High Throughput Screening Identifies Novel Lead Compounds with Activity against Larval, Juvenile and Adult Schistosoma mansoni.

An estimated 600 million people are affected by the helminth disease schistosomiasis caused by parasites of the genus Schistosoma. There is currently only one drug recommended for treating schistosomiasis, praziquantel (PZQ), which is effective against adult worms but not against the juvenile stage. In an attempt to identify improved drugs for treating the disease, we have carried out high throughput screening of a number of small molecule libraries with the aim of identifying lead compounds with balanced activity against all life stages of Schistosoma. A total of almost 300,000 compounds were screened using a high throughput assay based on motility of worm larvae and image analysis of assay plates. Hits were screened against juvenile and adult worms to identify broadly active compounds and against a mammalian cell line to assess cytotoxicity. A number of compounds were identified as promising leads for further chemical optimization.

Staphylococcus aureus haem biosynthesis: characterisation of the enzymes involved in final steps of the pathway.

Haem is a life supporting molecule that is ubiquitous in all major kingdoms. In Staphylococcus aureus, the importance of haem is highlighted by the presence of systems both for the exogenous acquisition and endogenous synthesis of this prosthetic group. In this work, we show that in S. aureus the formation of haem involves the conversion of coproporphyrinogen III into coproporphyrin III by coproporphyrin synthase HemY, insertion of iron into coproporphyrin III via ferrochelatase HemH, and oxidative decarboxylation of Fe-coproporphyrin III into protohaem IX by Fe-coproporphyrin oxidase/dehydrogenase HemQ. Together, this route represents a transitional pathway between the classic pathway and the more recently acknowledged alternative biosynthesis machinery. The role of the haem biosynthetic pathway in the survival of the bacterium was investigated by testing for inhibitors of HemY. Analogues of acifluorfen are shown to inhibit the flavin-containing HemY, highlighting this protein as a suitable target for the development of drugs against S. aureus. Moreover, the presence of a transitional pathway for haem biosynthesis within many Gram positive pathogenic bacteria suggests that this route has the potential not only for the design of antimicrobials but also for the selective discrimination between bacteria operating different routes to the biosynthesis of haem.

Drug discovery for human African trypanosomiasis: identification of novel scaffolds by the newly developed HTS SYBR Green assay for Trypanosoma brucei.

Human African trypanosomiasis (HAT) is a vector-transmitted tropical disease caused by the protozoan parasite Trypanosoma brucei. High-throughput screening (HTS) of small-molecule libraries in whole-cell assays is one of the most frequently used approaches in drug discovery for infectious diseases. To aid in drug discovery efforts for HAT, the SYBR Green assay was developed for T. brucei in a 384-well format. This semi-automated assay is cost- and time-effective, robust, and reproducible. The SYBR Green assay was compared to the resazurin assay by screening a library of 4000 putative kinase inhibitors, revealing a superior performance in terms of assay time, sensitivity, simplicity, and reproducibility, and resulting in a higher hit confirmation rate. Although the resazurin assay allows for comparatively improved detection of slow-killing compounds, it also has higher false-positive rates that are likely to arise from the assay experimental conditions. The compounds with the most potent antitrypanosomal activity were selected in both screens and grouped into 13 structural clusters, with 11 new scaffolds as antitrypanosomal agents. Several of the identified compounds had IC50 <1 µM coupled with high selectivity toward the parasite. The core structures of the scaffolds are shown, providing promising new starting points for drug discovery for HAT.

Repositioning: the fast track to new anti-malarial medicines?

