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Poisoning by organophosphate cholinesterase inhibitors results in a rapid rise in acetylcholine (ACh) in the synapse and many pronounced pharmacological effects in numerous tissues in the body. The treatment for organophosphorus poisoning involves administration of a cholinesterase—reactivating oxime to restore the activity of the cholinesterase and an antimuscarinic agent to block the response to the excess ACh. Presently atropine is the standard antimuscarinic agent used clinically as an antidote. This research was directed toward finding an antimuscarinic agent better than atropine as an antidote. Since caramiphen and aprophen have been reported to be effective antidotes we synthesized numerous structural variations of these molecules with the aim of enhancing the antimuscarinic and antidotal properties. Many of these compounds showed enhanced antimuscarinic properties. We synthesized analogs of caramiphen which have different substituents in the para position of the phenyl ring. The purpose of the set was to test the effect of different substituents on the antimuscarinic and antidotal activity and to look for any possible relationship of activity with substituent parameters such as Hammett’s sigma (σ) or Hansch’s pi (π) values. Four substituents were selected which have extreme values for sigma and pi in a positive or negative direction, in all combinations. The substituents chosen for use in this approach were the amino (-σ, -π); 1-‘ tetrazolyl (+σ, -π); l-pyrrolidinyl (-σ, +π); and the trifluoromethyl (+σ, +π). Some N-substituted-1,6-hexanediamines were synthesized to examine the SAR for antimuscarinic and antidotal properties of this series. In a binding assay these compounds showed moderate affinity with a preference for the M2 receptor subtype. It was determined the bis-quaternary structure was not mandatory for muscarinic activity. The optimum compound of this series was N,N’-dimethyl-N,N’-bis[3-(2- phthalimido)propyl]-1,6-hexanediamine. Also, a number of literature and currently manufactured antimuscarinics were obtained. All of the compounds are being screened in a number of in vitro and in vivo assays designed to give information on the SAR for the pharmacological properties which might be important as an antidote. Current information from this research indicates the best antidotes are not the best antimuscarinics in the pharmacological assays. Good central antimuscarinic activity is the primary property of a good antidote, although the antidotal effectiveness of an agent can not be described solely based on its antimuscarinic properties.

It is a fact that cigarette smoke (CS) has negative effects on patients with Crohn's disease (CD), whereas CS seems to provide protection to patients with Ulcerative Colitis (UC). The mechanism of how CS ingredients or nicotine modulate inflammatory phenotypic response in IBD subsets remained unclear. Unlike UC, CD has been associated with genetic disposition, immuno-dysregulation and infection by pathogens mainly Mycobacterium avium paratuberculosis (MAP). In this study, we investigated the cellular and molecular effects of pure nicotine and tobacco extracts from HLE-nicotine rich-plant and LAMD-nicotine less-plant in infected macrophages. Unlike LAMD extracts, Nicotine (4ug/mL) and HLE extracts (0.18%) significantly favored M2 polarization and phenotypic response. While macrophages infected with MAP or treated with LPS promoted pro-inflammatory response, treatment of infected macrophages with nicotine/HLE extracts further elevated pro-inflammatory response, and enhanced MAP burden. Pre-conditioning macrophages with nicotine or blocking alpha-7nAChR with antagonist reversed the effect of nicotine in infected macrophages. We demonstrated that MAP infection in macrophages was mediated through TLR2/MyD88 signaling; blocking TLR2 and TLR4 with antagonists significantly reduced the inflammatory effect of MAP and LPS. Interestingly, nicotine in infected macrophages significantly downregulated TLR2/TLR4 expression, activated MyD88, and increased expression of pro-inflammatory cytokines. Surprisingly, dual treatment of MAP-infected macrophages with MyD88 antagonist and nicotine absolutely impaired immune response during infection. The data shows a role for TLR2/MyD88 signaling in elevated inflammatory response during infection in CD smokers. Overall, we conclude that in absence of infection in UC, nicotine activates the cholinergic anti-inflammatory pathway through alpha-7nAChRs. However, in CD smokers with MAP infection, nicotine action is mediated through TLR2/MyD88 signaling leading to significant inflammatory response. The study is the first to unmask the mechanism involved in the contradictory effect of CS in IBD populations.

No description available.

