New Potent Inhibitors of Trypanosoma cruzi Sterol C14 Demethylase Based on a Substituted 2-quinolinone (R115777)

Frederick Buckner, Michael Gelb, James Kraus, Christophe Verlinde, Mandana Karimi, Pravin Kumar, Srinivas Olepu, and Jan Schoepe. Dept. of Medicine, Dept. of Chemistry, and Dept. of Biochemistry University of Washington, Seattle, WA

The primary goal of this project is to produce new chemotherapeutic agents for treatment of Chagas disease. Current drugs are toxic and have low cure rates for persons chronically infected with Trypanosoma cruzi. Based on extensive evidence that antifungal drugs (i.e. azoles) have moderate activity against T. cruzi, we are pursuing new compounds that are better optimized for inhibition of the target, sterol C14-demethylase. The starting point is a class of compounds based on R115777 (tipifarnib), a human protein farnesyltransferase (PFT) inhibitor developed by Johnson & Johnson, which is in Phase III clinical trials as an anti cancer drug. This compound has oral bioavailability, a good pharmacokinetic profile, and is fairly well tolerated in cancer patients. Earlier work by our group demonstrated that R115777 is very potent against T. cruzi grown in 3T3 fibroblasts (ED50 = 5-10 nM). Follow up investigations demonstrated that R115777 appears to mediate its toxic effects on T. cruzi by inhibiting sterol C14-demethylase rather than by inhibiting T. cruzi PFT. Using structural models of the T. cruzi sterol C14-demethylase and the mammalian PFT, we are making modifications to R115777 in order to optimize binding to sterol C14-demethylase while removing activity against mammalian PFT. The purpose will be to minimize any toxicity of the drug that may be mediated through inhibition of mammalian PFT. Simple changes to R115777 have led to a ~400-fold increase in IC50 against the mammalian PFT with a small change in anti-T. cruzi activity. Our goal is to design a potent anti-trypanosomal agent that can be taken orally for an extended period with minimal toxicity.

A metallopeptide mimic of the coiled coil domain of Class 1 viruses is a useful target for antiviral drugs

Lifeng Cai and Miriam Gochin. Department of Basic Sciences, Touro University – California, Vallejo, CA 94592 and Department of Pharmaceutical Chemistry, University of California San Francisco CA 94143

We have been working on a metallopeptide construct of the coiled coil domain of HIV-1 gp41, for use as a receptor to detect entry inhibitors through high throughput fluorescence screening. A metal coordination complex holds together three component peptides from the trimeric coiled coil and quenches fluorescently labeled peptide from a viral domain that binds to the coiled coil. This binding event precipitates the fusion process in the intact virus. Small molecules able to prevent the binding can act as fusion inhibitors, and can be detected by an increase in fluorescence in a ternary system containing the metallopeptide, the fluorescent peptide and the small molecule inhibitor. We have carried out studies to confirm that the metallopeptide is representative of the intact viral protein, including the ability of the protocol system to specifically and sensitively detect know gp41 fusion inhibitors. We will demonstrate a novel class of peptidomimetic fusion inhibitors that we have detected using this system. Although these only inhibit in the micromolar range, they are relatively small and amenable to modification, so that they may be developed into more potent fusion inhibitors. The fluorescence protocol is robust, quantitative and easily automated for high throughput screening. A similar fusion process occurs with other class 1 viruses including paramyxoviruses (RSV, Mumps, Measles, Sendai Virus), orthomyxoviruses (Influenza A), coronavirus (SARS), retroviruses (HIV, HTLV) and filovirus (Ebola), so that broader application of the method is expected.

