John K. Buolamwini - US grants

This proposal responds to the timely initiative of the National Heart Lung and Blood Institute to increase the number of underrepresented minorities in cardiovascular disease research. the proposed training will be expose, train and mentor minority students in various cardiovascular disease research areas. cardiovascular disease is the number cause of death among the American population, taking the lives of over 980,000 people a year, minorities being disproportionately affected, especially African-Americans. The proposal seeks to address the problem of minority underrepresentation through both the recruitment, and intensive training and mentoring of talented minority students in research projects that are focused on cardiovascular experimental therapeutics, and pharmacology. The School of Pharmacy will draw from its extensive experience in the NIH-funded Summer Minority Biomedical Research Internship Program, which has been in place for three consecutive years (1995-1998), to increase the pool of minorities entering into careers in the biomedical sciences. Each year, for a total of five years, eight (8) minority undergraduate students who have spent at least one year in college, will be recruited into the cardiovascular research programs at the University of Mississippi School of Pharmacy, in the Department of Medicinal Chemistry and Pharmacology. These trainees will spend three summer months immersed in training under a faculty mentor, on NIH, American Heart Association, and other funded research projects in adrenergic receptor pharmacology, biochemical mechanisms of alpha- and beta-adrenergic receptor specific agonists and antagonists, the design of novel adenosine transport blockers, nitric oxide synthase and complement system inhibitors for prevention of ischemia/reperfusion injury and/or cardiac xenograft rejection. Proactive steps will be taken to create interest in, and guide trainees to pursue careers in cardiovascular research through mentor encouragement and advising, field trips, invited speakers seminars and/or role models, and poster sessions. Plans will be put in place to track trainees to evaluate the program's success, and to effect changes as deemed necessary. Recruitment will be in- state, from community colleges and universities in Mississippi. Liaisons and direct contacts will be established with feeder institutions, building on already established strong networks like the Mississippi Alliance for Minority Participation of which the University of Mississippi is a member. The program will be evaluated by extensive surveys during and after each training period. Students and mentors will be given questionnaire. Participants will be tracked for 10 years to assess program.

DESCRIPTION (Adapted from applicant's abstract) This is a Mentored Minority Faculty Development Award Proposal designed to provide the necessary mentoring and research activities necessary to enable the PI achieve his career goals of becoming an established independent academic investigator in the cardiovascular research and drug discovery field. The proposal responds to the timely initiative of the National Heart Lung and Blood Institute to prepare underrepresented minorities scientists as independent investigators. Cardiovascular disease is the number cause of death among the American population, afflicting 1.5 million Americans with a new or recurrent heart attack, and killing over 980,000 people every year. Minorities especially African-Americans are disproportionately affected. There is definitely a need more minority researchers in the cardiovascular field especially in Mississippi, which houses a large African-American Population. This proposal seeks to provide intensive mentoring and research training for the PI who is a minority assistant professor at a predominantly white higher institution, the University of Mississippi. The overall career goals of the candidate are to acquire research capabilities in areas of experimental analysis of protein- ligand complex interactions at the molecular level using photo labeling, affinity purification and use them to structurally characterize the interaction of inhibitors with the es adenosine (nucleoside) transporter to obtain insights that will be used to design and develop more specific and potent adenosine transport inhibitors as potential cardioprotective and neuroprotective drugs. Adenosine is a physiological nucleoside that is released in ischemic conditions such as a heart attack or stroke to protect tissue injury. However, its rapid uptake by nucleoside transporters abrogates this protective action. Nucleoside transport inhibitors block adenosine uptake by cells, and therefore enhance its extracellular protective effects. The inhibition of adenosine transport has therapeutic potential in heart disease and stroke that has yet to be tapped by he design and discovery of inhibitors with the requisite pharmacological profiles. Designed compounds will be synthesized and tested as es transporter ligands and adenosine transport inhibitors by flow cytometry and radioisotope methods. The candidate will receive mentorship by established well-accomplished investigators, the primary and secondary sponsors, and a cadre of four senior faculty serving on the candidates advisory committee. Ancillary course work in advanced protein techniques, mass spectrometry and experimental design, as well as American Chemical Society techniques workshops, and our research office workshops. Biweekly research progress meetings will be held with sponsors and committee members.