Smart drug-design for antibody and nanomaterial-based therapies allows for optimization of drug efficacy and more efficient early-stage pre-clinical trials. The ideal drug must display maximum efficacy at target tissue sites, but to track and predict distribution to these sites, one must have a mechanistic understanding of the kinetics involved with the individual cells of the tissue itself. This process can be tracked through biological simulations coupled with in-vitro approaches, which result in a rapid and efficient in-depth understanding of drug transport within tissue vasculature and cellular environment. As a result, it becomes possible to predict drug biodistribution within live animal tissue cells without the need for animal studies. Herein, we use in-vitro assays to translate transport kinetics to whole-body animal simulations to predict drug distribution from vasculature into individual tissue cells for the first time. Our approach is based on rate constants obtained from an in-vitro assay that accounts for cell-induced degradation, which are translated to a complete animal simulation to predict nanomedicine biodistribution at the single cell level. This approach delivers predictions for therapies of varying size and type for multiple species of animals solely from in-vitro data. Thus, we expect this work to assist in refining, reducing, and replacing animal testing, while at the same time, giving scientists a new perspective during early stages of drug development.

The physicochemical features that affect cell-membrane permeability of bacteria are currently not well understood. Due to the lack of tools to predict penetration of the bacterial envelope, the discovery and development of novel antibiotics is at a standstill. For this reason, the increasing spread of resistant bacteria has become a major threat to public health because these pathogens remain unchallenged. To understand how intrinsic properties, such as porins and efflux pumps, of Gram-negative bacteria influence membrane permeability and consequently, antibiotic uptake, we have developed a systematic approach to evaluate the penetration of various compounds into several strains of Escherichia coli. We study Wild Type, a porin (ompR) knockout, and an efflux pump (tolC) knockout to measure the effects that these physicochemcial properties have on antibacterial uptake. Minimum inhibitory concentrations are calculated for eighteen different compounds when tested against these three strains of E. coli. Next, compound accumulation is assessed through LC-MS/MS analysis and killing kinetics are observed. Through this, a relationship between chemical structure and bacterial membrane permeability can be identified, thus a better understanding of the roles of porins and efflux pumps in the development of antimicrobial resistance is gained.

The peroxisome proliferator-activated receptors (PPARs) are the transcriptional regulators of glucose, lipids and cholesterol metabolisms. It has been established that PPAR-γ is the receptor for thiazolidinediones (TZDs) class of type II anti-diabetic drugs. These compounds act as agonists of PPAR-γ. They may delay the development of type II diabetes in individuals at high risk of developing the condition, and have been shown to have potentially beneficial effects on cardiovascular risk factors. PPAR-γ receptor activation by TZDs improves insulin sensitivity by promoting fatty acid uptake into adipose tissue, increasing production of adiponectin (responsible for glucose regulation and fatty acid metabolism) and reducing levels of inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha), plasminogen activator inhibitor-1(PAI-1) and interleukin-6 (IL-6). Our goal is to take advantage of the mode of binding of known PPAR-γ agonists, such as Rosiglitazone to PPAR-γ to rationally design novel agonists of PPAR-γ. Our long-term objective is to generate new and potent PPAR-γ agonists that could be used to treat diabetes. To achieve our goal the study was divided into five specific aims, including: Aim 1. Expression and purification of PPAR-γ ligand binding domain (LBD). Aim 2. Molecular modeling to design PPAR-γ receptor modulators. Aim 3. Synthesis of potential PPAR-γ receptor modulators. Aim 4. Functional studies to determine the binding affinity of PPAR-γ receptor modulators. Aim 5. Structural studies of PPAR-γ LBD in complex with PPAR-γ receptor modulators. We expressed the His-tagged PPAR-γ LBD protein in Rosetta DE3 cells, and used a one step affinity chromatography (Ni-NTA column) to obtain a significant yield of pure protein. Using the structural features and the known binding mode of Rosiglitazone to PPAR-γ LBD as a starting point, two classes of compounds (type-I and type-II compounds) were designed as potential PPAR-γ agonists. These novel compounds were rationalized to improve on the binding modes of Rosiglitazone via additional hydrogen-bonding and/or hydrophobic interactions to the protein. Five type-I and II compounds were synthesized and tested against PPAR-γ receptor for binding affinity, using fluorescent polarization assay. The IC-50 value of the most potent compound (compound B) was found to be ~ 7-fold lower than Rosiglitazone, significantly lower than the expected value. It seems that unlike Rosiglitazone which has free rotatable thiazolidinedione ring that can make optimal interactions with His323 and His449 (two critical residues that are important for binding affinity), the thiazolidinedione ring in our compounds are fixed in one position that may not lead to optimal contact with the protein. We are currently synthesizing analogs of our compounds with rotatable thiazolidinedione ring for further studies. X-ray crystallographic study has been initiated to determine the binding modes of our compounds with PPAR-γ LBD which would allow for structural modifications for improving existing interactions and/or formation of new favorable interactions that could lead to higher affinity and potency. We have also initiated testing of these compounds to determine their PPAR-γ agonistic effects.

