Worcester, MA, United States
Worcester, MA, United States
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Yanachkov I.B.,Glsynthesis, Inc. | Dix E.J.,Glsynthesis, Inc. | Yanachkova M.I.,Glsynthesis, Inc. | Wright G.E.,Glsynthesis, Inc.
Organic and Biomolecular Chemistry | Year: 2011

P 1,P 2-Diimidazolyl derivatives of pyrophosphate and halomethylene-bis-phosphonates have been synthesized and characterized, and the mechanism of their formation was studied. These reagents enable synthesis of dinucleoside tetraphosphates and tetraphosphonates conveniently and in high yields. © 2011 The Royal Society of Chemistry.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 243.27K | Year: 2015

DESCRIPTION provided by applicant The objective of the proposed project is to design and construct a highly specific and high throughput in vitro biological assay system to characterize the interaction of drugs and drug candidates with human BSEP Bile Salt Export Protein BSEP ABCB is an active ATP dependent transporter localized in the liver canicular membrane It mediates efflux of bile salts from hepatocytes into bile and is essential for normal bile formation and flow Inhibition of BSEP activity due to an administered xenobiotic or a mutation in the ABCB gene can lead to liver injury caused by intracellular accumulation of cytotoxic bile constituents Given that drug induced liver injury DILI is a major cause of serious illness in humans drug withdrawal post marketing regulatory failure and attrition during preclinical and clinical development regulatory agencies recommend in vitro measurement of the BSEP inhibitory potential of drug candidates The assay system will consist of a purified and functionally competent BSEP integrated into the novel assay platform the Fluorosome r Platform This assay system to be referred to as Fluorosome trans bsep will be marketed as a superior substitute for the only existing method which uses inverted vesicles generated from membranes of cells overexpressing BSEP The specific aims meant to achieve these goals include Production purification and reconstitution of active human BSEP these steps will be accomplished in the lab of subcontract P I Dr Balazs Sarkadi a known expert in the field of ABC transporters identification of an optimal test substrate tentatively vinblastin i e one that will be actively transported by the transporter and show significant quenching of the sensor employed in Fluorosome trans determination of reconstituted BSEP ATPase activities for demonstrated BSEP substrates and inhibitors and formation of Fluorosome trans bsep from reconstituted BSEP measurement of drug transport and its inhibition accompanied by parallel ATPase activity measurements The result of this project will be a simple to use commercial assay that is unambiguously specific for BSEP It will be rapid with a high signal to noise ratio provide real time results and will be applicable to a wide range of compounds at very small amounts It will only require a fluorescence plate reader rather than LC MS or radioactivity as is required for an existing assay The new assay will be amenable to moderate and high throughput screening It will be offered on the market both as a reagent and as an in house service PUBLIC HEALTH RELEVANCE This project will result in a unique technique to determine if drugs under development inhibit the bile salts export protein BSEP pump This pump is responsible for the transfer of bile salts from the liver to allow normal bile formation and flow Inhibition of this pump by a drug can lead to drug induced liver injury which is a serious cause of illness in humans


Grant
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.