DESCRIPTION (provided by applicant): Cancer is the second leading cause of death in the United States, contributing to one out of every four deaths. It is estimated that 555,500 people in the United States will die from cancer this year. Last year alone, cancer cost the United States' economy an amount of $156 billion in combined direct and indirect costs. This situation calls for the development of effective cancer therapies. The long-term goal of this research program is the discovery and preclinical development of compounds to target p53 pathway targets, specifically compounds that inhibit p53-Mdm2 interaction, using structure-based drug design and combinatorial chemistry in conjunction with mechanism-based assays. This grant proposal is designed to expand on biologically active lead compounds that were discovered through structure-based design using the reported X-ray crystallographic 3D atomic coordinates of the human p53 and Mdm2 binding complex. A series of compounds belonging to the beta-3-carboline class, that were among of lead compounds identified will be explore as potential novel molecularly target anticancer drugs, or as probes for cancer research. We have hypothesized that the observed effects of these compounds on elevation of cellular expression levels p53 and p21 stems from their inhibitory effect on p53-Mdm2 interaction. The specific aims of this two-year R15 proposal are: 1) to test the hypothesis that this novel series of compounds exert their effect on p53 and p21 levels by inhibiting p53-Mdm2 interaction, and 2) to conduct structure-activity relationship (SAR) studies and generate combinatorial libraries for high throughput screening to optimize 13-carboline lead compounds as novel p53 potentiating agents. Traditional and combinatorial medicinal chemistry approaches, as well as 3D quantitative structure-activity relationship analyses will be employed together with p53-Mdm2 binding assays and high throughput antitumor assays to optimize lead compounds. This research program is planned to involve the participation of undergraduate students for the purpose of their training in biomedical research.

DESCRIPTION (provided by applicant): Recent studies have shown an overexpression of nucleoside transporters in some human breast, liver, stomach and colorectal cancer tissues, as well as varying profiles of nucleoside transporter subtype gene overexpression among tumors. This might underscore the marked increases in the capacity to take up physiological nucleosides that has been observed as normal cells are transformed into tumor cells. It has also been revealed that nucleoside transporter genes belong to the delayed early proliferative genes class. Taken together, these observations strongly point to a possible role of nucleoside transporters in the carcinogenesis process, at least at the tumor promotion and progression stages. We have recently found that nucleoside transport inhibitors potently antagonize the inhibitory effect of exogenous nucleosides on the antitumor promotion activity of dehydroepiandrosterone (DHEA) against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion in the JB6 cell carcinogenesis model. This suggests that nucleoside transporters may be involved in the establishment of the tumorigenic phenotype, i.e. promotion of anchorage independent growth characteristics. This is consistent with reports that chemopreventive agents like tamoxifen downregulate nucleoside transporter gene expression. The long-term goal of this research program is to investigate the chemopreventive potential of nucleoside transport inhibitors. The specific aims of this project are: 1) to probe the changes in nucleoside transporter gene expression during JB6 cell tumorigenic transformation, 2) to determine whether nucleoside transport blockers can inhibit carcinogenesis compete against the inhibitory effects of exogenous nucleoside supplies on chemopreventive activity, and 3) to determine structure-activity relationships (SAR) regarding chemoprevention and inhibition of nucleoside transport. The research plan involves experiments to define the role of nucleoside transport inhibition in modulating the effects of chemopreventive agents like DHEA and tamoxifen, in the in vitro JB6 cell carcinogenesis model. Nucleoside transport inhibitors, namely, nitrobenzylthioinosine, dipyridamole and dilazep, as well as novel tetrahydroisoquinolinyl purine riboside inhibitors to be synthesized in the PI's laboratory will be used in these studies. The TPA-induced JB6 P+ cell transformation model using anchorage independent growth as end point, as well as JB6 P+/AP-l-luciferase reporter cell tumor promoter models will be employed for these proof-of-concept in vitro studies. Quantitative analysis of nucleoside transporter gene expression during JB6 tumorigenic transformation will also be carried out by means of real time quantitative RT-PCR. Ancillary studies involving analysis of cellular nucleotide levels by HPLC, and assays of G6PDH activity and its inhibition by DHEA will also be conducted. The mechanistic insights to be gained regarding the carcinogenesis process and its abrogation may lead to the identification of novel molecular targets and agents for cancer prevention.

