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Worcester, MA, United States

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 363.56K | Year: 2014

ABSTRACT In this project we propose to exploit the recent successful isolation and reconstitution of the important ATP- dependent drug efflux transporter, ABCG2 (BCRP, Breast Cancer Resistance Protein), and combine it with a new transport assay system, the Fluorosome platform, to characterize the interaction of ABCG2 with drugs and drug candidates. The result will provide a useful, novel, highly specific and rapid in vitro transport assay for future general use. We shall employ this assay together with ATPase studies to characterize a wide variety of ABCG2 substrates and modulators. Additionally, by means of this unique construct we shall screen the 89 anticancer drugs in the NCI Oncology Drug Set Plate series for inhibition of ABCG2-mediated transport. The ABCG2 protein is a ubiquitous high capacity drug transporter with wide substrate specificity. The White Paper recently issued by the International Transporter Consortium formed to propose industry and FDA standards for studies of drug:transporter inte

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 1.55M | Year: 2009

DESCRIPTION (provided by applicant): Phase I resulted in our discovery of low nanomolar level inhibitors of ADP-induced human platelet aggregation, with a unique mechanism of action, that warrant further development as first-in-class antithrombotic drugs. We developed a novel, efficient, high yield method to prepare a series of base and phosphate modified derivatives of diadenosine tetraphosphate (Ap4A). The proposed modifications revealed clear structure-activity trends, and led to the synthesis of highly potent inhibitors of human platelet aggregation that are also stable in plasma. We investigated for the first time the mechanism of action of this class at the level of the three platelet purinoreceptors (P2X1,P2Y1 and P2Y12), and showed that Ap4A and its analogs antagonize both P2Y1 and P2Y12. Moreover this unprecedented simultaneous inhibition of both platelet ADP dependent platelet receptors by a single ligand appears to be highly synergistic - IC50'sfor inhibition of ADP-induced platelet aggregation for the most active compounds is one to two order of magnitude lower than the corresponding IC50'sof inhibition of P2Y1 and P2Y12. Thus these compounds represent a new class of antiplatelet, and, ultimately, antithrombotic drugs with a novel mechanism of action. The specific aims are to 1, expand and tune the structure:activity relationship for inhibiting human platelet aggregation by synthesis and study of six additional Ap4A analogs, 2, evaluate the selectivity/specificity of the new class of Ap4A derivatives, by studying and quantifying the ability of 3-6 most active compounds to interact with non-platelet P2 receptors, utilizing tissue and recombinant receptor models, 3, optimize and scale up the chemical process of synthesis and purification of the class; 4, determine the basic pharmacokinetic parameters and identify metabolites of the 3 lead compounds candidates after IV administration in rats, and 5, study the aggregation properties of platelets after IV infusion of these candidates in catheterized rats, and 6, confirm the antithrombotic activity of the selected lead compound in the well-established Folts' canine thrombosis model. A single lead preclinical compound (plus backup) will be designated following these studies. Platelets play critical roles in hemostasis and its pathophysyology. Undesired platelet activation is a result of many common pathologies, e.g. hypertension and arteriosclerosis, and leads to excessive platelet aggregation and the generation of occlusive thrombi (thrombosis). The ischemic events that follow, such as myocardial infarction and stroke, are leading causes of death in the developed world, and antiplatelet drugs have been a major focus of drug development. Aspirin and clopidrogel (Plavix(R), 5.9B sales in 2005) are the most popular of the class today. Because of the vast interpatient variability in response to clopidogrel and newer single-receptor targeted drugs under clinical development, and because of the increased bleeding and morbidity in patients receiving clopidogrel, there is a need for the development of fast, direct acting, and rapidly cleared platelet ADP-receptor antagonists. The overall goal of this research is to identify a novel lead antithrombotic compound for human use, which can be licensed to a major pharmaceutical company for further pre-clinical and clinical development, or alternatively can be developed into a drug candidate by us after securing appropriate partnership or funding. PUBLIC HEALTH RELEVANCE: This project will result in an effective antithrombotic drug that will be used to treat arterial thrombosis in the acute setting. The candidate drug will directly and reversibly inhibit two important receptors involved in platelet aggregation, and will not have the drawbacks of slow and variable action of current drugs such as clopidogrel. The new drug will complement related drugs under development for arterial thrombosis.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 300.00K | Year: 2012

