Ion channels as drug targets in central nervous system disorders

Ion channel targeted drugs have always been related with either the central nervous system (CNS), the peripheral nervous system, or the cardiovascular system. Within the CNS, basic indications of drugs are: sleep disorders, anxiety, epilepsy, pain, etc. However, traditional channel blockers have multiple adverse events, mainly due to low specificity of mechanism of action. Lately, novel ion channel subtypes have been discovered, which gives premises to drug discovery process led towards specific channel subtypes. An example is Na+ channels, whose subtypes 1.3 and 1.7-1.9 are responsible for pain, and 1.1 and 1.2 - for epilepsy. Moreover, new drug candidates have been recognized. This review is focusing on ion channels subtypes, which play a significant role in current drug discovery and development process. The knowledge on channel subtypes has developed rapidly, giving new nomenclatures of ion channels. For example, Ca2+ channels are not any more divided to T, L, N, P/Q, and R, but they are described as Cav1.1-Cav3.3, with even newer nomenclature α1A-α1I and α1S. Moreover, new channels such as P2X1-P2X7, as well as TRPA1-TRPV1 have been discovered, giving premises for new types of analgesic drugs

SYNTHESIS AND EVALUATION OF PYRIDINIUM DERIVATIVES AS CENTRAL NERVOUS SYSTEM NICOTINIC ACETYLCHOLINE RECEPTOR LIGANDS

This project utilized synthesis and in vitro assays to generate antagonist SARs at various nAChR subtypes. Alkylation of the pyridino nitrogen of the nicotine molecule afforded subtype specific antagonists at a42* nAChR subtypes and nAChR subtypes that mediate nicotine-evoked dopamine release. Using this data, a series of mono-azaaromatic quaternary salts were produced and evaluated in binding and functional assays for a42* and a7* nAChR subtypes and nAChR subtypes that mediate nicotine-evoked dopamine release. Additionally, bis-azaaromatic quaternary salts were synthesized and evaluated in the same assays. Two potent lead compounds were identified. N-n-dodecylnicotinium iodide (NDDNI) was found to be very potent at both a42* nAChR subtypes and nAChR subtypes that mediate nicotine-evoked dopamine release. And the most promising candidate was N-N-bisdodecylpicolinium dibromide (bDDPiB), which was selective for the nAChR subtypes that mediate nicotine-evoked dopamine release (IC50 = 9 nM). Additionally, using the data from the SARs, predictive computer models were generated to assist in future compound assessment without in vitro assays. Three self-organizing map (SOMs) models were generated from three different sets of compounds. The groups consisted of the mono-substituted compounds, the bissubstituted compounds, and both sets combined. The models were able to successfully bin the test set of compounds after developing a model from a similar set of training compounds. Additionally, using genetic functional activity (GFA) algorithms an evolutionary approach to generating predictive model equations was applied to the compounds. Three separate equations were generated in order to form a predictive method for evaluating affinities at the a4b2* receptor subtype. In addition to the modeling and SAR work of the quaternary ammonium compounds, novel synthetic methods were also employed to develop enantiomerically pure nicotine analogs. Efficient enantioselective syntheses of (S)- and R-(+)-nornicotine, (S)-and R-(+)-anabasine, and (S)-and R-(+)-anatabine have been developed, affording isomers in high enantiomeric excess

Multi-target selection and high throughput quantitative structure-activity relationship model development

Ph.DDOCTOR OF PHILOSOPH

Dopamine Transporter (DAT), Nicotinic Acetylcholine Receptor (nAChR), and Metabotropic Glutamate Receptor 2 (mGlu2) Irreversible Probes For Identifying Anti-Psychostimulant Therapeutics