DESCRIPTION (provided by applicant): The introduction of highly active antiretroviral therapy (HAART) has been successful in prolonging the lives of HIV/AIDS patients. However, long-term use of HAART, which is a combination therapy that almost invariably contains nucleoside HIV reverse transcriptase inhibitors (NRTIs), is associated with major toxicities including liver damage, neuoropathy, pancreatitis, myopathy, neuropathy, lactic acidemia and lipodystrophy, some of which could result in patient fatalities. The culprits for these toxicities are believed to be the NRTIs in HAART. These nucleoside drugs, including zidovudine (AZT), stavudine (d4T), didanosine (DDI), zalcitabine (DDC) and lamivudine (3TC) are believed to cause mitochondria! depletion to varying extents, leading to cell death and tissue toxicity. This mitochondrial depletion stems mainly from the inhibition of mitochondrial (mt) DNA polymerase gamma by the triphosphate metabolites of NRTIs. The entry of nucleosides into mitochondria and cells occurs through specialized membrane carrier proteins termed nucleoside transporters. Upon entry into mitochondria, nucleosides are sequentially phosphorylated by mitochondrial kinases such as the mitochondrial-specific thymidine kinase (TK-2) and others to yield the active triphosphate metabolites, which then inhibit mtDNA synthesis. It has recently been shown that mitochondria in mammalian cells express the equilibrative nucleoside transporter 1 (ENT1) in their membranes and that this expression enhances mitochondrial toxicity of antiviral nucleoside drugs. In light of this observation, we hypothesize that selective inhibition of mitochondrial nucleoside transporters can prevent entry of NRTIs into mitochondria of patients undergoing HAART, can be used as an approach to reduce the mitochondrial toxicity of these anti-HIV drugs. The following specific aims will be pursued in exploring this strategy for reducing NTRI mitochondrial toxicity. 1) Synthesize and characterize novel ester prodrugs of the nucleoside transporter inhibitor dipyridamole in terms of ENT transporter inhibition, cellular permeation and enzymatic ester hydrolysis. 2) Determine the ability of the appropriate dipyridamole ester prodrug to protect against mitochondrial toxicity of anti-HIV nucleosides. Methods will include synthetic and analytical chemistry, nucleoside transporter binding and nucleoside uptake assays, flow cytometry and real-time PCR. If successful, this research will increase our understanding of the role of nucleoside transporters in mitochondrial toxicity and could lead to a new strategy for reducing or preventing NRTI toxicities of long-term use of HAART in HIV/AIDS patients, and provide a novel pharmacological approach to addressing mitochondrial toxicity of nucleoside analog antiviral or anticancer agents, as well as furnish new knowledge on the effects of inhibiting mitochondrial nucleoside transport in situ.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] Novel agents are needed for achieving the goals of the growing interest and promise in developing chemoprevention approaches to cancer control (Sporn and Liby, 2005, Smith et al., 2005; Lippman and Lee, 2006; Francis et al., 2006). In pursuing nucleoside transport inhibitors as potential chemopreventive agents, we have discovered that dipyridamole is a potent chemopreventive agent against 12-O-phorbol-13-myristylacetate (TPA) tumor promotion in JB6 P+ cells. With an IC50 of 10 nM, dipyridamole is a much more potent than the known chemopreventive agent dehydroepiandrosterone (DHEA), which exhibited an IC50 of 1.0 [unreadable]M in the same JB6 P+ cell carcinogenesis assay. This application is thus planned to capitalize on this novel finding to conduct a structure-activity relationship (SAR) study, and use it to select compounds that will be evaluated as novel cancer chemoprevention agents in an in vivo animal model. New compounds for which synthetic methods have already been established in the applicant's laboratory will be synthesized and evaluated against 12-O-phorbol-13- myristylacetate (TPA)-induced tumor promotion in JB6 P+ cells. This application fits well with the timely program announcement (PAR-06-313), which has two of its objectives as the "pilot testing and development of new methods of