BACKGROUND: Repositioning of existing drugs has been suggested as a fast track for developing new anti-malarial agents. The compound libraries of GlaxoSmithKline (GSK), Pfizer and AstraZeneca (AZ) comprising drugs that have undergone clinical studies in other therapeutic areas, but not achieved approval, and a set of US Food and Drug Administration (FDA)-approved drugs and other bio-actives were tested against Plasmodium falciparum blood stages. METHODS: Molecules were tested initially against erythrocytic co-cultures of P. falciparum to measure proliferation inhibition using one of the following methods: SYBR®I dye DNA staining assay (3D7, K1 or NF54 strains); [(3)H] hypoxanthine radioisotope incorporation assay (3D7 and 3D7A strain); or 4',6-diamidino-2-phenylindole (DAPI) DNA imaging assay (3D7 and Dd2 strains). After review of the available clinical pharmacokinetic and safety data, selected compounds with low µM activity and a suitable clinical profile were tested in vivo either in a Plasmodium berghei four-day test or in the P. falciparum Pf3D7(0087/N9) huSCID 'humanized' mouse model. RESULTS: Of the compounds included in the GSK and Pfizer sets, 3.8% (9/238) had relevant in vitro anti-malarial activity while 6/100 compounds from the AZ candidate drug library were active. In comparison, around 0.6% (24/3,800) of the FDA-approved drugs and other bio-actives were active. After evaluation of available clinical data, four investigational drugs, active in vitro were tested in the P. falciparum humanized mouse model: UK-112,214 (PAF-H1 inhibitor), CEP-701 (protein kinase inhibitor), CEP-1347 (protein kinase inhibitor), and PSC-833 (p-glycoprotein inhibitor). Only UK-112,214 showed significant efficacy against P. falciparum in vivo, although at high doses (ED90 131.3 mg/kg [95% CI 112.3, 156.7]), and parasitaemia was still present 96 hours after treatment commencement. Of the six actives from the AZ library, two compounds (AZ-1 and AZ-3) were marginally efficacious in vivo in a P. berghei model. CONCLUSIONS: Repositioning of existing therapeutics in malaria is an attractive proposal. Compounds active in vitro at µM concentrations were identified. However, therapeutic concentrations may not be effectively achieved in mice or humans because of poor bio-availability and/or safety concerns. Stringent safety requirements for anti-malarial drugs, given their widespread use in children, make this a challenging area in which to reposition therapy.

Non-benzimidazole containing inhibitors of respiratory syncytial virus.

Several non-benzimidazole containing inhibitors of respiratory syncytial virus are described. Core template modification, analysis of antiviral activity, physicochemistry and optimisation of properties led to the thiazole-imidazole 13, that showed a good potency and pharmacokinetic profile in the rat.

Selective inhibitors of protozoan protein N-myristoyltransferases as starting points for tropical disease medicinal chemistry programs.

Inhibition of N-myristoyltransferase has been validated pre-clinically as a target for the treatment of fungal and trypanosome infections, using species-specific inhibitors. In order to identify inhibitors of protozoan NMTs, we chose to screen a diverse subset of the Pfizer corporate collection against Plasmodium falciparum and Leishmania donovani NMTs. Primary screening hits against either enzyme were tested for selectivity over both human NMT isoforms (Hs1 and Hs2) and for broad-spectrum anti-protozoan activity against the NMT from Trypanosoma brucei. Analysis of the screening results has shown that structure-activity relationships (SAR) for Leishmania NMT are divergent from all other NMTs tested, a finding not predicted by sequence similarity calculations, resulting in the identification of four novel series of Leishmania-selective NMT inhibitors. We found a strong overlap between the SARs for Plasmodium NMT and both human NMTs, suggesting that achieving an appropriate selectivity profile will be more challenging. However, we did discover two novel series with selectivity for Plasmodium NMT over the other NMT orthologues in this study, and an additional two structurally distinct series with selectivity over Leishmania NMT. We believe that release of results from this study into the public domain will accelerate the discovery of NMT inhibitors to treat malaria and leishmaniasis. Our screening initiative is another example of how a tripartite partnership involving pharmaceutical industries, academic institutions and governmental/non-governmental organisations such as Medical Research Council and Wellcome Trust can stimulate research for neglected diseases.

An integrated transcriptomic and meta-analysis of hepatoma cells reveals factors that influence susceptibility to HCV infection.

Hepatitis C virus (HCV) is a global problem. To better understand HCV infection researchers employ in vitro HCV cell-culture (HCVcc) systems that use Huh-7 derived hepatoma cells that are particularly permissive to HCV infection. A variety of hyper-permissive cells have been subcloned for this purpose. In addition, subclones of Huh-7 which have evolved resistance to HCV are available. However, the mechanisms of susceptibility or resistance to infection among these cells have not been fully determined. In order to elucidate mechanisms by which hepatoma cells are susceptible or resistant to HCV infection we performed genome-wide expression analyses of six Huh-7 derived cell cultures that have different levels of permissiveness to infection. A great number of genes, representing a wide spectrum of functions are differentially expressed between cells. To focus our investigation, we identify host proteins from HCV replicase complexes, perform gene expression analysis of three HCV infected cells and conduct a detailed analysis of differentially expressed host factors by integrating a variety of data sources. Our results demonstrate that changes relating to susceptibility to HCV infection in hepatoma cells are linked to the innate immune response, secreted signal peptides and host factors that have a role in virus entry and replication. This work identifies both known and novel host factors that may influence HCV infection. Our findings build upon current knowledge of the complex interplay between HCV and the host cell, which could aid development of new antiviral strategies.