Numerous in vitro and in vivo studies implicate that certain ligands that interact with DAT, nAChRs, and mGlu2 have tremendous potential as anti-addiction therapeutics. However, understanding how these promising anti-addiction compounds interact with their major drug targets at the molecular level is limited because of the absence of human DAT, nAChRs, and mGlu2 x-ray crystal structures. This knowledge gap is important towards rationally designing new therapeutics for psychostimulant abuse and addiction. The objective of this research was to develop irreversible chemical probes based on promising anti-addiction lead compounds (i.e., pyrovalerone, bupropion, BINA, etc) to map their binding sites and poses within the DAT, select nAChR subtypes, or mGlu2. The central hypothesis was that these compounds could be rationally derivatized, without significant alteration in their pharmacological activity, with a photoreactive group capable of forming a covalent bond to their target protein and a tag for application of a Binding Ensemble Profiling with (f)Photoaffinity Labeling (BEProFL) experimental approach. BEProFL rationally couples photoaffinity labeling, chemical proteomics, and computational molecular modeling to allow structure-function studies of the target proteins. This central hypothesis was tested by pursuing three specific aims: 1.) Identification of non-tropane photoprobes based on pyrovalerone (PV) suitable for DAT structure-function studies, 2.) Identification of bupropion (BP)-based photoprobes suitable for DAT, and nAChR structure-function studies, and 3.) Identification of irreversible mGlu2 PAM ligands as chemical probes suitable for mGlu2 structure-function studies. In the first aim, PV, a non-tropane DAT inhibitor, was structurally modified to contain a photoreactive group (i.e., an aryl azide) and a tag (i.e., 125I). These photoprobes were then pharmacologically evaluated to identify suitable candidates for DAT structure-function studies. In the second aim, BP was structurally modified to contain an aryl azide and 125I. This probe successfully identified the exact location of the bupropion-binding site within the Torpedo nAChR. Under the third aim, biphenyl-carboxylic acid indanone- and pyridone-based mGlu2 PAMs were structurally modified to contain a photoreactive group (e.g., aryl azide, acetophenone) and a tag (e.g., terminal alkyne, aliphatic azide). These compounds, at present, are being subjected to mGlu2 pharmacological evaluation to identify suitable chemical probe candidates for mGlu2 structure-function studies

Spider venoms and chronic pain – developing novel pharmacological tools from the spider venoms to target P2X4 in microglia

Today, one in five adults experience chronic pain and this figure increases for those over 65 years old. However, frustration is mounting over the inadequate treatment for chronic neuropathic pain since its symptoms are challenging to treat and often resistant to opioids. Processing of pain signals relies on the activities of ion channels with the microglial P2X4 receptor being an important player. Animal venoms play an essential role in drug discovery as they contain a rich source of bioactive molecules evolutionarily fine-tuned to target ion channels such as P2X receptors. First, we have established and validated several fluorescent-based high throughput screening assays for assessing the activity of venom toxins at P2X receptors. Second, a diverse selection of 180 crude venoms has been screened against human P2X4 in HEK293 and 1321N21 cells, resulting in several venoms containing inhibitors against hP2X4. Two of them, LK-601 and LK-729, were confirmed to be structurally uncharacterized acylpolyamines, which potently inhibited hP2X4 with the apparent IC50 values between 1.1 – 4.5 μM, however only LK-601 showed a relatively high level of selectivity over hP2X3, hP2X7 and NMDA 1a/2a. Species differences were evident with no effect at rat P2X4, however, blocking the mouse P2X4. Using LK-601 as a structural guide, the fragment-based screening was carried out and five smaller toxin analogues chemically synthesized. One of them, LA-3, was found to block the hP2X4 (IC50 of 9.7 – 18.6 μM) and showed selectivity to hP2X4 over hP2X3, hP2X7 and rP2X4 with a modest inhibition at mP2X4. Due to the differential sensitivity of LA-3 to block P2X4 orthologues, the potential binding site were identified, and the validation showed that two crucial amino acid residues, D220 and N238, might be involved in LA-3 binding to hP2X4; however, more experiments are needed to confirm that effect fully. In summary, we discovered a novel toxin from a spider venom with inhibitory activity at human P2X4 ion channels that shows selectivity at hP2X4 over other P2X receptors. Further characterization and validation are required to understand whether these novel compounds could be useful as analgesics

Part I, Unified Pharmacophore Protein Models of the Benzodiazepine Receptor Subtypes ; Part II, Subtype