chemoprevention..." and the "development and evaluation of molecular targets to prevent, reverse, or retard progression of precancerous lesions (and, hence, the cancer process) by natural, synthetic, chemopreventive agents." We are developing synthetic chemopreventive agents. We seek to achieve two specific aims in this application. Specific Aim 1. To evaluate novel nucleoside transport inhibitors synthesized in the applicant's laboratory as antitumor promotion agents in vitro, and select potent and "drug-like" compounds for in vivo animal testing. Specific Aim 2. To investigate the chemopreventive ability of compounds selected from Specific Aim 1 in an in vivo mouse skin carcinogenesis model. A new AP-1-SEAP JB6 P+ cell reporter assay developed in the applicant's laboratory will be used in conjunction with an anchorage independent clonogenic assay to assess the in vitro chemopreventive activities of target compounds. Ancillary studies will also be undertaken to establish a rational basis for chemopreventive agent development. Thus, the determination of the levels and of activation of mitogen activated protein kinases (MAPKs) such as ERKs, p38 and JNK, which have been shown to be involved in tumor promotion signal transduction pathways in the JB6 P+ mouse epidermal carcinogenesis model, will be undertaken. The SENCAR mouse DMBA-initiation-TPA-promotion skin carcinogenesis model will be used for the in vivo studies in Specific Aim 2 to identify potential candidates for preclinical development as chemopreventive agents. Tumor incidence and multiplicity will be Histopathological and immunohistochemical analysis will be performed, as well as biomarker analysis of tumors that will be induced in the in vivo studies. The success of this project will expand our armamentarium of chemopreventive agents and uncover novel chemoprevention molecular targets. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Our studies on novel small molecules inhibitors of HIV integrase have led to the identification of new potent phenanthrene diketoacid (DKA) HIV integrase inhibitors that also inhibit HIV replication in cell culture, with a selectivity index up to 10. In light of the need for more effective and less toxic anti- HIV drugs, these results appear promising and warrant follow-up optimization and preclinical studies. However, since phenanthrenes are polyaromatic hydrocarbons (PAHs) with potential carcinogenicity we propose to investigate these novel phenanthrene diketoacids for carcinogenicity potential. Our hypothesis going forward is that appropriate substitution with electron withdrawing groups can minimize mutagenicity and produce useful anti-HIV agents. This is supported by the example of the new anti-malarial drug halofantrine, a substituted phenanthrene that is effective against multidrug resistant malaria, and is neither mutagenic nor teratogenic. We thus believe that appropriate substitution of our HIV integrase inhibitory phenanthrene DKAs will make them non-mutagenic and non-carcinogenic. Thus the specific aims of this proposal are: 1) to synthesize and test the IN inhibitory activity of new phenanthrene diketoacids with multiple electron withdrawing substituents and 2) to determine the carcinogenicity potential of the phenanthrene DKAs synthesized in Specific Aim 1. We will use the Ames test for in vitro carcinogenicity testing. The success of this project will provide vital information to decide whether or not to go ahead with optimization of the potent phenanthrene DKA HIV integrase inhibitors that we discovered recently and shown to selectively inhibit HIV replication in human peripheral blood mononuclear cells. If this approach to reducing carcinogenicity is successful it will not only provide a means of eliminating carcinogenicity of the phenanthrene DKAs, but may also provide a general method for reducing the carcinogenicity of PAH containing drugs. PUBLIC HEALTH RELEVANCE: This project is aimed at investigating whether or not a novel class of potent phenanthrene diketo acid HIV integrase inhibitors that have been shown to suppress HIV viral replication in cell culture, are carcinogenic. The results of the research will determine whether to carry on with further work on optimizing this class of new anti-HIV agents towards AIDS therapeutics development.