Simple spectroscopic monitoring of liquid surface curvature changes in thin wells

Lifeng Cai and Miriam Gochin. Department of Basic Sciences, Touro University – California, Vallejo, CA 94592 and Department of Pharmaceutical Chemistry, University of California San Francisco CA 94143

The boundary between liquid and solid is often characterized by a curved liquid surface in thin tubes or in small liquid drops on a solid surface. The surface curvature is a manifestation of the equilibrium between solid surface tension, liquid surface tension, solid-liquid interface tension and gravity. Knowledge of these forces is important in many technological processes, including surfactant development in drug delivery formulation and physiology, micro- or nano-manufacturing, microfluidics and surface fabrication. Classical liquid surface tension measurements are very precise, but require complex preparation and expertise in sample handling. Few simple and high-throughput (HT) methods are currently available to study liquid-solid-surface interactions. Here we report a simple fluorescence method to study liquid surface curvature changes in thin-well plates, with a dynamic range of 60% and a relative sensitivity of 2%. It exploits the effect of total internal reflection on fluorescence emission through a curved liquid surface. Application of the effect is demonstrated by a method for rapid and universal determination of surfactant critical micelle concentration (CMC) and by a simple protocol for promiscuous inhibitors resulting from micelle formation. Plate-based measurement of surface curvature is an efficient method for investigating systems where colloidal properties, surface tension or interfacial tension are paramount.

Reduction in pathology and parasitologic cure of mice infected with Schistosoma mansoni using a peptidomimetic cysteine protease inhibitor

C.R. Caffrey, M. Abdulla, K.C. Lim, M. Sajid, and J.H. McKerrow. Sandler Center for Basic Research in Parasitic Diseases, California Institute for Quantitative Biomedical Research, Box 2550, University of California, San Francisco, San Francisco, CA 94143

Small-molecule inhibitors of Clan CA cysteine proteases are in pre-clinical development for treatment of a number of diseases caused by parasitic protozoa, including Falciparum malaria, African Sleeping Sickness and Chagas’ disease. Similar research for parasitic helminth diseases lags behind, but given the well-documented central functions of Clan CA proteases in such parasites, a similar therapeutic strategy may prove beneficial. Schistosomiasis is second only to malaria in numbers infected and, in the absence of a vaccine, just one drug is recommended for chemotherapy with the continual concern of the emergence of resistance. We tested the efficacy of the peptidomimetic vinyl sulfone inhibitor, K11777, in the murine model of Schistosomiasis mansoni. Disease parameters measured were worm and egg burdens, and organ pathology, including hepato- and splenomegaly, presence of parasite egg-induced granulomas in the liver and levels of circulating alanine aminotransferase activity as a marker of hepatocellular function. K11777 (25mg/kg BID), administered intra-peritoneally at the time of parasite migration through the skin and lungs (days 1 – 14 post-infection), resulted in parasitologic cure (elimination of parasite eggs) in 70% of infections and a resolution of other disease parameters. K11777 (50mg/kg BID), administered at the commencement of egg-laying by mature parasites (days 30 – 37 post-infection), reduced worm and egg burdens and ameliorated organ pathology. Using protease class-specific substrates and active site-labeling, one molecular target of K11777 was identified as a cathepsin B cysteine protease associated with the parasite gut. K11777 has completed pre-clinical tests for the treatment of Chagas’ disease and the inhibitor is non-toxic and non-mutagenic with an acceptable pharmacokinetic profile. The pronounced ameliorative impact on parasite burden and pathology demonstrated here for K11777 suggests that a similar therapeutic strategy may prove valuable in the treatment of human schistosomiasis.