Part I. New models of unified pharmacophore/receptors have been constructed guided by the synthesis of subtype selective compounds in light of recent developments both in ligand synthesis and structural studies of the binding site itself. The evaluation of experimental data in combination with comparative models of the α1β2γ2, α2β2γ2, α3β2γ2 and α5β2γ2 GABA(A) receptors has led to an orientation of the pharmacophore model within the benzodiazepine binding site (Bz BS). These results not only are important for the rational design of new selective ligands, but also for the identification and evaluation of possible roles which specific residues may have within the benzodiazepine binding pocket. More importantly, the process summarized here may be used as a general template to help scientists develop novel ligands for receptors for which the three dimensional structure has not yet been confirmed by X-ray crystallography or cryo-electron microscopy. Presented here are new models of the α1β2γ2, α2β2γ2, α3β2γ2 and α5β2γ2 GABA(A) receptors which have incorporated homology models built based on the acetylcholine binding protein. These new models will further our ability to understand structural characteristics of ligands which act as agonists, antagonists, or inverse agonists to the Bz BS of the GABA(A) receptor. This approach will also serve as a powerful model for structure based approaches carried out using ligand-protein docking methods. Part II. An effective strategy to alleviate memory deficits would be to enhance memory and cognitive processes by augmenting the impact of acetylcholine released from cholinergic neurons of the hippocampus. Using the included volume pharmacophore presented in Part I, a number of a5 selective compounds were synthesized, notably PWZ-029. PWZ-029 was examined in rats in the passive and active avoidance, spontaneous locomotor activity, elevated plus maze and grip strength tests which are indicative of the effects on memory acquisition, locomotor activity, anxiety, and muscle tone. Improvement of task learning was shown at a dose of 5mg/kg in passive avoidance test without effect on anxiety or muscle tone. Moderate negative modulation at GABA(A) receptors containing the α5 subunit using a moderate inverse agonist such as PWZ-029, is a sufficient condition for eliciting enhanced encoding/consolidation of declarative memory. Using low temperature NMR and X-ray analysis, it was shown that enhanced selectivity and potent in vitro affinity of α5 selective benzodiazepine dimers was possible with aliphatic linkers of 3 to 5 carbons in length. Although originally proposed to enhance solubility, oxygen-containing linkers caused the dimer to fold back on itself leading to the inability of dimers to enter the binding pocket. In addition, studies of a series of PWZ-029 analogs found that the electrostatic potential near the ligands\u27 terminal substituent correlated with its binding selectivity toward the α5β2γ2 versus α1β2γ2 Bzr/GABA(A) ergic isoform. Investigations further found that compound PWZ-029, which exhibits reasonable binding selectivity toward GABA(A) receptors containing the a5 subunit and possesses a favorable electrophysiological profile, was able to attenuate scopolamine induced contextual memory impairment in mice. This compound appears to be useful (Harris, Delorey et al.) for the treatment of cognitive deficits in rodents as well as primates (Rowlett et al.) and may well be a compound for the treatment of patients with Alzheimers disease

Mechanistic insights and in silico studies on selected G protein-coupled receptors implicated in HIV and neurological disorders.

Doctoral Degree. University of KwaZulu-Natal, Durban.G protein-coupled receptors (GPCRs) are the largest membrane protein receptor superfamily involved in a wide range of physiological processes. GPCRs form the major class of drug targets for a diverse array of pathophysiological conditions. Consequently, GPCRs are recognised as drug targets for the treatment of various diseases, including neurological disorders, cardiovascular conditions, oncology, diabetes, and HIV. The recent advancement in GPCR structure resolutions has provided novel avenues to understand their molecular basis of signal transduction, ligand recognition and ligand-receptor interactions. These advances provide a framework for the structure-based discovery of new drugs in targeting GPCRs implicated in the pathogenesis of various human diseases. In this thesis, the interactions of inhibitors at two dopamine receptor subtypes and C-C chemokine receptor 5 (CCR5) of the Class A GPCR family were investigated. Dopamine receptors and CCR5 are validated GPCR targets implicated in neurological disorders and HIV disease, respectively. The lack of structural information on these receptors limited our comprehension of their antagonists’ structural dynamics and binding mechanisms. The recently solved crystal structures for these receptors have necessitated further investigations in their ligand-receptor interactions to obtain novel insights that may assist drug discovery towards these receptors. This thesis comprehensively investigated the binding profiles of atypical antipsychotics (class I and class II) at the first crystal structure of the D2 dopamine receptor (D2DR). The class I antipsychotics exhibited binding poses and dynamics different from the class II antipsychotics with disparate interaction mechanistic at D2DR active site. The class II antipsychotics were remarkably observed to establish a recurrent and vital interaction with Asp114 via strong hydrogen bond interactions. Furthermore, compared to class I antipsychotics, the class II antipsychotics were found to engage favourably with the deep hydrophobic pocket of D2DR. In addition, the structural basis and atomistic binding mechanistic of the preferential selective inhibition at D3DR over D2DR were explored. This study investigated two small molecules (R-VK4-40 and Y-QA31) with substantial selectivity (> 180-fold) for D3DR over D2DR. The selective antagonists adopted shallow binding modes at D3DR while demonstrating a deep hydrophobic pocket binding at D2DR. Also, the vital roles and contribution of critical residues to the selective binding of R-VK4-40 and Y-QA31were identified in D3DR. Structural and binding free energy analyses further discovered distinct stabilising effects of the selective antagonists on the secondary architecture and binding profiles of D3DR relative to D2DR. Furthermore, the atomistic molecular interaction mechanism of how slight structural modification between novel derivatives of 1-heteroaryl-1,3-propanediamine (Compd-21 and - 34) and Maraviroc significantly affects their binding profiles toward CCR5 were elucidated. This study utilised explicit lipid bilayer molecular dynamics (MD) simulations and advanced analyses to explore these inhibitory disparities. The thiophene moiety substitution common to Compd-21 and -34 was found to enhance their CCR5-inhibitory activities due to complementary high-affinity interactions with residues critical for the gp120 V3 loop binding. The study further highlights the structural modifications that may improve inhibitor competitiveness with the gp120 V3 loop. Finally, structure-based virtual screening of antiviral chemical database was performed to identify potential compounds as HIV-1 entry inhibitors targeting CCR5. The identified compounds made pertinent interactions with CCR5 residues critical for the HIV-1 gp120-V3 loop binding. Their predicted in silico physicochemical and pharmacokinetic descriptors were within the acceptable range for drug-likeness. Further structural optimisations and biochemical testing of the proposed compounds may assist in the discovery of novel HIV-1 therapy. The studies presented in this thesis provide novel mechanistic and in silico perspective on the ligand-receptor interactions of GPCRs. The findings highlighted in this thesis may assist in further research towards the identification of novel drug molecules towards CCR5 and D2-like dopamine receptor subtypes.List of thesis publications on page vi-vii. Research Output on page viii-ix