DESCRIPTION (provided by applicant): Drugs targeting the HIV virus transcriptase or protease are used in highly active antiretroviral therapy (HAART) cocktails that have prolonged the lives of AIDS patients. Unfortunately, these drugs are associated with severe toxicities, unable to control viral replication in some patients, and are rendered in effective by viral drug resistance. The absolute requirement of the integrase enzyme for HIV replication, and the fact that it has no host cell counterpart, make it an attractive drug development target. The recent introduction of the integrase targeted drug raltegravir has validated this enzyme as a very promising HIV/AIDS therapeutic target. Our studies on small molecule inhibitors of HIV integrase with novel bioisosteric replacement of the beta-diketo acid motive have led to the identification of novel HIV integrase inhibitors with single digit micromolar potency. We propose to undertake lead optimization to increase the potency and assess their potential to treat HIV infection. We will combine medicinal chemistry with computer-aided drug design and biological testing for potency, selectivity and toxicity. An innovative integration of docking, molecular dynamics simulations, structure-based design, chemoinformatics, pharmacophore mapping and three dimensional quantitative structure-activity relationships (3D-QSAR), parallel combinatorial synthesis, bioassays using recombinant wild type and mutant HIV integrase variants, cell culture testing against viral replication including drug resistant isolates from AIDS patients, will be applied. HIV-1 infected human peripheral blood mononuclear cell (PBMC) cultures will be used to test the ability of compounds to block HIV replication. Toxicity of compounds will be tested using uninfected PBMCs as well as CEM leukemia and Vero cells to assess therapeutic index. The success of this project will provide novel "drug- like" integrase inhibitors as preclinical HIV/AIDS drug development candidates;and increase our knowledge in the design of novel HIV integrase targeted antiviral agents. PUBLIC HEALTH RELEVANCE: This grant proposal seeks to develop new compounds for inhibiting HIV integrase to treat AIDS. The PI has discovered potent integrase inhibitors. The objectives of the grant proposal are to optimize activity and test toxicity.

DESCRIPTION (provided by applicant): The advances in treatment notwithstanding, heart disease remains the number one cause of death in the Nation, claiming the lives of nearly one million Americans each year, and running up medical costs estimated at $286.5 billion per year. Ischemia-reperfusion injury is the major cause of complications of cardiac surgery;morbidity and mortality, but current cardioprotective options are very limited and suboptimal in high-risk patients. This leaves a dire need for effective treatments. Approaches to augment the cardioprotective actions of the physiological nucleoside adenosine (Ado) continue to hold promise and remain to be exploited for novel cardioprotective therapies. This project will pursue the identification of high-affinity specific inhibitors of a recently characterized novel pH-dependent cardiac adenosine transporter, ENT4. ENT4 selectively transports adenosine relative to other physiological nucleosides, making it an intriguing transporter to study with respect to adenosine modulation in myocardial ischemia and reperfusion. Currently, there are no known potent or specific inhibitors of ENT4 Ado transport. Thus, the identification of potent specific inhibitors is of high priority, not only for cardiac studies but for studying adenosine regulation by ENT4 in other tissues as well. They will facilitate studies of its role in cardiac ischemia-reperfusion, and show whether or not ENT4 could be a cardioprotection drug target for combination regimens with other cardioprotective agents like ENT1 nucleoside transport inhibitors. In line with our pursuit of adenosine uptake inhibitors as cardioprotective agents for the treatment of ischemia- reperfusion injury, we have cloned and stably expressed the human ENT4 transporter (hENT4) in a nucleoside transporter deficient porcine PK15 (PK15NTD) cell line. We subsequently used this expression system to screen, and identify small molecules inhibitors of ENT4 Ado transport with IC50s values down to submicromolar level, and selectivity up to 78-fold relative to one or the other human plasma membrane equilibrative nucleoside transporters, hENT1 or hENT2. Thus we aim to: 1) optimize the potency and selectivity of these lead compounds through structure- activity relationship (SAR), pharmacophore mapping and 3D-QSAR studies, and to 2) explore the cardioprotective effects of ENT4 inhibitors in an isolated rat heart global ischemia model of myocardial infarction. The new ENT4 inhibitors will be compared with our recently discovered cardioprotective ENT1 inhibitors (Zhu et al., Am. J. Physiol. Heart Circ. Physiol. 2007, 292, H2921-2926). We will explore the possibility that ENT4 inhibitors can be additive or synergistic in combination with ENT1 inhibitors for cardioprotection. A multidisciplinary approach combining parallel combinatorial synthesis, chemoinformatics/3D-QSAR, biochemistry, physiology and cell biology methods will be applied to achieve the aims of the project. The results will provide insights on the role of ENT4 in cardiac ischemia, and its inhibitors in cardioprotection, and provide potential lead compounds for the development of new cardioprotective drugs and/or use as much needed novel research tools to probe ENT4 physiology. The adenosine potentiation approach to cardioprotection could not be more timely, in light of recent clinical results showing that statins, which increase interstitial Ado levels by activating ecto-5'-nucleotidase, can reduce heart attack and stroke risks in people with normal cholesterol levels (Ridker et al., N. Engl. J. Med. 2008;AHA, 2008);and the demonstration of synergism between low dose statin and Ado uptake inhibitor for cardioprotection, when used in combination (Ye et al., 2007). PUBLIC HEALTH RELEVANCE: In this proposal, we seek to study a new class of adenosine uptake inhibitors as cardioprotective agents. We will investigate structure-activity relationships (SARs) and test the cardioprotective properties of the compounds in an isolated rat heart ischemia model.

