Synthetic trehalose glycolipids (TGLs) that modulate the Macrophage Inducible C-Type Lectin receptor (Mincle) are attractive targets for adjuvant development. However, natural ligands such as trehalose dimycolate (TDM), as well as existing synthetic mimetics, are limited by poor physicochemical properties and reactogenicity, motivating the development of rationally designed synthetic TGLs. Herein, we investigated the biophysical properties of amphiphilic TGLs featuring hydrophobic, aromatic moieties attached to the polar trehalose headgroup via ester, amide, or triazole linkages. The formation of homogeneous and colloidally stable aqueous nanodispersions was readily achieved through focused ultrasonication, eliminating the need for cosolvents and/or surfactants that might interfere with the self-assembling and biological properties of these compounds. These self-assembled particles were characterized using DLS, cryo-TEM, and synchrotron small-angle X-ray scattering to gain insight into their particle morphology and liquid crystalline phase behavior. Additionally, molecular dynamics (MD) simulations were conducted to evaluate the critical packing parameter (CPP), a predictive descriptor of the supramolecular structures formed by self-assembling amphiphiles, under conditions that closely mimic experimental environments. The synthetic Mincle ligands formed lyotropic liquid crystals, including cubosomal or hexagonal phases as well as lamellar phases. In vitro and in vivo testing of these self-assemblies revealed that all tested TGLs exhibited measurable immunological activity. Analogues forming cubic phases were associated with comparatively stronger cytokine production and enhanced antigen-specific humoral and cell-mediated immune responses within this compound set. Taken together, the data support a model in which both molecular structure and supramolecular organization contribute to immunostimulatory activity, although these effects cannot be fully decoupled in the present study. Additionally, we developed an in silico model capable of predicting supramolecular assembly, providing insight into the self-assembly tendencies of trehalose-based amphiphiles, and offering broader relevance to other biologically active, self-assembling systems.

NMDA-type ionotropic glutamate receptors are critical for normal brain function and are implicated in central nervous system disorders. Structure and function of NMDA receptors composed of GluN1 and GluN3 subunits are less understood compared to those composed of GluN1 and GluN2 subunits. GluN1/3 receptors display unusual activation properties in which binding of glycine to GluN1 elicits strong desensitization, while glycine binding to GluN3 alone is sufficient for activation. Here, we explore mechanisms by which GluN1-selective competitive antagonists, CGP-78608 and L-689,560, potentiate GluN1/3A and GluN1/3B receptors by preventing glycine binding to GluN1. We show that both CGP-78608 and L-689,560 prevent desensitization of GluN1/3 receptors, but CGP-78608-bound receptors display higher glycine potency and efficacy at GluN3 subunits compared to L-689,560-bound receptors. Furthermore, we demonstrate that L-689,560 is a potent antagonist of GluN1(FA+TL)/3A receptors, which are mutated to abolish glycine binding to GluN1, and that this inhibition is mediated by a non-competitive mechanism involving binding to the mutated GluN1 agonist binding domain (ABD) to negatively modulate glycine potency at GluN3A. Molecular dynamics simulations reveal that CGP-78608 and L-689,560 binding or mutations in the GluN1 glycine binding site promote distinct conformations of the GluN1 ABD, suggesting that the GluN1 ABD conformation influences agonist potency and efficacy at GluN3 subunits. These results uncover the mechanism that enables activation of native GluN1/3A receptors by application of glycine in the presence of CGP-78608, but not L-689,560, and demonstrate strong intra-subunit allosteric interactions in GluN1/3 receptors that may be relevant to neuronal signaling in brain function and disease.

The identification of Mincle as the C-type lectin receptor on innate immune cells responsible for binding TDM and the realization that this receptor could be key to productive vaccines for mycobacterial infection has raised interest in the development of synthetic Mincle ligands as novel adjuvants. We recently reported on the synthesis and evaluation of Brartemicin analog UM-1024 that demonstrated Mincle agonist activity, exhibiting potent Th1/Th17 adjuvant activity that was greater than that of trehalose dibehenate (TDB). Our pursuit to understand Mincle/ligand relationships and improve the pharmacologic properties of the ligands has expanded and continues to reveal new and exciting structure activity relationships. Herein we report the synthesis of novel bi-aryl trehalose derivatives in good to excellent yields. These compounds were evaluated for their ability to engage the human Mincle receptor and tested for the induction of cytokines from human peripheral blood mononuclear cells. A preliminary structure-activity relationship (SAR) of these novel bi-aryl derivatives revealed that bi-aryl trehalose ligand 3D showed relatively high potency in cytokine production in comparison to trehalose glycolipid adjuvant TDB and the natural ligand TDM and induced dose-dependent, Mincle selective stimulation in hMincle HEK reporter cells. Also, through computational studies, we provide an insight into the potential mode of binding of 6,6′-Biaryl trehalose compounds on human Mincle receptor.