DESIGN, SYNTHESIS, AND PHARMACOLOGICAL EVALUATION OF A SERIES OF NOVEL, GUANIDINE AND AMIDINE-CONTAINING NEONICOTINOID-LIKE ANALOGS OF NICOTINE: SUBTYPE-SELECTIVE INTERACTIONS AT NEURONAL NICOTINIC-ACETYLCHOLINE RECEPTOR.

The current project examined the ability of a novel series of guandine and amidine-containing nicotine analogs to interact with several native and recombinantlyexpressed mammalian neuronal nicotinic-acetylcholine receptor (nAChR) subtypes. Rational drug design methods and parallel organic synthesis was used to generate a library of guanidine-containing nicotine (NIC) analogs (AH compounds). A smaller series of amidine-containing nicotine analogs (JC compounds) were also synthesized. In total, \u3e150 compounds were examined. Compounds were first assayed for affinity in a high-throughput [3H]epibatidine radioligand-binding screen. Lead compounds were evaluated in subtype-selective binding experiments to probe for affinity at the α4β2* and α7* neuronal nAChRs. Several compounds were identified which possess affinity and selectivity for the α4β2* subtype [AH-132 (Ki=27nm) and JC-3-9 (Ki=11nM)]. Schild analysis of binding suggests a complex one-site binding interaction at the desensitized high-affinity nAChR. Whole-cell functional fluorescence (FLIPR) assays revealed mixed subtype pharmacology. AH-compounds were identified which act as activators and inhibitors at nAChR subtypes, while lead JC-compounds were found which possess full agonist activity at α4β2* and α3β4* subtypes. Compounds were identified as partial agonists, full agonists and inhibitors of multiple nAChR subtypes. Several SAR-based, ligand-receptor pharmacophore models were developed to guide future ligand design. Second-generation lead compounds were identified

Study and Design of Kynurenine Aminotransferase-II Inhibitors for the Treatment of Neurological Conditions

The majority of tryptophan metabolism passes through the kynurenine pathway. Metabolic imbalances in this pathway are implicated disease. KYNA, transaminated by the kynurenine aminotransferase (KAT) enzymes, is elevated in patients with schizophrenia. Schizophrenia is a neuropsychiatric disease with limited treatment options and debilitating symptoms. Glutamatergic systems are thought to have a significant role in its pathogenesis, providing a basis by which KYNA, an endogenous glutamate antagonist, is implicated in the disease. Four pyridoxal 5’-phosphate-dependent homologues of KAT are reported. KAT-II is primarily responsible for KYNA production in the human brain. KAT-II inhibitors reduce KYNA production, increase neurotransmitter release and elicit pro-cognitive effects, indicative of their potential as novel therapies in treating schizophrenia. In this work, surface plasmon resonance has been employed to screen a fragment library, from which two fragments, F6037-0164 and F0037-7280 were pursued (IC50 of 524.5 μM and 115.2 μM, respectively). Another strategy was to consider estrogen compounds as schizophrenia is a sexually dimorphic condition, in which female patients have reduced estrogen levels. Enzyme inhibitory assays displayed estradiol disulfate as a strong inhibitor of KAT-I and KAT-II (IC50: 291.5 μM and 26.3 μM, respectively), with estradiol, estradiol 3-sulfate and estrone sulfate inhibiting weakly. Molecular modelling suggests that the 17-sulfate moiety in estradiol disulfate improves its potency by 10-100 fold compared to estradiol. This 17-sulfate moiety was mimicked on existing KAT-II inhibitor scaffolds to develop two novel inhibitors, JN-01 and JN-02, with improved potencies (IC50: 73.8 μM and 112.8 μM, respectively). Co-crystallisation studies resulted in the determination of a human KAT-II crystal structure (PDB ID: 6D0A) with 1.47 Å resolution, the highest resolution structure provided for KAT-II, with the least structural inconsistencies