Grant
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


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 597.91K | Year: 2016

DESCRIPTION provided by applicant In Phase I as proposed we successfully demonstrated proof of principle for the Flurosome trans abcg assay This innovative assay characterizes how compounds interact with the important ATP dependent drug efflux transporter ABCG BCRP Breast Cancer Resistance Protein i e the susceptibility of drug candidates to be extruded from their target tissue A successful Phase I forms the basis for this Phase II proposal The goal of Phase II is to bring the Fluorosome trans abcg assay to commercialization providing a fully validated high throughput solution for ABCG transporter studies to drug development groups worldwide Our ultimate goal is to commercialize a complete suite of assay reagents for all FDA and EMA mandated transporters enabling the pharmaceutical industry to efficiently evaluate the suitability of drug candidates for continued development The andquot Fluorosome trans abcg andquot reagent will be one of this suite of reagents We were able to successfully accomplish all the proof of principle goals of our Phase I proposal In brief we were able to produce purified human ABCG reconstitute the ABCG transporter in a functional form into lipid bilayers test and confirm the reconstituted ABCG andapos s ATPase activity use the reconstituted ABCG to construct lipid vesicles containing encapsulated drug sensor andquot Fluorosome trans abcg andquot demonstrate that ABCG is functional in the resulting Fluorosome trans abcg construct demonstrate the ability of Fluorosome trans abcg to detect the active transport of test substrates and demonstrate that the assay is sensitive to known inhibitors of ABCG In addition to succeeding in these initial goals we have made a significant advance over what was proposed in Phase I in that instead of relying on an external vendor for the purification and reconstitution of the ABCG protein we have both improved these procedures and brought them entirely in house For Phase II we will bring the Fluorosome trans abcg assay to a commercial stage When successful this assay will be the first off the shelf high throughput assay available to drug developers that is unambiguously specific for the ABCG transporter The assay uses small amounts of test material and is simple to use With respect to instrumentation it requires only a standard injecting multiwell fluorescence plate reader which generates real time data in under a minute To bring this assay to commercialization we propose the development of various in house processes technologies that enable the large scale production of purified ABCG protein and its incorporation into our Flurosome trans abcg reagent the necessary extensive validation of the assay using drugs known to interact with ABCG establishment of rigorous quality control methods required for reagent manufacture and storage and finally the implementation of an extensive plan which includes an Early Adopter Program followed by product launch and revenue generation Accomplishing these goals will result in a unique easy to use rapid cost effective and highly specific assay for characterizing the interaction of drugs and drug candidates with the ABCG transporter The Fluorosome trans abcg assay will be poised to enter the market as a highly competitive and useful product This will greatly facilitate us in bringing our other two current Fluorosome assays those for the human pgp and BSEP into the marketplace PUBLIC HEALTH RELEVANCE This project will result in a commercial level technique to determine if drugs will be expelled from their target tissue by the Breast Cancer Resistance Protein andquot ABCG andquot The test will allow the pharmaceutical industry to evaluate at an early stage the suitability of drug candidates for continued development The test will be highly specific reliable simple rapid inexpensive and amenable to robotics


Grant
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.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 3.00M | Year: 2010

ABSTRACT Among inhibitors of Gram+ DNA polymerases IIIC and IIIE, several compounds are active against multiple strains of the anaerobic Gram+ bacterium Clostridium difficile (Cdiff). The compounds appear to be selective for Cdiff compared with other Gram+ anaerobes and aerobes, and a lead compound - 2-(3,4-dichlorobenzyl)- 7-(5-morpholinylpentyl)guanine or 359E - is active orally in protecting hamsters from lethal Cdiff infection. The compounds of interest are poorly absorbed orally and too weak to be developed for systemic use against Gram+ aerobe infections. Compound 359E and analogs are tertiary amines, readily form water soluble salts, and are highly effective against Cdiff in vitro and in vivo. Given the increasing prevalence of Clostridium difficile-associated diarrhea (CDAD), including that from highly virulent, toxin-overproducing and/or antibiotic- resistant strains, the need for new and selective antibacterials to treat this disease is growing. Our phase II results strongly suggest that development of the lead compound or an alternative as an oral treatment for Cdiff diarrhea in human patients will result in a novel, first in class drug to treat this emerging infectious disease. The specific aims of the competing renewal of this project are focused on preclinical development of 359E or a closely related lead compound (LC). The aims are to: 1, scale up and begin process development for 359E; 2, determine the mechanism of action, selectivity, anticlostridial spectrum, and resistance development of 359E; 3, determine oral safety, anti-clostridial efficacy, and absorption of 359E in the hamster; 4, synthesize and screen analogs of 359E as backup LC compounds, and designate a candidate for development (CD). Once this has occurred, IND-enabling studies will commence. These include: 5, in vitro ADME studies; 6, preclincal analytical, toxicology and toxicokinetic studies; 7, safety pharmacology and genotoxicity studies; 8, cGMP production of the CD. Once all preclinical studies have been completed, aim 9 will encompass preparation an IND application for the CD as oral treatment for CDAD. Incidence of CDAD is on the rise in the United States and Europe, and Cdiff is the major identified infectious cause of nosocomial diarrhea in patients to whom antibiotics had been previously administered. Vancomycin and metronidazole are first-line therapy for treatment of CDAD, but there have been reports of treatment failure and CDAD recurrence after treatment with metronidazole, and the Centers for Disease Control and Prevention (CDC) has discouraged vancomycin for treatment of CDAD in hospitals to minimize the risk of vancomycin-resistant enterococci and staphylococci. Various treatments are in clinical trials and preclinical development for Cdiff infections, ranging from direct-acting antibacterials to vaccines and compounds to neutralize Cdiff toxins. The results of our phase II studies indicated the strong likelihood that a DNA polymerase III inhibitor will be an effective and non-toxic oral treatment of CDAD.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 3.40M | Year: 2013