Discovery of a highly potent series of TLR7 agonists.

The discovery of a series of highly potent and novel TLR7 agonist interferon inducers is described. Structure-activity relationships are presented, along with pharmacokinetic studies of a lead molecule from this series of N9-pyridylmethyl-8-oxo-3-deazapurine analogues. A rationale for the very high potency observed is offered. An investigation of the clearance mechanism of this class of compounds in rat was carried out, resulting in aldehyde oxidase mediated oxidation being identified as a key component of the high clearance observed. A possible solution to this problem is discussed.

Trypanosoma brucei glycogen synthase kinase-3, a target for anti-trypanosomal drug development: a public-private partnership to identify novel leads.

BACKGROUND: Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (HAT), expresses two proteins with homology to human glycogen synthase kinase 3ß (HsGSK-3) designated TbruGSK-3 short and TbruGSK-3 long. TbruGSK-3 short has previously been validated as a potential drug target and since this enzyme has also been pursued as a human drug target, a large number of inhibitors are available for screening against the parasite enzyme. A collaborative industrial/academic partnership facilitated by the World Health Organisation Tropical Diseases Research division (WHO TDR) was initiated to stimulate research aimed at identifying new drugs for treating HAT. METHODOLOGY/PRINCIPAL FINDINGS: A subset of over 16,000 inhibitors of HsGSK-3 ß from the Pfizer compound collection was screened against the shorter of two orthologues of TbruGSK-3. The resulting active compounds were tested for selectivity versus HsGSK-3ß and a panel of human kinases, as well as in vitro anti-trypanosomal activity. Structural analysis of the human and trypanosomal enzymes was also performed. CONCLUSIONS/SIGNIFICANCE: We identified potent and selective compounds representing potential attractive starting points for a drug discovery program. Structural analysis of the human and trypanosomal enzymes also revealed hypotheses for further improving selectivity of the compounds.

Design and optimisation of orally active TLR7 agonists for the treatment of hepatitis C virus infection.

The synthesis and structure-activity relationships of a series of novel interferon inducers are described. Pharmacokinetic studies and efficacy assessment of a series of 8-oxo-3-deazapurine analogues led to the identification of compound 33, a potent and selective agonist of the TLR7 receptor with an excellent in vivo efficacy profile in a mouse model.

HCV resistance to cyclosporin A does not correlate with a resistance of the NS5A-cyclophilin A interaction to cyclophilin inhibitors.

BACKGROUND & AIMS: The cyclophilin (Cyp) inhibitors - cyclosporine A (CsA), NIM811, Debio 025, and SCY 635 - block HCV replication both in vitro and in vivo, and represent a novel class of potent anti-HCV agents. We and others showed that HCV relies on cyclophilin A (CypA) to replicate. We demonstrated that the hydrophobic pocket of CypA, where Cyp inhibitors bind, and which controls the isomerase activity of CypA, is critical for HCV replication. Recent studies showed that under Cyp inhibitor selection, mutations arose in the HCV nonstructural 5A (NS5A) protein. This led us to postulate that CypA assists HCV by acting on NS5A. METHODS: We tested this hypothesis by developing several interaction assays including GST pull-down assays, ELISA, and mammalian two-hybrid binding assays. RESULTS: We demonstrated that full-length NS5A and CypA form a stable complex. Remarkably, CsA prevents the CypA-NS5A interaction in a dose-dependent manner. Importantly, the CypA-NS5A interaction is conserved among genotypes and is interrupted by CsA. Surprisingly, the NS5A mutant protein, which arose in CsA-resistant HCV variants, behaves similarly to wild-type NS5A in terms of both CypA binding and CsA-mediated release from CypA. This latter finding suggests that HCV resistance to CsA does not correlate with a resistance of the CypA-NS5A interaction to Cyp inhibitors. Moreover, we found that CypA, devoid of its isomerase activity, fails to bind NS5A. CONCLUSIONS: Altogether these data suggest that CypA, via its isomerase pocket, binds directly to NS5A, and most importantly, that disrupting this interaction stops HCV replication.

The use of AlphaLISA technology to detect interaction between hepatitis C virus-encoded NS5A and cyclophilin A.