DESCRIPTION (provided by applicant): There is no cure for herpes simplex virus (HSV) latent infections of neurons, and the possibility of asymptomatic reactivation and simultaneous transfer of infectious virus via mucous membranes is a significant drawback of current therapies. We have pioneered the study of selective inhibitors of the herpes virus-specific thymidine kinase (TK), the enzyme responsible both for activation of anti-herpes nucleoside analogs and for reactivation of virus from the latent state in sensory nerve ganglia. Animal studies, however, have required intraperitoneal dosing of test compounds because the compounds are poorly soluble in water and not sufficiently orally available to warrant study of their effectiveness by the oral route. Among strategies to enhance oral absorption of candidate drugs, we have synthesized the 6-deoxy analogs of two promising compounds, and have found that one of these, which we call SacrovirTM, has moderate oral absorption in the mouse, which is enhanced by mixing with the solubilizing polymer Soluplus, and is converted nearly completely into the ultimate drug N2-[m-(trifluoromethyl) phenyl] guanine, mCF3PG. Thus, we propose to develop one or more formulations that will greatly increase the oral absorption of Sacrovir and show oral efficacy in animal models of HSV reactivation. To achieve this goal, our specific aims are to: 1. formulate the pro-drug Sacrovir (6-deoxy-mCF3PG) to enhance its intestinal absorption; strategies include emulsions, small molecule adjuvants, and solubility enhancers; 2. Compare oral bioavailability of Sacrovir formulations in the mouse, by analyzing plasma samples by LC-MS methods for both the pro-drug and the ultimate drug mCF3PG; 3. determine the oral bioavailability of mCF3PGin rabbits and guinea pigs after oral treatment with Sacrovir; and 4) test, through collaborators, the efficacy of oral Sacrovir in rabbit and guinea pi models of HSV reactivation. Following successful demonstration of an optimal oral formulation of Sacrovir, the experiments of aim 4 to test efficacy in rabbit and guinea pig models, respectively, will be conducted by subcontractor Dr. James M. Hill at LSU Health Sciences Center, New Orleans, and by Drs. David Bernstein and Rhonda Cardin of Cincinnati Children's Hospital Medical Center, under contract from NIAID (Dr. Heather Greenstone, Antiviral Branch). Positive results will be used as background to secure additional funding, for example through a phase II small business grant, and ultimately from a corporate sponsor or licensee, to fully develop the drug. The pro-drug Sacrovir will represent a first-in-class, novel oral treatment for HSV latent infections of neurons and, importantly, will prevent both asymptomatic virus shedding and the resulting transmission of infection to sex partners. PUBLIC HEALTH RELEVANCE: There is no cure for herpes simplex virus infections; asymptomatic reactivation of virus from the latent state in neurons and its transfer via mucous membranes is a significant drawbackof current therapies. We have synthesized a pro-drug SacrovirTM that is both orally absorbed in mice and converted metabolically to the ultimate anti-reactivation drug, mCF3PG . In this project we will develop a formulation of Sacrovir that will increase its oral absorption in mice, and show that this formulation will have efficacy in rabit and guinea pigs models of herpes simplex reactivation. Sacrovir represents a first-in-class, novel oral treatment for latent herpes simplex infections of neuronsand, importantly, will prevent transmission of infectious virus.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 299.85K | Year: 2010

