a yeast-based high-throughput screening assay that avoids this problem. Using a simple growth-based functional readout, we screened a library of 106,281 small molecules and identified two new inhibitors and three new activators of the mammalian K2P channel K2P2.1 (KCNK2, TREK-1). By combining biophysical, structure-activity, and mechanistic analysis, we developed a dihydroacridine analogue, ML67-33, that acts as a low micromolar, selective activator of temperature- and mechano-sensitive K2P channels. Biophysical studies show that ML67-33 reversibly increases channel currents by activating the extracellular selectivity filter-based C-type gate that forms the core gating apparatus on which a variety of diverse modulatory inputs converge. The new K2P modulators presented here, together with the yeast-based assay, should enable both mechanistic and physiological studies of K2P activity and facilitate the discovery and development of other K2P small molecule modulators.

Mechanistic and structural understanding of uncompetitive inhibitors of caspase-6.

Inhibition of caspase-6 is a potential therapeutic strategy for some neurodegenerative diseases, but it has been difficult to develop selective inhibitors against caspases. We report the discovery and characterization of a potent inhibitor of caspase-6 that acts by an uncompetitive binding mode that is an unprecedented mechanism of inhibition against this target class. Biochemical assays demonstrate that, while exquisitely selective for caspase-6 over caspase-3 and -7, the compound's inhibitory activity is also dependent on the amino acid sequence and P1' character of the peptide substrate. The crystal structure of the ternary complex of caspase-6, substrate-mimetic and an 11 nM inhibitor reveals the molecular basis of inhibition. The general strategy to develop uncompetitive inhibitors together with the unique mechanism described herein provides a rationale for engineering caspase selectivity.

Diverse inhibitor chemotypes targeting Trypanosoma cruzi CYP51.

BACKGROUND: Chagas Disease, a WHO- and NIH-designated neglected tropical disease, is endemic in Latin America and an emerging infection in North America and Europe as a result of population moves. Although a major cause of morbidity and mortality due to heart failure, as well as inflicting a heavy economic burden in affected regions, Chagas Disease elicits scant notice from the pharmaceutical industry because of adverse economic incentives. The discovery and development of new routes to chemotherapy for Chagas Disease is a clear priority. METHODOLOGY/PRINCIPAL FINDINGS: The similarity between the membrane sterol requirements of pathogenic fungi and those of the parasitic protozoon Trypanosoma cruzi, the causative agent of Chagas human cardiopathy, has led to repurposing anti-fungal azole inhibitors of sterol 14α-demethylase (CYP51) for the treatment of Chagas Disease. To diversify the therapeutic pipeline of anti-Chagasic drug candidates we exploited an approach that included directly probing the T. cruzi CYP51 active site with a library of synthetic small molecules. Target-based high-throughput screening reduced the library of ∼104,000 small molecules to 185 hits with estimated nanomolar K(D) values, while cross-validation against T. cruzi-infected skeletal myoblast cells yielded 57 active hits with EC(50) <10 µM. Two pools of hits partially overlapped. The top hit inhibited T. cruzi with EC(50) of 17 nM and was trypanocidal at 40 nM. CONCLUSIONS/SIGNIFICANCE: The hits are structurally diverse, demonstrating that CYP51 is a rather permissive enzyme target for small molecules. Cheminformatic analysis of the hits suggests that CYP51 pharmacology is similar to that of other cytochromes P450 therapeutic targets, including thromboxane synthase (CYP5), fatty acid ω-hydroxylases (CYP4), 17α-hydroxylase/17,20-lyase (CYP17) and aromatase (CYP19). Surprisingly, strong similarity is suggested to glutaminyl-peptide cyclotransferase, which is unrelated to CYP51 by sequence or structure. Lead compounds developed by pharmaceutical companies against these targets could also be explored for efficacy against T. cruzi.

A high-throughput drug screen for Entamoeba histolytica identifies a new lead and target.