Cyclosporine A (CsA) is an immunosuppressive molecule that also impedes replication of hepatitis C virus (HCV). CsA inhibits isomerase activity of cellular-encoded cyclophilin proteins, of which cyclophilin A (CypA) in particular is required for HCV replication. Evidence suggests that the HCV-encoded NS5A and NS5B proteins may govern dependence of the virus on CypA-mediated isomerase activity, although the molecular mechanisms involved are unclear. However, association of NS5A and NS5B, with CypA has been reported, raising the possibility that direct interaction between these proteins facilitates HCV replication. In the present study, mammalian two-hybrid and AlphaLISA technologies were utilized to detect interactions between NS5A and NS5B, with CypA. AlphaLISA analysis revealed associations between NS5A and CypA using purified proteins, and in cell lysates prepared from co-transfected cells. Importantly, the NS5A-CypA interactions were sensitive to CsA in a dose-responsive manner and an isomerase mutant of CypA interacted with NS5A less efficiently than wild-type CypA. These findings correlate the anti-HCV properties of CsA with an ability of the compound to disrupt NS5A-CypA interactions in vitro and in vivo, whilst providing the basis for development of assay platforms suitable to screen compound libraries for novel inhibitors of the NS5A-CypA interaction.

Small molecule drug discovery for Dengue and West Nile viruses: applying experience from hepatitis C virus.

There are currently no specific treatments for infection with Dengue virus (DENV) and West Nile Virus (WNV). Drug-discovery programs are underway for both viruses, but as yet no small molecules have advanced to clinical trials. Hepatitis C virus (HCV) is a related flavivirus that has been the focus of intense drug discovery efforts for the last two decades. Many approaches currently being pursued for DENV and WNV have been previously attempted for HCV with varying degrees of success. The experience with HCV may direct DENV and WNV efforts towards approaches with the best chance of success. Based on experience with HCV, the viral polymerase and protease are attractive targets to focus on since these have been most successful to date. Cell-based phenotypic screening may also yield attractive inhibitors. The helicase and methyltransferase enzymes are likely to prove difficult targets and host target approaches are fraught with safety concerns.

Design and optimisation of potent gp120-CD4 inhibitors.

The synthesis and structure-activity relationship of a series of novel gp120-CD4 inhibitors are described. Pharmacokinetic studies and antiviral spectrum assessment of lead compounds led to the identification of compound 36, a potent gp120-CD4 inhibitor which exhibited antiviral potency across a spectrum of 25 clade B isolates.

Discovery of a small molecule inhibitor through interference with the gp120-CD4 interaction.

A series of piperazine derivatives were designed and synthesised as gp120-CD4 inhibitors. SAR studies led to the discovery of potent inhibitors in a cell based anti viral assay represented by compounds 9 and 28. The rat pharmacokinetic and antiviral profiles of selected compounds are also presented.

The isomerase active site of cyclophilin A is critical for hepatitis C virus replication.

Cyclosporine A and nonimmunosuppressive cyclophilin (Cyp) inhibitors such as Debio 025, NIM811, and SCY-635 block hepatitis C virus (HCV) replication in vitro. This effect was recently confirmed in HCV-infected patients where Debio 025 treatment dramatically decreased HCV viral load, suggesting that Cyps inhibitors represent a novel class of anti-HCV agents. However, it remains unclear how these compounds control HCV replication. Recent studies suggest that Cyps are important for HCV replication. However, a profound disagreement currently exists as to the respective roles of Cyp members in HCV replication. In this study, we analyzed the respective contribution of Cyp members to HCV replication by specifically knocking down their expression by both transient and stable small RNA interference. Only the CypA knockdown drastically decreased HCV replication. The re-expression of an exogenous CypA escape protein, which contains escape mutations at the small RNA interference recognition site, restored HCV replication, demonstrating the specificity for the CypA requirement. We then mutated residues that reside in the hydrophobic pocket of CypA where proline-containing peptide substrates and cyclosporine A bind and that are vital for the enzymatic or the hydrophobic pocket binding activity of CypA. Remarkably, these CypA mutants fail to restore HCV replication, suggesting for the first time that HCV exploits either the isomerase or the chaperone activity of CypA to replicate in hepatocytes and that CypA is the principal mediator of the Cyp inhibitor anti-HCV activity. Moreover, we demonstrated that the HCV NS5B polymerase associates with CypA via its enzymatic pocket. The study of the roles of Cyps in HCV replication should lead to the identification of new targets for the development of alternate anti-HCV therapies.

Targeting the non-structural proteins of hepatitis C virus: beyond hepatitis C virus protease and polymerase.

