1 APPLICATION FOR PROVOST’S FUND RESEARCH SUPPORT FALL RETREAT, 2014 Lactams as Neuroprotective Agents Monika I. Konaklieva and Victoria Connaughton Departments: Chemistry and Biology Requested funds: Funds are requested to purchase zebrafish, supplies for behavioral observations (experimental containers, light box), glutamate agonists to examine the ability of -lactams, known protective agents, to protect against glutamate-induced toxicity in an animal model and student research assistance. The protective effects will be determined by examining behavioral and physiological (heart rate) changes in exposed larvae. Our results will document these effects in a vertebrate model (zebrafish) and build on existing literature showing neuroprotection in in vitro cell culture studies. Project description Glutamate is the principal excitatory neurotransmitter in the nervous system. Although crucial for normal neuronal excitability, high doses of glutamate in the synapse (often induced by stress) can lead to nervous cell damage and cell death (excitotoxicity). Neuronal damage due to excess of glutamate is associated with a broad spectrum of diseases including Parkinson’s [1, 2], Alzheimer’s [3-6], depressive-like behaviors and possibly major depressive disorder (MDD) [7-10] amyotrophic lateral sclerosis (ALS) [11], brain tumors [12], epilepsy [13], stroke [14], and others. A blinded screening of 1,040 FDA approved drugs and nutritional compounds has demonstrated the utility of lactam antibiotics (penicillins and cephalosporins) in both in vitro and in vivo preparations to protect against excitotoxicity [15]. This neuroprotection is associated with increases in the glial glutamate reuptake protein, GLT1, suggesting that -lactam’s neuroprotective effects is mediated by an increase in glutamate removal from the synapse. Ceftriaxone, a cephalosporin antibiotic, has been used mainly in the recent studies on evaluating the neuroprotective capabilities of -lactam antibiotics against glutamate neurotoxicity since it is the drug widely used for clinical treatment of bacterial meningitis. Ceftriaxone is currently in Phase III clinical trials for ALS [16]. There are challenges, however, to using ceftriaxone as a neuroprotective agent. Ceftriaxone is only available as an intravenous formulation and it is likely that chronic use would promote antibiotic resistance to itself and other -lactam antibiotics. Therefore, developing oral agents that are able to maintain ceftriaxone neuroprotective abilities, preferably without the antibiotic properties, is necessary [17]. We have designed and synthesized a library of novel lipophilic monocyclic lactams lacking the ionizable group that is characteristic for the currently available -lactam antibiotics and necessary for binding to the transpeptidases, which are the cellular target of the penicillins and cephalosporins. Our lactams are neuroprotective against glutamate-induced excitotoxicity in fetal rat cerebellar cells, similar to the action of ceftriaxone, and the presence of the thiophenol moiety in our compounds appears to be necessary for neuroprotective activity. We hypothesize that the sulfur atom of our lactams interacts with an intracellular thiol to form a mixed disulfide, which in turn leads to neuroprotection. The lactam moiety serves as a carrier of the aromatic thiol into the neuronal cell. A one-way ANOVA revealed a significant effect on cell viability when cells were pre-treated with some of our compounds, and then treated with appropriate dose of glutamate, e.g. MK35, F (4,115) = 28.81, p<0.05. Post hoc analyses with Dunnett’s multiple comparison test revealed that pre-treatment with our compounds at concentrations of 250 µM, 500 µM and 1000µM, rescued neurons from glutamate neurotoxicity, compared to untreated controls, p<0.01, with complete protection at the highest concentration. These experiments were performed in cell culture experiments and we would like to test these compounds in an in vivo system. 