The serotonin (5-HT) receptors, with seven subtypes and at least fifteen distinct members, mediate a wide range of physiological functions both in the central nervous system and in the periphery. All members of the 5-HT family except the 5-HT3 subtype belong to the family of aminergic G protein-coupled receptors (GPCRs). Over the years, various molecules have been reported which act selectively at 5-HT2 receptors. However, there are no ligands that exhibit complete selectivity for one subpopulation of 5-HT2 receptors. Insight into how drugs bind to 5-HT2 receptors could contribute significantly to the development of subtype-selective agents with enhanced therapeutic effects. We have begun to address this challenge by the combined approach of chemical synthesis and molecular modeling. 9-(Aminomethyl)-9,10-dihydroanthracene (AMDA) a novel, selective 5-HT2 antagonist that also has modest affinity for the histamine (H1) receptor has been reported by Westkaemper et al. A structure-affinity relationships (SAFIR) study of AMDA and its analogs was carried out by studying the effects of N-alkylation, variation of the amine-ring system linker chain length and constraint of the aromatic rings on the binding affinities of the compounds for the 5-HT2A and H1 receptors. The results of the docking studies carried out on the homology models of 5-HT2A and H1 receptors were consistent with the observed binding affinity data for both receptors. In order to explore the additional binding site interactions of 5-HT2A receptor, synthesis and testing of the ring-annulated analogs of AMDA were carried out. A 3-methoxytetraphen analog of AMDA (26) showed high affinity (Ki = 21 nM) and selectivity (126-fold) for 5-HT2A receptor as compared to H1 receptor (Ki = 2640 nM). Further, to test the utility of our homology models, and investigate the binding site specific interaction, a compound was synthesized and tested that lacks a basic amine and contains an acidic functionality designed specifically to interact with lysine K1915.39 found in H1 but not in 5-HT2A receptor. This compound would thus be both H1-selective and demonstrate that a basic amine-D3.32 interaction is not necessary for high affinity. The synthesized compound (34) lacking the nitrogen atom showed moderate affinity at the H1 receptor (Ki = 250 nM), and lacked affinity for 5-HT2A receptors. The modeled ligand orientations in combination with the observed affinity data provide another example of a successful structure-based design strategy.

Pyridoxal kinase (PL kinase) and pyridoxine 5’-phosphate oxidase (PNP oxidase) are the two vitamin B6 salvage enzymes involved in metabolism of the primary inactive vitamin B6 (pyridoxal, pyridoxine and pyridoxamine) into the active cofactor form, pyridoxal 5’-phosphate (PLP). PLP, arguably the most important vitamin, is required by numerous vitamin B6 (PLP-dependent) enzymes as a co-factor. These enzymes serve vital roles in the metabolism of glucose, lipids, amino acids, heme, DNA/RNA and many neurotransmitters. High levels of vitamin B6 are linked to neurotoxicity, due to the non-specific interactions of PLP with non-B6 proteins. This problem is controlled, in part, by maintaining a low in vivo concentration of free PLP (~1 μM); raising the intriguing question of how the cell regulates, as well as, supplies sufficient PLP to meet the requirements of B6 enzymes. Similar to PLP excess, PLP deficiency, due to mutations in PL kinase and PNP oxidase or drug-induced inhibition of their activity, has been implicated in many pathological conditions. The objective of this study is to elucidate the mechanisms underlying PLP regulation by PL kinase, and its subsequent transfer to dozens of PLP-dependent enzymes. A second objective is to gain valuable information into whether a missense mutation (S261F) in PL kinase could affect the enzyme activity and/or structure. A third objective is to understand how vitamin B6 metabolism by PL kinase is disrupted by the neurotoxic compound, ginkgotoxin. The mutant (hPL kinase S261F) was obtained using site-directed mutagenesis. It was then expressed, purified and analyzed by circular dichroism, fluorescence spectroscopy, enzyme kinetics and native-PAGE. Our results showed no considerable differences between wild-type enzyme and the mutant, suggesting the mutation to be non-pathogenic. PLP was found to inhibit PL kinase by binding to the substrate PL site in the presence of substrate MgATP to form an abortive ternary complex (PL kinase-PLP-MgATP). The physiological significance of this ternary complex was also analyzed and it was found to be a source of PLP transfer to apo B6 enzymes. Enzyme kinetics, affinity chromatography and fluorescence polarization techniques were used to test our hypothesis that the reactive PLP is transferred from PL kinase to apo-B6 enzymes via channeling. Channeling should provide an efficient and protected way for PLP transfer from the kinase or oxidase to apo-B6 enzymes. Our results provide a strong support to the channeling mechanism. Ginkgotoxin was found to be a competitive inhibitor of PL kinase with a Ki of 18 μM. X-ray crystallographic analysis of its binding mode to PL kinase confirmed its binding to the substrate PL site of the enzyme. A unique hydrophobic interaction between its lipophilic side chain 4’-OCH3 and nearby Tyr127 and Val231, in addition to the conserved PL binding interactions, was found to be responsible for its higher affinity to the enzyme.