BACKGROUND: Chronic hepatitis C virus (HCV) infection is a main cause of cirrhosis of the liver and hepatocellular carcinoma. The standard of care is a combination of pegylated interferon with ribavirin, a regimen that has undesirable side effects and is frequently ineffective. Compounds targeting HCV protease and polymerase are in late-stage clinical trials and have been extensively reviewed elsewhere. OBJECTIVE: To review and evaluate the progress towards finding novel HCV antivirals targeting HCV proteins beyond the already precedented NS3 protease and NS5B polymerase. METHODS: Searches of CAplus and Medline databases were combined with information from key conferences. This review focuses on NS2/3 serine protease, NS3 helicase activity and the non-structural proteins 4A, 4B and 5A. CONCLUSIONS: Use of the replicon model of HCV replication and biochemical assays of specific targets has allowed screening of vast libraries of compounds, but resulted in clinical candidates from only NS4A and NS5A. The field is hindered by a lack of good chemical matter that inhibits the remaining enzymes from HCV, and a lack of understanding of the functions of non-structural proteins 4A, 4B and 5A in the replication of HCV.

Comparison of rat and human responses to toll-like receptor 7 activation.

Toll-like receptors recognize invading microorganisms and activate innate immune responses. Their discovery has opened up a range of therapeutic possibilities, in particular for infectious diseases. Responses to TLR agonists have been largely studied in mice and little information exists in other species. Given that rats are commonly used for pharmacokinetic and toxicology studies in drug development, we compared TLR7 responses in rat and human. Stimulation of rat and human peripheral blood mononuclear cells with the TLR7 agonist SM360320 showed that in rat cells, the interferon-induced gene, 2', 5' oligoadenylate synthase and tumor necrosis factor alpha were induced at lower concentrations and to a greater degree compared with human cells. Both human and rat cells demonstrated tolerance and could not be restimulated following initial treatment with high concentrations of SM360320. Reducing the concentration of the initial treatment allowed cells to be restimulated following a period of recovery. The initial treatment concentration had to be reduced to a greater extent to enable restimulation of rat cells compared with human cells. Dosing whole rats repeatedly with different concentrations of SM360320 confirmed the in vitro results. Treatment of human cells with high concentrations of interferon alpha did not induce tolerance to subsequent treatment with SM360320 indicating that tolerance occurs in the TLR7 signaling pathway, rather than the interferon signaling pathway. We conclude that rat and human cells respond differently to TLR7 activation and that these differences should be considered when using rat as a model to study TLR7 agonists.

The future of toll-like receptor therapeutics.

Since the discovery of human TLRs, manipulating the activity of these receptors to modulate immune responses for therapeutic purposes has initiated intense activity in the pharmaceutical industry. The focus of these activities has been largely in the areas of infectious diseases, cancer, allergic diseases and vaccine adjuvants. Although initial clinical trials for infectious diseases and cancer showed early promise, subsequent longer-term trials have been disappointing and more research is required to find dosing regimes that balance efficacy with acceptable side-effect profiles. As only a small number of doses are given, TLR agonists as vaccine adjuvants appear to hold greater potential and have less safety concerns than for other applications. Several innovative strategies for using TLR agonists in vaccine development are currently being pursued, and these are discussed in more detail in this review.

Investigating Toll-like receptor agonists for potential to treat hepatitis C virus infection.

Toll-like receptors (TLRs) are key mediators of innate immunity, and their activation by microbial components leads to the production of cytokines and interferons. Recombinant alpha interferon has been used to treat several viral diseases and is the current standard of care for hepatitis C virus (HCV) infection. Recently, agonists of TLR7 and TLR9 have been shown to have clinical efficacy in HCV patients, and this is correlated with their ability to induce endogenous type I interferon production. We have carried out a comprehensive study of agonists of TLRs 1 to 9 to determine if any additional TLRs can induce antiviral molecules from human peripheral blood mononuclear cells (PBMCs). The agonists were incubated with PBMCs, and the supernatant was then removed and added to HCV replicon cells to assess antiviral activity. Agonists of TLRs 3, 4, 7, 8, and 9 were found to be potent inducers of antiviral activity in PBMC supernatants, and the activity correlated with the induction of alpha interferon and the interferon-induced antiviral biomarker 2',5'-oligoadenylate synthase. Antiviral activity of TLR7 and TLR8 agonists was blocked by an antibody that binds to the type I interferon receptor, confirming that the antiviral activity results from type I interferon induction. TLR4 and TLR8 agonists were found to strongly induce the proinflammatory cytokines interleukin 1beta and tumor necrosis factor alpha at concentrations similar to those inducing antiviral activity. This raises concerns about adverse side effects if these were to be used as antiviral agents. We therefore conclude that TLRs 3, 7, and 9 represent the most attractive targets for the development of new HCV therapies.