The Trypanosoma cruzi protease cruzain mediates immune evasion

Patricia S. Doyle, Juan C. Engel, Yuan M. Zhou, Ivy Hsieh, Pn’g Loke, Doron Greenbaum, and James H. McKerrow. Tropical Disease Research Unit, and Sandler Center, Department of Pathology, University of California, San Francisco, CA 94121

The biological role for the Trypanosoma cruzi cysteine protease cruzain in immune evasion was elucidated in a comparative study of parental wild type-parasites and parasites resistant to the cysteine protease inhibitor N-Pip-F-hF-Vsφ that results in protease-deficiency. Wild type T. cruzi does not activate the host macrophage following infection. Cruzain and the signaling factor NF-κB P65 co-localize to the cell surface of intracellular parasites. P65 is proteolytically cleaved. No significant IL-12 expression occurs in macrophages infected with wild type parasites and subsequently activated with LPS confirming macrophage unresponsiveness. In contrast, cysteine protease inhibitor-resistant, and cruzain-deficient parasites induce macrophage activation and nuclear NF-κB P65 localization. Thus, cruzain hinders macrophage activation allowing T. cruzi survival and replication, and expansion of infection in Chagas’ disease.

A Cysteine Protease Inhibitor Cures Chagas’ disease in an Immunodeficient Murine Model of Infection

Patricia S. Doyle, Juan C. Engel, Yuan M. Zhou, and James H. McKerrow. Tropical Disease Research Unit, and Sandler Research Institute, Department of Pathology, University of California, San Francisco, CA 94121

Chagas’ disease remains the leading cause of heart disease in Latin America with 12 to 16 million people estimated to be infected, and over 90 million at risk. Chagas’ disease is caused by the protozoan parasiteTrypanosoma cruzi that infects the human host most commonly through an insect vector or blood transfusion. Classic clinical manifestations derive from parasite infection of cardiac muscle, leading to progressive cardiomyopathy. Some patients develop megacolon or megaesophagus due to infection of plexus nerve ganglia. A very aggressive clinical course including fulminant meningoencephalitis has been reported in patients who contract Chagas’ disease in the background of immunodeficiency. This includes patients with HIV infection as well as patients receiving immunosuppressive therapy for cardiac transplant. Currently, only two drugs are approved for the treatment of Chagas’ disease, Nifurtimox and Benznidazole. Both have significant limitations due to common and serious side effects, as well as limited availability. One promising group of new drug leads for Chagas’ disease is cysteine protease inhibitors targeting cruzain, the major protease of Trypanosoma cruzi. At least one of these inhibitors is in late-stage preclinical development. Cruzain plays a number of roles during the lifecycle of T. cruzi, including a role in immune evasion. Therefore, the question arises whether protease inhibitors targeting cruzain would have efficacy in Chagas’ disease, occurring in the background of immunodeficiency. To address this question, we studied the course of infection in recombinase deficient (Rag1 knockout) mice infected with T. cruzi. We show that a vinyl sulfone protease inhibitor administered at a daily dose of 100mg/kg weight can cure animals even in the absence of a functioning adaptive immune response.

Docking based discovery of non covalent cruzain inhibitors

Rafaela S. Ferreira, Alan P. Graves, Brian K. Shoichet, and James H. McKerrow. Department of Pharmaceutical Chemistry and Department of Pathology, University of California San Francisco, CA 94158

Cruzain, the major cysteine protease of Trypanosoma cruzi, is an essential enzyme for this parasite and a therapeutic target for Chagas’ disease. Different classes of cruzain inhibitors have been developed, and in several cases co-crystallized with the enzyme, providing a platform to structure-based drug design. Current cruzain inhibitors are mostly peptidic compounds which bind irreversibly to the enzyme. In an attempt to find new classes of inhibitor with better pharmacological properties, we used DOCK to search for reversible, non-covalent cruzain inhibitors. The lead-like subset of the ZINC database (~about 450,000 compounds), was docked and the top 500 scoring molecules were inspected. 17 compounds were purchased and tested for inhibition of cruzain activity. 2 compounds were shown to be competitive inhibitors with Ki values of 64 μM (compound 1) and 125 μM. A series of commercially available analogs of compound 1 was purchased and tested against cruzain, providing information about the pharmacophore and insights for structure-based design of new inhibitors.