DESCRIPTION (provided by applicant): Promotion agents with a structure-activity relationship (SAR) that points to some mechanism(s) other than NT inhibition. For example, among novel tetrahydroisoquinoline riboside NT inhibitors tested as anti-tumor promotion agents in the JB6 P+ carcinogenesis model, the relative order of potency with regard to anti-promotion activity was: Compound 23 >Compound 3 >Compound 4, whereas the NT inhibitory potency order was the opposite, with Compound 4 being the most potent (Ki = 0.45 nM) followed by Compound 3 (Ki = 15 nM) and then followed by Compound 23, the least potent (Ki = 300 nM). Upon further investigation with Compound 23, we have shown that in addition to inhibiting TPA-induced tumorigenic transformation, it also inhibits TPA-induced AP-1 transcription factor activation, which has been shown to have a major role in TPA-induced tumor promotion in the JB6 mouse epidermal cell carcinogenesis model, as well as in many other in vitro and in vivo carcinogenesis processes. Thus, in this application, we propose to initiate an investigation of the mechanisms of action of compound 23, as well as conduct antitumor promotion SAR and test the compounds'cancer chemopreventive potential in an in vivo mouse skin carcinogenesis model. The specific aims are: 1) to synthesize and test new aromatic and heterocyclic analogs of Compound 23, 2) to investigate interference with AP-1 DNA binding, and MAP kinase signaling pathways that might account for the possible AP-1-dependent antitumor promotion activity of the compounds, and 3) to evaluate the best compound from Aim 2 in an in vivo mouse skin carcinogenesis model. AP-1-SEAP JB6 reporter cells created by us will be used for the in vitro investigations of SAR and mechanism. We will use the AP-1-SEAP reporter and anchorage independent cell growth clonogenic transformation assays to test the potential chemopreventive activity of new compounds to be synthesized activity. Interference with AP-1 DNA binding, protein kinase C, as well as the role of the mitogen-activated protein kinases (MAPKs) ERK, p38 and JNK, known to be upstream of AP-1, will be analyzed. The SENCAR mouse skin two-stage carcinogenesis model will be used to assess the in vivo anti-tumor promotion activity of this class of compounds in attempts to identify potential lead compounds for preclinical optimization. The success of this research program may lead to the identification of novel promising agents for skin cancer chemoprevention.

DESCRIPTION (provided by applicant): Although the introduction of highly active antiretroviral therapy (HAART) targeting the human immunodeficiency virus (HIV) revolutionized acquired immunodeficiency disease (AIDS) therapy, nucleoside reverse transcriptase inhibitors (NRTIs) that are a cornerstone of HAART cocktails have been found to cause severe mitochondrial toxicities that have limited their use. NRTIs cause toxicity by inhibiting mitochondrial DNA (mtDNA) synthesis through inhibition of polymerase g (Pol g), which is responsible for mtDNA synthesis. Although anti-HIV NRTIs have drawn the most attention to nucleoside drug-induced mitochondrial toxicity, this toxicity has also been seen with other antiviral nucleoside drugs such as fialuridine (FIAU) and clevudine (L-FMAU) that were being developed to treat hepatitis B (HBV) infections, and gemcitabine, the drug of choice for the treatment of pancreatic cancer. Moreover, the problem is likely to become more widespread as nucleoside drugs are used outside the antiviral and anticancer fields to disease areas such as multiple sclerosis. Unfortunately, there is no current proven general treatment of prevention strategy for nucleoside drug-induced mitochondrial toxicity. Thus, the development of innovative approaches to preempt or treat mitochondrial toxicity will be an important advance in the field of nucleoside drug therapy. NRTI triphosphates, the metabolites that inhibit Pol g can be synthesized inside mitochondria from imported NRTIs from the cytosol. Imported nucleosides are sequentially phosphorylated starting with rate-limiting monophosphorylation by mitochondrial-specific thymidine kinase 2 (TK-2) and/or deoxyguanosine kinase (dGK). Mitochondrial membrane nucleoside transporters (NTs) import NRTIs from the cytoplasm. The NT isoforms that have been implicated in mitochondrial nucleoside uptake are the equilibrative nucleoside transporters (ENTs), particularly ENT1 and ENT3. The transgenic overexpression of ENTs has been shown to enhance the mitochondrial toxicity of the former anti-hepatitis B