DESCRIPTION (provided by applicant): The bacterial second messenger c-di-GMP (cyclo-(GMP)2) is responsible for inducing certain pathogenic bacteria to form biofilms, complex structures of one or more bacterial strains that resist conventional antibiotics and are associated with numerous infectious diseases such as bacterial pneumonia, stent blockage, catheter colonization, etc. The second messenger c-di-GMP plays an essential role in the regulation of biofilm formation of important pathogens as Vibrio cholerae, Yersinia pestis, P. aeruginosa, E. coli, S. enteric and S. aureus. The biosynthesis of c-di-GMP from two molecules of GTP is catalyzed by highly conserved enzymes known as diguanylate cyclases (DGCs). A few studies have been published describing the effects of analogs of c-di-GMP on its functions, but no reports are available describing the effects of GTP analogs on inhibition of its synthesis or as alternate substrates. One GTP analog, (Rp) 1-thio-GMP, has been cocrystallized with the catalytic form of Caulobacter crescentus DGC. We hypothesize that competitive inhibitors of conversion of GTP to c-di-GMP could be developed as novel drugs to inhibit biofilm formation or stability. We will focus on the structure-activity relationships (SAR) of GTP analogs as competitive inhibitors of c-di-GMP biosynthesis for this phase I feasibility study. We will capitalize on the applicant's background in synthesis and study of guanine nucleotides as inhibitors/alternate substrates of nucleic acid metabolizing enzymes and that of our subcontractor in enzymatic and cell-based study of DGCs to discover novel compounds that will inhibit the utilization of GTP by relevant bacteria. We recognize that this is but the first step in the discovery of new anti-biofilm antibacterials, because the nucleotides so identified will not likely be drug-like . Consequently, phase II of this project will be devoted to chemical modification of selected GTP analogs to discover leads for anti-biofilm antibacterials. The specific aims of this project are to: 1, synthesize or procure 29 analogs of GTP in which modifications in the base (N2- and 8-substitution, aza and deaza isosteres), sugar (O-methyl, arabino, acyclo) and phosphate moieties (methylene, difluoromethylene, imido, thio) are made. All new compounds will be purified by reverse phase or ion exchange chromatography, and characterized by NMR and LCMS methods; 2, test GTP analogs for their ability to inhibit cyclization of GTP to c-di- GMP by the Pseudomonas aeruginosa DGC PA3702 ; a coupled assay measuring pyrophosphate release and a HPLC assay measuring product formation will be used for this purpose. Where it is suggested that the analog is actually a substrate for the enzyme, i.e. where a modified c-di-GMP has been produced, LCMS will be used to quantitate and determine the molecular structure of the product(s); and 3, selectively test potent inhibitors for effects on c-di-GMP production and biofilm formation in P. aeruginosa assays, and selectivity in P. aeruginosa and mammalian cell (HeLa) proliferation assays. Results of the above experiments will be the first detailed characterization of the SAR for binding of substrate analogs to an important DGC and will be used to design more drug-like compounds for synthesis and testing, likely in Phase II of the project. The impacts of this project will be at least twofold. First, the substrate binding site and SAR for a CDG will be described for the first time. Second, lead generation for drugs that can be developed to interfere with biofilms and virulence of important pathogenic bacteria will be developed. The proposed study will result in modified substrate analogs as new research tools in the field and lead to development of novel therapeutic agents for treatment of biofilm-related diseases. PUBLIC HEALTH RELEVANCE: Certain pathogenic bacteria form biofilms, complex structures that resist conventional antibiotics and are associated with numerous infectious diseases such as pneumonia, stent blockage, catheter colonization, etc. One molecule c-di-GMP plays an essential role in the regulation of biofilm formation, and the inhibition of its biosynthesis is the basis for this project. We will synthesize or procure 29 analogs of the substrate GTP, and determine their structure-activity relationships (SAR) as competitive inhibitors and possible alternate substrates of the enzyme from Pseudomonas aeruginosa responsible for c-di-GMP production. Results of these studies will have impact in two areas. First, the substrate binding site and SAR for a biofilm-related enzyme will be described for the first time. Second, lead generation for drugs that can be developed to interfere with biofilms and virulence of important pathogenic bacteria will be developed.

Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: STTR | Phase: Phase I | Award Amount: 320.47K | Year: 2015

DESCRIPTION provided by applicant This phase project has as its overall objectives to design engineer optimize and implement a novel genetically encoded fluorescent drug sensor FDS based on F rster Resonance Energy Transfer FRET by exploiting conformational rearrangements induced by drug interactions with human serum albumin SA SA is uniquely suited for a general drug sensor because it possesses multiple binding sites for small molecules We have acquired the ORF open reading frame encoding human SA and will insert this ORF into Gateway vectors purify the chimeric proteins from E coli and analyze fluorescence responses to drugs using a well microplate spectrofluorimeter During the first year we will focus on design and construction of sensors once a successful sensor has been obtained we will optimize and fine tune the sensor to different ligand groups The following specific aims are proposed to achieve the goals of phase I of this project Convert the SA gene into Gateway compatible format insert into a suite of Gateway vectors transform E coli and grow cultures test responses in crude lysates to drugs and monitor spectra of promising affinity purified sensor proteins Obtain binding isotherms and determine affinities compare to published data for drug binding to SA constructs in comparison with standard SA fluorescein Optimize signal to noise ratio by linker mutagenesis and create a series of specificity and affinity mutants Screen NCI Oncology Drug Set compounds to confirm sensorandapos s general sensing capacity Development of a successful new drug requires identifying those that show maximal ability to reach target cells permeability while having minimal effects on drug transporters drug drug interactions For this reason the Fluorosome division of GLSynthesis Inc has established collaboration with Prof W Frommer at the Carnegie Institution for Science Stanford University to create genetically encoded FRET drug sensors FDS from SA This novel drug sensor technology is expected to enhance the commercial use of Fluorosome r based assays and lead to the development of novel assay systems PUBLIC HEALTH RELEVANCE We will engineer optimize and implement a novel genetically encoded fluorescent drug sensor that will assist in identifying drug candidates that show maximal ability to reach target cells permeability while having minimal effects on drug transporters drug drug interactions

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