Cyclooxygenase-1 as an anti-stroke target: potential inhibitor identification and non-synonymous single nucleotide polymorphism analysis

Stroke is the third leading cause of death worldwide, with 87% of cases being ischemic stroke. The two primary therapeutic strategies to reduce post-ischemic brain damage are cellular and vascular approaches. The vascular strategy aims to rapidly re-open obstructed blood vessels, while the cellular approach aims to interfere with the signalling pathways that facilitate neuron damage and death. Unfortunately, popular vascular treatments have adverse side effects, necessitating the need for alternative chemotherapeutics. In this study, cyclooxygenase-1 (COX-1), which plays a significant role in the post- ischemic neuroinflammation and neuronal death, was targeted for identification of novel drug compounds and to assess the effect of nsSNPs on its structure and function. In a drug discovery part, ligands from the South African Natural Compounds Database (SANCDB-https://sancdb.rubi.ru.ac.za/) and ZINC database (http://zinc15.docking.org/) were used for high-throughput virtual screening (HVTS) against COX-1. Additionally, five nsSNPs were being investigated to assess their impact on protein structure and function. Three of these SNPs were in the COX-1 dimer interface. Molecular docking and molecular dynamics simulations revealed asymmetric nature of the protein. Several ligands, peculiar to each monomer, exhibited favourable binding energies in the respective active sites. SNP analysis indicated effects on inter-monomer interactions and protein stability

Cyclooxygenase-1 as an anti-stroke target: potential inhibitor identification and non-synonymous single nucleotide polymorphism analysis

Stroke is the third leading cause of death worldwide, with 87% of cases being ischemic stroke. The two primary therapeutic strategies to reduce post-ischemic brain damage are cellular and vascular approaches. The vascular strategy aims to rapidly re-open obstructed blood vessels, while the cellular approach aims to interfere with the signalling pathways that facilitate neuron damage and death. Unfortunately, popular vascular treatments have adverse side effects, necessitating the need for alternative chemotherapeutics. In this study, cyclooxygenase-1 (COX-1), which plays a significant role in the post- ischemic neuroinflammation and neuronal death, was targeted for identification of novel drug compounds and to assess the effect of nsSNPs on its structure and function. In a drug discovery part, ligands from the South African Natural Compounds Database (SANCDB-https://sancdb.rubi.ru.ac.za/) and ZINC database (http://zinc15.docking.org/) were used for high-throughput virtual screening (HVTS) against COX-1. Additionally, five nsSNPs were being investigated to assess their impact on protein structure and function. Three of these SNPs were in the COX-1 dimer interface. Molecular docking and molecular dynamics simulations revealed asymmetric nature of the protein. Several ligands, peculiar to each monomer, exhibited favourable binding energies in the respective active sites. SNP analysis indicated effects on inter-monomer interactions and protein stability

THE ROLE OF CONFORMATIONAL DYNAMICS IN ISOCYANIDE HYDRATASE CATALYSIS

Post-translational modification of cysteine residues can regulate protein function and is essential for catalysis by cysteine-dependent enzymes. Covalent modifications neutralize charge on the reactive cysteine thiolate anion and thus alter the active site electrostatic environment. Although a vast number of enzymes rely on cysteine modification for function, precisely how altered structural and electrostatic states of cysteine affect protein dynamics, which in turn, affects catalysis, remains poorly understood. Here we use X-ray crystallography, computer simulations, site directed mutagenesis and enzyme kinetics to characterize how covalent modification of the active site cysteine residue in the enzyme, isocyanide hydratase (ICH), affects the protein conformational ensemble during catalysis. Our results suggest that cysteine modification may be a common and likely underreported means for regulating protein conformational dynamics. This thesis will also include ongoing work with ICH homologs, showing that Cys covalent modification-gated helical dynamics in Cys dependent enzymes is common to enzymes of this family. Advisor: Dr. Mark. A. Wilso

Publications of the Jet Propulsion Laboratory July 1965 through July 1966

Bibliography on Jet Propulsion Laboratory technical reports and memorandums, space programs summary, astronautics information, and literature searche