DESCRIPTION Undesired platelet activation can be result of many common pathologies or interventions Arterial thrombosis and the acute ischemic events that follow such as myocardial infarction and stroke are among the leading causes of death incapacitation and rising health care costs in the developed world Therefore antiplatelet drugs have significant market share and clinical importance and advances in this area can have a significant social impact In the previous phases of this project we developed a series of new bis adenosine tetraphosphate Ap A analogs which inhibit platelet aggregation by a unique new mechanism targeting both platelet ADP receptors P Y and P Y Our studies have indicated that this simultaneous targeting might have a synergistic effect on platelet aggregation inhibition Simultaneous targeting of two synergistic targets can also provide for an additional margin of safety against undesired off site target side effects In phase II we studied the specificity and selectivity of he class and identified a candidate with efficacy in vivo for further development This candidate CD inhibits ADP and collagen induced platelet aggregation in the low nanomolar range Furthermore it showed high antithrombotic activity in the well established preclinical canine model of recurrent thrombosis mimicking unstable angina andquot Folts modelandquot In this model it completely abolished the cyclic flow variation recurrent thrombosis in injured and restricted coronary artery at an infusion dose of g kg min In a dose escalation study in rats the CD had a profound effect on platelet reactivity and relatively small increase of the bleeding time only at the highest dose studied times higher than the maximum efficacious dose It also showed no effect on hemodynamic parameters at any dose It did not act as agonist or antagonist of human P Y P Y P Y or P X receptors and had good safety margin in the endothelium P Y dependent guinea pig aorta relaxation model Moreover the antiplatelet effect of the CD is reversible has very fast onset and offset of its in vivo activity and is quickly clered from the systemic circulation We also demonstrated that the CD can be synthesized and purified in multi gram scales In view of the unique properties of the class and the promising results obtained in phase II we propose to continue the preclinical development of the CD in order to obtain the package of data which would enable us to secure investor or partner funding for initiation of clinical testing after IND submission or alternatively to license the CD to a maor pharmaceutical company for further clinical development Despite the recent advances in development of new types of antithrombotics and because of the high mortality and recurrence rate and major bleeding events among acute coronary syndrome patients there is a significant need for new antiplatelet agents that a can reduce ischemic events without causing excessive bleeding b have low interpatient variability of the effect c are fast acting assuring immediat patient protection d have quick reversibility of the effect so the treatment can start before fial diagnosis and intervention strategy are in place The main antiplatelet drugs in clinical use today aspirin and clopidogrel target platelet COX and P Y platelet receptors respectively Limited response to both drugs often referred to as andquot resistanceandquot occurs in significant numbers of patients and is associated with poor clinical outcomes No drugs have yet been identified that inhibit both P Y and P Y receptors No rapidly reversible within hours injectable antiplatelet drugs are currently available for clinical use for patients who are in need of antiplatelet therapy and may be at risk for bleeding complications or may need surgical intervention Therefore we expect the further development of the CD to result in a new antithrombotic drug with superior safety and efficacy and therapeutic properties that will satisfy important unmet clinical needs and reduce the mortality rate among patients with acute coronary syndrome PUBLIC HEALTH RELEVANCE Platelets play critical roles in hemostasis but their aggregation can cause thrombosis Antiplatelet drugs such as clopidogrel Plavix R are slow to exert effects and cause irreversible inhibition of platelets We have synthesized novel compounds that have a unique mechanism of action and fast onset offset of action and could be used effectively during acute events such as stroke and during surgery