Deficiency of vitamin B6 due to mutations in key B6 metabolizing enzymes is suspected to contribute to several pathologies. Vitamin B6 in its active form, pyridoxal 5’-phosphate (PLP) is a cofactor for over 140 known B6 requiring (or PLP-dependent) enzymes, that serve vital roles in many biochemical reactions. There are three primary vitamin B6 forms, pyridoxine (PN), pyridoxamine (PM) and pyridoxal (PL) which are phosphorylated to pyridoxine 5’-phosphate (PNP), pyridoxamine 5’-phosphate (PMP) and PLP respectively. Pyridoxal kinase (PLK) and pyridoxine 5’-phosphate oxidase (PNPO) are the key enzymes involved in both salvage and de novo pathways of PLP biosynthesis. Mutations in these enzymes are one of the most important causes of PLP deficiency, apart from dietary insufficiency of vitamin B6 and drug inhibition of PLK and PNPO. One of our objectives is to understand the molecular basis of reduced catalytic activity of PNPO in case of the R95C homozygous missense natural mutant, which leads to the PLP deficiency and the debilitating disease, neonatal epilepsy encephalopathy. Using site-directed mutagenesis, circular dichroism, enzyme kinetics and fluorescence spectroscopy, we have shown that the reduced enzymatic activity exhibited by PNPO R95C mutant is due to reduced binding affinity of the oxidase cofactor, flavin mononucleotide (FMN), which is required by the enzyme for oxidizing the inactive B6 vitamers into the active PLP. High concentrations of B6 are linked to neurotoxic effects, which can be attributed to the highly reactive aldehyde group of PLP which reacts with many nucleophiles in the cell. This reactivity is most likely why the in vivo concentration of “free” PLP is about 1 μM, raising the intriguing question of how the cell supplies sufficient PLP to meet the requirements of the numerous B6 dependent enzymes. Our second objective is to determine how despite the low in vivo concentration of free PLP, enough of this co-factor is made available to activate PLP-dependent enzymes. We have used affinity pull down assays, fluorescence polarization and enzyme kinetics to show that PNPO forms specific interactions with B6 enzymes with dissociation constants less than 1 µM. We also show that transfer of PLP from PNPO possibly occurs by compartimentalization or channeling. Although, channeling is a controversial subject, it offers an efficient, exclusive, and protected means of delivery of the highly reactive PLP. High concentrations of B6 are linked to neurotoxic effects, which can be attributed to the highly reactive aldehyde group of PLP which reacts with many nucleophiles in the cell. This reactivity is most likely why the in vivo concentration of “free” PLP is about 1 ?M, raising the intriguing question of how the cell supplies sufficient PLP to meet the requirements of the numerous B6 dependent enzymes. Our second objective is to determine how despite the low in vivo concentration of free PLP, enough of this co-factor is made available to activate PLP-dependent enzymes. We have used affinity pull down assays, fluorescence polarization and enzyme kinetics to show that PNPO forms specific interactions with B6 enzymes with dissociation constants less than 1 µM. We also show that transfer of PLP from PNPO possibly occurs by compartimentalization or channeling. Although, channeling is a controversial subject, it offers an efficient, exclusive, and protected means of delivery of the highly reactive PLP.