Archiving and Mining Neglected-Infectious-Disease Screening Data for Web-based Collaborative Drug Discovery

E. Hansell, K. Gregory, B. Bunin, P. Doyle, D. Ruelas, M. Abdulla, N. Tucker, Z. Mackey, C. Caffrey, and J. McKerrow. Department of Pathology, Sandler Center for Basic Research in Parasitic Diseases, University of California, San Francisco CA 94158 and Collaborative Drug Discovery (CDD, Inc., 1633 Bayshore Highway, Suite 342, Burlingame, CA 94010

The Sandler Center for Basic Research in Parasitic Diseases at UCSF has been using a new type of web-based database to study neglected-infectious diseases such as Malaria, Chagas Disease, Leishmaniasis, African Sleeping Sickness and Schistosomiasis. This web-based database has been created by Collaborative Drug Discovery (CDD) to help scientists more effectively develop new drug candidates for commercial and humanitarian markets. This database can be used to archive, mine, and collaborate around drug discovery data. The CDD technology uses automated data “mappers and slurpers” to upload the protocol data that researchers typically have in Excel and sd file formats. This technology is designed to provide gated access for private versus public groups at the individual experimental data level. The data can be “mined” at the molecular level, using keywords or structural elements. It can also be mined at the protocol level, by selecting all or a subset of protocols, with the option of setting cut-off limits for viewing. These elements can be combined to mine a subset of structures. Data reports can be exported for local use. Additional tools are being developed for viewing structure protocol relationships, and improving ease of use. The Sandler Center has successfully used the CDD database to accumulate information on 8000 molecular structures from more than 20 collaborators (academic and industrial), and protocol data from enzyme, cell, and animal assays.

The ZINC database of commercially available compounds for virtual screening

John J Irwin and Brian K Shoichet. Department of Pharmaceutical Chemistry, University of California, San Francisco CA 94158

An important problem in virtual screening is the quality of the database being screened. Careful database creation and curation are ongoing problems for experts in the field and barriers to entry for non-experts. This has led us to create ZINC, a free database of commercially available compounds for virtual screening, on the web at ZINC includes multiple representations of molecules which attempt to capture the physiologically relevant protonation state, tautomeric, regio- and stereo-isomeric forms. ZINC is available in pH-range-specific subsets, so as to be able to cope with both low charge sites as well as more extreme charge situations as found in metalloezymes, for example. ZINC has been broadly sub-categorized into several libraries, such as ‘drug like’, ‘lead-like’ and ‘fragment-like’ molecules, for different applications that we and our colleagues find useful. Our quest to create biologically relevant representations has led us to prepare a database of high energy intermediates for substrate discovery. Experimentally tested prospective substrate will be described.

Community-based approach to research on infectious diseases of the developing world and global health

Rita Stanikunaite, Arnas Palaima, and Barry A. Bunin. Collaborative Drug Discovery (CDD, Inc.), 1633 Bayshore Highway, Suite 342, Burlingame, CA 94010

Currently, infectious diseases of the developing world (e.g., malaria, tuberculosis) represent a global health challenge of the 21st century and require new approaches that would allow scientists to do research more effectively. As a result of the development of web-database technologies, recently a community-based approach to research on infectious diseases of the developing world and global health has emerged. The major components of effective scientific community include: (1) unifying goal, or focus on common therapeutic areas/diseases; (2) multiple research areas/expertise; (3) uniform database platform that allows effective data accumulation and management; (4) easy access and sharing of information; (5) potential for unlimited growth. The Collaborative Drug Discovery (CDD) database built by utilizing community-based web technologies currently provides a platform that allows scientists to archive, mine, and share research data with a focus on infectious diseases of the developing world. This new collaborative technology allows researchers to build up networks of technical experts around therapeutic or target areas thus facilitating discovery of new drug candidates. It allows scientists to speed up the research by sharing unpublished data providing new hope in the race to overcome drug resistance. An example illustrating how potential chemosensitizers to address chloroquine resistance could be identified by using the CDD database platform is presented.