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 237.36K | Year: 2013

DESCRIPTION: Androgen action is the net result of opposing pathways of steroid biosynthesis and metabolism. It is now widely recognized that testosterone (T) is an androgenic precursor of the more potent 5 -dihydrotestosterone (DHT), produced by 5 -reductase type 2. Circulating T and DHT also serve as precursors of estrogens through their conversion to estradiol and estrone by aromatase. Thus, regardless of gender, these secreted steroids are converted peripherally into divergent signals for two members ofthe steroid hormone nuclear receptor family. The overproduction of androgens in both sexes is the root cause of most acne (acne vulgaris), alopecia and seborrhea, and the practice of using antiandrogenic therapy in these disorders is accepted. However, the systemic antiandrogen side effects of marketed nonsteroidal androgen antagonists (flutamide, nilutamide, bicalutamide) and 5 -reductase type 2 inhibitors (finasteride) are serious drawbacks to their repeated use, and some are contraindicated in pregnantwomen. While acne vulgaris is the most prevalent skin disorder in humans, the other diseases are also relatively common. All of the current therapies for these disorders, including over the counter medications, treat the symptoms but not the excess skin androgens. In addition, all current treatments for these disorders have local and systemic side effects. Severe forms of these skin disorders, e.g. cystic acne, are largely untreatable and represent a significant unmet medical need. We and our collaborators at Hygeia Therapeutics Inc. have identified a synthetic antiandrogenic compound - (S)-HYG-440 - a chiral ester that potently binds the androgen receptor and reduces androgenic functional activity in cell cultures. In contrast, its hydrolysis product - (S)-HYG-441 - is devoid of both activities. (S)-HYG-440, applied topically to one hamster flank, reduced the size of this organ but only on the ipsilateral side These results suggest that this drug is a soft antiandrogen , expected to be active topically inandrogen-dependent maladies of the skin and scalp, but to be hydrolyzed by plasma or tissue enzymes, thus terminating its systemic action. Indeed, (S)-HYG-440 is readily hydrolyzed after incubation in animal plasmas. The specific aims to answer the fundamental question is (S)-HYG-440 actually a soft drug? include: synthesis by yet newer methods of both (S)-HYG-440 and (S)-HYG-441 as candidate drug and metabolic product, respectively; the latter will serve also as toxicology sample and marker for further analyses; application of (S)-HYG-440 to the skin of hamsters, and analysis of plasma and tissue extracts for uptake and distribution of the parent compound and its conversion to (S)-HYG-441;and toxicity testing of the hydrolysis product (S)-HYG-441 in vitro and in vivo the hamster. Although inhibition of androgen action to treat acne, seborrhea and alopecia in men and women (and hirsutism in women) is efficacious, it can carry serious systemic risks. However, because all of these maladies are localizedto the skin, it follows that local treatment would carry a higher benefit-to-risk ratio compared to orally administered therapies. Unlike all other antiandrogens or 5 -reductase inhibitors on the market or in development, (S)-HYG-440 was designed to avoidsystemic activity by taking advantage of esterases and hydrolases found in most tissues in the body for metabolic deactivation, i.e. as a metabolically soft drug. The antiandrogenic activity of the prototype compound (S)-HYG-440 may be limited to the parent drug because its putative hydrolysis product has no detectable affinity for the androgen receptor. An antiandrogen specifically designed to act locally would minimize or avoid unwanted systemic antiandrogen effects. The synthesis and discovery of an optimal locally active androgen antagonist to be applied to the skin to treat acne, alopecia, seborrhea (and hirsutism in women) are the subjects of this application. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: Secreted androgenic steroids are converted into male and female hormones in both sexes. Because the androgenic hormones are responsible for acne, alopecia and seborrhea, the rationale for using antiandrogenic therapy for their treatment is strong. Our lead compound (S)-HYG-440 has shown efficacy in vivo after topical application in the hamster, and may behave as a metabolized soft drug. The purpose of this project is to obtain direct evidence for this hypothesis and to obtain toxicity of the presumed metabolite (S)-HYG-441.


Grant
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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