Entamoeba histolytica, a protozoan intestinal parasite, is the causative agent of human amebiasis. Amebiasis is the fourth leading cause of death and the third leading cause of morbidity due to protozoan infections worldwide(1), resulting in ~70,000 deaths annually. E. histolytica has been listed by the National Institutes of Health as a category B priority biodefense pathogen in the United States. Treatment relies on metronidazole(2), which has adverse effects(3), and potential resistance of E. histolytica to the drug is an increasing concern(4,5). To facilitate drug screening for this anaerobic protozoan, we developed and validated an automated, high-throughput screen (HTS). This screen identified auranofin, a US Food and Drug Administration (FDA)-approved drug used therapeutically for rheumatoid arthritis, as active against E. histolytica in culture. Auranofin was ten times more potent against E. histolytica than metronidazole. Transcriptional profiling and thioredoxin reductase assays suggested that auranofin targets the E. histolytica thioredoxin reductase, preventing the reduction of thioredoxin and enhancing sensitivity of trophozoites to reactive oxygen-mediated killing. In a mouse model of amebic colitis and a hamster model of amebic liver abscess, oral auranofin markedly decreased the number of parasites, the detrimental host inflammatory response and hepatic damage. This new use of auranofin represents a promising therapy for amebiasis, and the drug has been granted orphan-drug status from the FDA.

Quantification and clustering of phenotypic screening data using time-series analysis for chemotherapy of schistosomiasis.

BACKGROUND: Neglected tropical diseases, especially those caused by helminths, constitute some of the most common infections of the world's poorest people. Development of techniques for automated, high-throughput drug screening against these diseases, especially in whole-organism settings, constitutes one of the great challenges of modern drug discovery. METHOD: We present a method for enabling high-throughput phenotypic drug screening against diseases caused by helminths with a focus on schistosomiasis. The proposed method allows for a quantitative analysis of the systemic impact of a drug molecule on the pathogen as exhibited by the complex continuum of its phenotypic responses. This method consists of two key parts: first, biological image analysis is employed to automatically monitor and quantify shape-, appearance-, and motion-based phenotypes of the parasites. Next, we represent these phenotypes as time-series and show how to compare, cluster, and quantitatively reason about them using techniques of time-series analysis. RESULTS: We present results on a number of algorithmic issues pertinent to the time-series representation of phenotypes. These include results on appropriate representation of phenotypic time-series, analysis of different time-series similarity measures for comparing phenotypic responses over time, and techniques for clustering such responses by similarity. Finally, we show how these algorithmic techniques can be used for quantifying the complex continuum of phenotypic responses of parasites. An important corollary is the ability of our method to recognize and rigorously group parasites based on the variability of their phenotypic response to different drugs. CONCLUSIONS: The methods and results presented in this paper enable automatic and quantitative scoring of high-throughput phenotypic screens focused on helmintic diseases. Furthermore, these methods allow us to analyze and stratify parasites based on their phenotypic response to drugs. Together, these advancements represent a significant breakthrough for the process of drug discovery against schistosomiasis in particular and can be extended to other helmintic diseases which together afflict a large part of humankind.

Mining a cathepsin inhibitor library for new antiparasitic drug leads.

The targeting of parasite cysteine proteases with small molecules is emerging as a possible approach to treat tropical parasitic diseases such as sleeping sickness, Chagas' disease, and malaria. The homology of parasite cysteine proteases to the human cathepsins suggests that inhibitors originally developed for the latter may be a source of promising lead compounds for the former. We describe here the screening of a unique ∼ 2,100-member cathepsin inhibitor library against five parasite cysteine proteases thought to be relevant in tropical parasitic diseases. Compounds active against parasite enzymes were subsequently screened against cultured Plasmodium falciparum, Trypanosoma brucei brucei and/or Trypanosoma cruzi parasites and evaluated for cytotoxicity to mammalian cells. The end products of this effort include the identification of sub-micromolar cell-active leads as well as the elucidation of structure-activity trends that can guide further optimization efforts.