2 The specific focus of this proposal is to assess the effect of these compounds on a live model organism, the zebrafish, Danio rerio. Zebrafish have been used in various studies determining the effect of small molecules because of their small size, rapid external development, and the ability to administer compounds directly to the fish through the water. The effects of the chemicals can then be determined using a variety of techniques, including visual examination, assays of behavioral changes, measured changes in heart rate, and observations of morphological changes. More specifically, the proposed studies will involve inducing excitotoxicity in zebrafish and assessing the ability of various monocyclic -lactams to abate this damage. Two experiments are proposed. In Experiment 1, zebrafish larvae will be exposed to four different doses of glutamate, or a glutamate agonist, (50, 100, 200, and 500uM), to determine a dose of glutamate effective in inducing excitotoxicity. In Experiment 2, larvae will be pre-treated with either vehicle, penicillin or several of our lactams for 30-60 minutes prior to glutamate exposure. This second study should allow us to see any neuroprotective effect of the compounds (relative to vehicle) on glutamate excitotoxicity. Each experiment will used zebrafish larvae, aged 6-8dpf, with 3-5 larvae per treatment, with 2 replicates per treatment. Excitotoxicity will be assayed through examination of larval behavior, using EthoVision tracking software, changes in heart rate, and overall survival. This work will be significant as it will identify neuroprotective effects of our -lactam compounds in vivo, extending cell culture experiments to a living model organism. If we are able to identify neuroprotective effects of these compounds in this initial screen, subsequent experiments will examine the mechanism of this neuroprotection using physiological techniques targeting specific nerve cells. Our lactams differ from the traditional -lactam antibiotics while still maintaining a narrow cidal activity against phylogenetically unrelated Mycobacterium tuberculosis and Moraxella catharralis. Specifically, they lack an ionizable group at the lactam nitrogen within 3.6 Å of the β-lactam carbonyl carbon, which prevents them from binding to and impairing the bacterial transpeptidases, the molecular target of clinically relevant -lactam antibiotics, such as ceftriaxone. Therefore, their mechanism of action does not include the expected cell wall inhibition and is rather likely associated with coenzyme A and lipid biosynthesis. This difference in the bacterial mechanism of action of our compounds, as well as the fact that the cerebellum lacks GLT1 transporters, leads us to expect the neuroprotective mechanism of our compounds to be unique. Anticipated outcomes This work is a collaboration between Monika Konaklieva (synthesis and knowledge of -lactam compounds) and Vikki Connaughton (development and physiology of zebrafish). Data obtained from this research will not only expand existing research foci in both our laboratories, but also provide preliminary data for further experiments involving graduate and/or undergraduate students and for a grant proposal to extend the demonstrations of neuroprotection as well as to determine its mechanism. References [1]. Hazell, A. S.; Itzhak, Y.; Liu, H.; Norenberg, M. D. J. Neurochem. 1997, 68, 2216. [2]. Berman, S. B.; Hastings, T.G. J. Neurochem. 1997, 69, 1185. [3]. Masliah, E.; Alford, M.; DeTeresa, R.; Mallory, M.; Hansen, L. Ann. Neurol., 1996, 40, 759. [4]. Masliah, E.; Alford, M.; Mallory, M.; Rockenstein, E.; Moechars, D.; Van Leuven, F. Exp. Neurol., 2000, 163, 381. [5]. Masliah, E.; Raber, J.; Alford, M.; Mallory, M.; Mattson, M. P.; Yang, D.; Wong, D.; Mucke, L. J. Biol. Chem. 1998, 273, 12548. [6]. Scott, H. L.; Pow, D. V.; Tannenberg, A. E.; Dodd, P. R. J. Neurosci. 2002, 22, RC206. [7]. Sapolsky, R. M. BiolPsichiatry, 2000, 48, 755. [8]. Lowy, M.T.; Wittenberg, L.; Yamamoto, B. K. J. Neurochem. 1995, 65, 268. [9]. Moghaddam, B. J. Neurochem. 1993, 60, 1650-1657 [10]. Mineur, Y. S.; Picciotto, M. R.; Sanacora, G. BiolPsichiatry, 2007, 61, 250. [11]. Rothstein, J. D.; Van Kammen, M.; Levey, A. I.; Martin, L. J.; Kuncl, R. W. Ann. Neurol. 1995, 38, 73. [12]. Ye, Z. C. ; Rothstein, J. D. ; Sontheimer, H. J. Neurosci. 1999, 19, 10767. [13]. Sepkuty, J. P. ; Cohen. S.; Eccles C.; Rafiq A.; Behar K.; Ganel R.; Coulter D. A.; Rothstein J. D. J. Neurosci. 2002, 22, 6372. 3 [14]. Rao, V.; Dogan A.; Todd K. G.; Bowen K. K.; Kim B. T.; Rothstein J. D.; Dempsey R. J. J. Neurosci. 2001, 21, 1876. [15]. Rothstein, J. D.; Patel, S.; Regan, M. R.; Haenggell, C.; Huang Y. H.; Bergles, D. E.; Jin, L., Hoberg, M. D.; Vidensky, S.; Chung, D. S.; Toan, S. V.; Bruijn, L. I.; Su, Z-z.; Gupta, P.; Fischer, P. B. Nature, 2005, 433, 73. [16]. Berry, J. D.; Shefner, J. M.; Conwit, R.; Schoenfeld, D.; Keroack, M.;Felsenstein, D.; Krivickas, L.; David, W. S.; Vriesendorp, F.; Pestronk, A.; Caress, J. B.; Katz, J.; Simpson, E.; Rosenfeld, J.; Pascuzzi, R.; Glass, J.; Rezania, K.; Rothstein, J. D.; Greenblatt, D. J.; Cudkowicz, M. E. PLOS ONE, 2013, 8, e61177. [17]. Konaklieva, M.I.; Plotkin, B. J.; Herbert, T. Anti-Infective agents in Medicinal Chemistry, 2009, 8, 28-35.