Mechanisms of Feedback in the Visual System

Feedback is an ubiquitous feature of neural systems though there is little consensus on the roles of mechanisms involved with feedback. We set up an in vivo preparation to study and characterize an accessible and isolated feedback loop within the visual system of the leopard frog, Rana pipiens. We recorded extracellularly within the nucleus isthmi, a nucleus providing direct topographic feedback to the optic tectum, a nucleus that receives the vast majority of retinal output. The optic tectum and nucleus isthmi of the amphibian are homologous structures to the superior colliculus and parabigeminal nucleus in mammals, respectively. We formulated a novel threshold for detecting neuronal spikes within a low signal-to-noise environment, as exists in the nucleus isthmi due to its high density of small neuronal cell bodies. Combining this threshold with a recently developed spike sorting procedure enabled us to extract simultaneous recordings from up to 7 neurons at a time from a single extracellular electrode. We then stimulated the frog using computer driven dynamic spatiotemporal visual stimuli to characterize the responses of the nucleus isthmi neurons. We found that the responses display surprisingly long time courses to simple visual stimuli. Furthermore, we found that when stimulated with complex contextual stimuli the response of the nucleus isthmi is quite counter-intuitive. When a stimulus is presented outside of the classical receptive field along with a stimulus within the receptive field, the response is actually higher than the response to just a stimulus within the classical receptive field. Finally, we compared the responses of all of the simultaneously recorded neurons and, together with data from in vitro experiments within the nucleus isthmi, conclude that the nucleus isthmi of the frog is composed of just one electrophysiological population of cells

A Peptide-Based Platform for Displaying Antibodies to Engage T Cells

This study investigated a strategy by which antibodies were displayed on a gel-like substance to engage T cells. The substance, a peptidic composite, was characterized in vitro and explored as an injectable system in vivo. The composite consists of two amphiphilic peptides, AEAEAKAKAEAEAKAK (referred to as EAK ) and AEAEAKAKAEAEAKAKHHHHHH ( EAKH6 ). Spectroscopic analysis showed the two peptides integrated into a single structure. Prior to combination, conformational analysis revealed that EAKH6 adopts a mixed alpha-helix/bata-strand conformation. In the presence of EAK, EAKH6 exists predominantly in a beta;-strand conformation. Using nickel-bound horseradish peroxidase as a probe, the composite of EAK-EAKH6 was found to display His-tags. T-cell-specific antibodies were found stably displayed on the EAK-EAKH6 assembly using recombinant protein A/G and anti-hexahistidine antibody as an adaptor. When mounted with an anti-CD4 antibody, the system was shown to capture CD4 T cells in a mixed population of lymphocytes. Antibodies were concentrated in the subcutaneous space in mice when co-administered with EAK and EAKH6 along with protein A/G and anti-hexahistidine antibody as an aqueous (deionized water

Mapping Functionally Important and Stabilising Regions in Biotherapeutic Proteins, using NMR and Mutagenesis

Structure-function relationships in proteins refer to a trade-off between stability and bioactivity, moulded by evolution of the molecule. Identifying which protein amino acid residues jeopardise global or local stability for the benefit of bioactivity would reveal residues pivotal to this structure-function trade-off. Demonstrated here is the use of varied-temperature 15N-1H heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy to probe the microenvironment and dynamics of residues in granulocyte-colony stimulating factor (G-CSF). This experimental approach was also used to investigate (de-) stabilising mechanisms of action for previously studied excipients with G-CSF. Combining NMR with in silico analysis revealed four structural clusters that are subject to localised conformational changes (some of which are key to bioactivity) or partial unfolding prior to global unfolding at higher temperatures. Mechanisms by which excipients influence these important structural changes and implement their own structural clusters reflects their impact on stability and function. This approach was leveraged for semi-rational mutant/formulation design. These mutants were tested for fitness with respect to thermostability and functionality. The Mutants P65V and E45Q were constructed to elicit mutation-excipient interactions, and presented the largest impact on the respective fitness. Hence, this study proposes an approach to profile residues, thus highlighting their roles in stability and bioactivity while exposing potential mutation-excipient interactions. This permits a semi-rational protein engineering approach to optimise desirable protein fitness characteristics