nucleoside drug fialuridine (FIAU) and the NRTI AIDS drug AZT. We have obtained preliminary data showing that a prodrug approach could be used to protect against NRTI-induced mitochondrial toxicity; and we will test that hypothesis by pursuing the following specific aims: 1) synthesize and characterize novel prodrugs for lack of NT inhibition and cellular release of active drug, 2) evaluate the abiliy of prodrugs to protect mitochondria from the toxicity of nucleoside analog drugs, and 3) study biopharmaceutic and pharmacokinetic properties of selected prodrugs and their influence on the pharmacokinetics of nucleoside drugs of interest. A multidisciplinary approach integrating synthetic medicinal chemistry, biochemical and molecular biological assays, laser scanning confocal microscopy, chromatography and tandem mass spectrometry will be employed. The success of the project will confirm the possibility of protecting against nucleoside analog mitochondrial toxicity through the inhibition of nucleoside transporters, as an innovative general preemptive means of protection against toxicities of anti-HIV NRTIs and other nucleoside drugs like the anticancer nucleoside analog gemcitabine, which is associated with rare but potentially fatal liver toxicity and myopathy. Novel research tools will also be developed.

? DESCRIPTION (provided by applicant): Currently, there are no effective strategies or treatments to preserve cognitive function in AD patients. The recent series of disappointing clinical trials highlight the need to explore alternative pathways. Novel compounds that can preserve cognitive function and prevent disease progression in a manner distinct from previous approaches could provide new therapeutic opportunities. To this end, we are developing and testing small molecule compounds designed as allosteric modulators of the ryanodine receptor (RyR), a large conductance calcium channel found on the ER membrane, as candidates for clinical testing in early AD or MCI patients. In both human AD patients and AD mouse models, increased RyR2 expression precedes the amyloid deposition, tau histopathology, neuronal loss, and cognitive impairments. In AD mouse models, increased RyR-evoked calcium release is greatest in dendritic spines and synaptic compartments, and contributes to synaptic pathology and dysfunction, increased amyloid and tau pathology, disrupted memory function, and other AD-defining features. We and others have recently demonstrated that treating AD mice with dantrolene, a RyR channel stabilizer, resulted in exciting therapeutic effects. Although our treatment regimens differed, the consistent results demonstrate normalized calcium signaling (Chakroborty et al., 2012a; Oule et al., 2012; Stutzmann et al., 2006), normal synaptic transmission and plasticity expression (Chakroborty et al., 2012a), restored synaptic structure and integrity (Briggs et al., 2014), reduced A? levels (Chakroborty et al., 2012a; Oule et al., 2012; Peng et al., 2012), restored RyR isoform levels (Chakroborty et al., 2012a; Oule et al., 2012), and improved performance on memory tests (Oule et al., 2012; Peng et al., 2012; Stutzmann lab, unpublished data). These data support a strong case for stabilizing RyR function, with a focus on RyR2, as a novel therapeutic strategy for AD. The objective of this study is to design, test, and optimize compounds that will function as RyR channel negative allosteric modulators, serving to suppress excessive calcium release while maintaining physiological functions. The central hypothesis is that development and optimization of small molecule RyR stabilizers will generate therapeutic leads for clinical testing in early AD and MCI patients, and through the preservation of calcium homeostasis and synaptic function, will protect cognitive abilities. This will be accomplished with the following Aims: 1. Compound development and medicinal chemistry optimization. This will use iterative medicinal chemistry procedures and bioactivity assays in mice. 2. Rapid screening assay in cell culture systems and neurons from AD mice. Initial screening will use automated fluorometric testing of RyR-evoked calcium signals in cultured N2A cells in 96-well plates, followed by screening in primary neurons from control and AD mice. 3. In vivo verification in mouse models. Sub chronic treatment in AD and control mice, followed by physiological and biochemical assays, will then be used to identify and finalize the optimal compounds. The significance to public health is the availability of an effective and novel treatment for AD.