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In vitro effects of organophosphorus compounds (OP), such as malathion (M), methyl parathion (MP) and ethyl parathion (EP), on calmodulin (CaM) activity and its active conformation were studied to understand the mechanism(s) of neurotoxicity, since CaM is known to regulate Ca2+ transport and the enzymes involved in signal transduction and nucleotide metabolism. The biological activity of CaM was assessed as a measure of phosphodiesterase (PDE) stimulation. The effect of OP compounds on the active conformation of CaM was determined by studying the binding of fluorescence probes, namely N-phenyl-1-naphthylamine (NPN), and changes in dansyl-calmodulin fluorescence. Dansylated calmodulin was also used to study the effect of OP compounds on complex formation between CaM and PDE. All three OP compounds inhibited the CaM activity and its active conformation in a concentration-dependent manner. Malathion was less effective in comparison to EP and MP, with IC50 values of 37 microM, 34.5 microM and 32 microM, respectively, for CaM activity. EP and MP significantly altered NPN and dansyl-calmodulin fluorescence (50 microM concentrations of OP compounds), whereas M did not show any significant effect on NPN fluorescence. All these compounds significantly affected complex formation between the dansylated CaM and PDE. These results suggest that OP compounds may be interacting with CaM, altering its active conformation, and thus may be inhibiting its biological activity.

Poster Sessions structures and discuss further design strategies for highly selective family 3 glycoside hydrolase inhibitors.Keywords: glycosyl hydrolases, antibiotic resistance, structure-based drug design P04.15.355Acta Cryst. (2008). A64, C342The crystal structure of AKR1C1 in complex with an active-site inhibitor Hydroxysteroid dehydrogenases (HSDs) regulate a wide range of physiological processes including reproduction, development and homeostasis. AKR1C1, is a 20α-HSD involved in the conversion of progesterone to 20-hydroxyprogesterone. Increased activity of AKR1C1 in the endometrium and in breast tissues leads to the formation of tumor-promoting metabolites and to the development of endometriosis, breast cancer and endometrial cancer. At present, there are few known inhibitors that specifically bind and inhibit the adverse actions of AKR1C1. Here we present the first crystal structure of AKR1C1 in complex with potent inhibitor 3,5-dichlorosalicylic acid (IC50 = 44 nM). The crystal structure was solved at a resolution of 1.8 Å, with clear electron density corresponding to the inhibitor bound in the active site. The details of the enzyme-inhibitor interactions and selectivity against members of the AKR1C subfamily will also be discussed. The structural information obtained from this study will help speed up the drug design process for the development of more selective and potent compounds that can be used in the treatment of endometriosis and cancer. Eimeria species are parasitic Apicomplexa protozoans that cause gastrointestinal coccidiosis infections in birds, and are associated with general morbidity and intestinal lesions. These parasitic infections lead to major losses within the broiler industry, a situation that is deteriorating due to emerging resistance to available therapeutics. By analogy with other Apicomplexa obligate parasites, in the intracellular stages of its lifecycle the Eimeria parasite relies heavily on glycolysis for ATP production, and hence on homolactic fermentation -the action of lactate dehydrogenase (LDH) -to restore the NADH/NAD + balance. These parasites are therefore extremely sensitive to LDH inhibition. In common with the LDH of the malaria causing parasite Plasmodium falciparum (PfLDH), Eimeria tenella LDH (EtLDH) has a characteristic five amino acid insert in a loop directly adjacent to the active site. As a consequence, we reasoned that compounds we have previously designed to specifically inhibit PfLDH should cross-react with EtLDH. We are therefore undertaking crystallisation and structural analysis of EtLDH and its inhibitory complexes in order to explore the possibility of targeting EtLDH for novel veterinary therapeutics. HU is an architectural protein for bacterial chromosome compaction and organization.HU is one of the most ubiquitous proteins in the bacterial cell.It is a small basic protein, binding non-specifically throughout the nucleoid. While the sequence of HU is highly conserved through most of the bacterial species, the HU in M.tuberculosis has a sequence...

Calmodulin (CaM), a calcium-binding protein, is found in high concentrations in mammalian brain where it plays a pivotal role in a large number of cellular functions. Protein kinase C (PKC), a multifunctional cytosolic enzyme, in the presence of both Ca2+ and phospholipids, transduce extracellular signals into intracellu-lar events. Both CaM and PKC are partially involved in maintaining Ca2+ homeostasis in the cell. Any fluctuations in the intracel-lular Ca2+ can modulate cellular functions and may contribute to neuronal dysfunction. Hence, the present investigation was initiated to study the effects of some selected penicillium (naturally occurring tremorgenic) mycotoxins like secalonic acid, citreoviridin, and verruculogen on CaM activity, active conformation of CaM and PKC activity. Stimulation of CaM-deflcient bovine brain 3′-5′ phosphodieste rase (PDE) indicated CaM activity. The modification of CaM active conformation was studied by the binding of fluorescent probe N-phenyl-1-napthylamine (NPN) to CaM. Alterations in the fluorescence of dansyl-CaM was used to study the effect of these compounds on complex formation between CaM and PDE. Rat brain cytosolic PKC was studied using 32P-ATP as a measure of altered protein phosphorylation. The concentrations of mycotoxins used were in the range of 10 to 50 μM. All three mycotoxins inhibited CaM-stimulated PDE activity in a concentration-dependent manner. Citreoviridin and secalonic acid inhibited NPN fluorescence and Ca2+-dependent complex formation of dansyl-CaM and PDE. The IC50 values for NPN fluorescence of citreoviridin and secalonic acid were 13 μM and 19 μM respectively. However, verruculogen showed little effect on NPN fluorescence and the Ca2+-dependent complex formation of dansyl-CaM and PDE. These mycotoxins also inhibited PKC activity in a concentration-dependent manner with IC50 values of 19.8, 25.7, and 38.4 μM for secalonic acid, citreoviridin, and verruculogen, respectively. The results of our study suggest that these mycotoxins at very low concentrations are interacting with CaM and PKC. Such an effect could lead to impairment of neurotransmission and result in neurotoxicity.