Functional Response of Neutrophils in the Presence of LPS: Aspects of Locomotion and Metabolism in Normal and Diabetic Cells

This thesis reports a study of effects of bacterial lipopolysaccharides (LPS) on morphological polarization, locomotion and chemiluminescence of human neutrophi. leukocytes (polymorphonuclear leukocytes:PMN). Change from a spherical to a polarized morphology by a cell is the initial event in cell locomotion. PMN in suspension were shown to change shape even in the presence of small amounts of LPS (≥100 ng/ml) in the medium. Exposing PMN to LPS from rough strains of bacteria resulted in slow shape change (60-90 minutes), unlike chemotactic factors which cause shape change within a few minutes. This suggests an indirect effect. Rough strains were more active than the smooth chemotypes in inducing polarization of PMN. The number of polarized cells increased as the period of incubation increased implying the possible presence of a second (non-LPS) agonist that promoted further polarizing activity. Polymyxin B sulfate (10mug/ml) was added to the cells prior to challenging with LPS (10mug/ml) in order to inhibit this biological effect. However, polymyxin in this system did not reduce the number of polarized PMN to a statistically significant extent. To test if a chemotactic-type factor might have been released by the cells, supernatant was prepared from PMN (10e6 cells/ml) which had been incubated with LPS for 90 minutes. The supernatant (but not LPS alone) was shown to cause the shape change within 10 minutes in a shortterm polarization assay on a new batch of PMN. By reducing the cell density in the presence of constant amounts of LPS (10 mug/ml), it could be shown that polarizing activity of the supernatant was reduced. To assess the relationship of shape change to that of locomotory ability, three additional leukocyte locomotion assays were employed. With a micropore filter assay, migration of the cells in different concentration gradients of the factor showed that the released material behaved like a chemotactic factor as analyzed by the checkerboard system. A collagen gel assay was used to study detailed cell locomotion quantitatively (population of cells migrating) and also qualitatively (morphological changes during locomotion). Detailed behavioural analysis of the PMN response to LPS was obtained from visual studies. Quantitative measurements of shape change showed a slowly developing increase in elongation of the cells exposed to LPS for up to 90 minutes, in contrast to rapid elongation of cells exposed to the chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP). One of the ways a neutrophil might release a stimulant is through arachidonic acid metabolism. Steroidal and non-steroidal antiinflammatory drugs used to modulate the shape change process of the leukocytes displayed varied effects. Two drugs, with reported inhibitory effects on lipoxygenase activity, BW755C and Revlon 5901A reduced the number of PMN polarized by LPS. Conversely, neither indomethacin nor dexamethasone had any effect. Preliminary characterization of the cell released factor was also conducted. The factor was heat stable and non-dialyzable. Hexane extraction, thin layer chromatography and column gel filtration suggested that the released material was heterogenous. A low molecular weight factor (about 2000 kD) was identified by gel filtration on Sephadex G-25. Monoclonal antibodies against the CD11/CD18 family of cell surface receptors (shown by other researchers to be responsible for the binding of LPS to PMN) were used as inhibitors of LPS binding to PMN. Some antibodies were relatively effective in inhibiting LPS-induced PMN polarization particularly those against CD11b/CD18. Combinations of monoclonals against three antigens (CD11a, b and c) were more effective than any one alone. The study using these antibodies unexpectedly showed that the binding site of LPS to the phagocyte is highly affected by antibodies to the alpha-chain and not the beta-chain as reported by previous investigators. Competitive inhibitory binding of LPS to PMN was observed with synthetic peptides containing an RGD-sequence. The observation points to a probable involvement of the sequence in LPS-induced neutrophil polarization. Chemiluminescence was used an additional test of LPS activation of PMN. On addition of LPS (10 mug/ml) , slow but progressive light emission was noted. Normal peaking time ranged from 30 to 60 minutes with different intensities from different donors. Addition of platelet activating factor (PAF) as a primer did not enhance the response but instead slightly depressed the activity. LPS on its own can also act as a priming agent