Mycobacterium tuberculosis (Mtb) continues to be a major health threat worldwide, and the development of Mtb vaccines could play a pivotal role in the prevention and control of this devastating epidemic. Th17-mediated immunity has been implicated in disease protection correlates of immune protection against Mtb. Currently, there are no approved adjuvants capable of driving a Th17 response in a vaccine setting. Recent clinical trial results using trehalose dibehenate have demonstrated a formulation-dependant proof of concept adjuvant system CAF01 capable of inducing long-lived protection. We have discovered a new class of Th17-inducing vaccine adjuvants based on the natural product Brartemicin. We synthesized and evaluated the capacity of a library of aryl trehalose derivatives to drive immunostimulatory reresponses and evaluated the structure-activity relationships in terms of the ability to engage the Mincle receptor and induce production of innate cytokines from human and murine cells. We elaborated on the structure-activity relationship of the new scaffold and demonstrated the ability of the lead entity to induce a pro-Th17 cytokine profile from primary human peripheral blood mononuclear cells and demonstrated efficacy in generating antibodies in combination with tuberculosis antigen M72 in a mouse model.

The cystine/glutamate antiporter system xc- (Sxc-) mediates the exchange of intracellular l-glutamate (l-Glu) with extracellular l-cystine (l-Cys(2)). Both the import of l-Cys(2) and the export of l-Glu take on added significance in CNS cells, especially astrocytes. When the relative activity of Sxc- overwhelms the regulatory capacity of the EAATs, the efflux of l-Glu through the antiporter can be significant enough to trigger excitotoxic pathology, as is thought to occur in glioblastoma. This has prompted considerable interest in the pharmacological specificity of Sxc- and the development of inhibitors. The present study explores a series of analogues that are structurally related to sulfasalazine, a widely employed inhibitor of Sxc-. We identify a number of novel aryl-substituted amino-naphthylsulfonate analogues that inhibit Sxc- more potently than sulfasalazine. Interestingly, the inhibitors switch from a competitive to noncompetitive mechanism with increased length and lipophilic substitutions, a structure-activity relationship that was previously observed with aryl-substituted isoxazole. These results suggest that the two classes of inhibitors may interact with some of the same domains on the antiporter protein and that the substrate and inhibitor binding sites may be in close proximity to one another. Molecular modeling is used to explore this possibility.

A series of analogues based on serine as lead structure were designed, and their agonist activities were evaluated at recombinant NMDA receptor subtypes (GluN1/2A-D) using two-electrode voltage-clamp (TEVC) electrophysiology. Pronounced variation in subunit-selectivity, potency, and agonist efficacy was observed in a manner that was dependent on the GluN2 subunit in the NMDA receptor. In particular, compounds 15a and 16a are potent GluN2C-specific superagonists at the GluN1 subunit with agonist efficacies of 398% and 308% compared to glycine. This study demonstrates that subunit-selectivity among glycine site NMDA receptor agonists can be achieved and suggests that glycine-site agonists can be developed as pharmacological tool compounds to study GluN2C-specific effects in NMDA receptor-mediated neurotransmission.

Cytochrome c can acquire peroxidase activity when it binds to cardiolipin in mitochondrial membranes. The resulting oxygenation of cardiolipin by cytochrome c provides an early signal for the onset of apoptosis. The structure of this enzyme substrate complex is a matter of considerable debate. We present three structures at 1.7-2.0 angstrom resolution of a domain-swapped dimer of yeast iso-1-cytochrome c with the detergents, CYMAL-5, CYMAL-6, and omega-undecylenyl-beta-D-maltopyranoside, bound in a channel that places the hydrocarbon moieties of these detergents next to the heme. The heme is poised for peroxidase activity with water bound in place of Met80, which serves as the axial heme ligand when cytochrome c functions as an electron carrier. The hydroxyl group of Tyr67 sits 3.6-4.0 angstrom from the nearest carbon of the detergents, positioned to act as a relay in radical abstraction during peroxidase activity. Docking studies with linoleic acid, the most common fatty acid component of cardiolipin, show that C11 of linoleic acid can sit adjacent to Tyr67 and the heme, consistent with the oxygenation pattern observed in lipidomics studies. The well-defined hydrocarbon binding pocket provides atomic resolution evidence for the extended lipid anchorage model for cytochrome c/cardiolipin binding. Dimer dissociation/association kinetics for yeast versus equine cytochrome c indicate that formation of mammalian cytochrome c dimers in vivo would require catalysis. However, the dimer structure shows that only a modest deformation of monomeric cytochrome c would suffice to form the hydrocarbon binding site occupied by these detergents.

Mammals express seven transporters from the SLC1 (solute carrier 1) gene family, including five acidic amino acid transporters (EAAT1-5) and two neutral amino acid transporters (ASCT1-2). In contrast, insects of the order Diptera possess only two SLC1 genes. In this work we show that in the mosquito Culex quinquefasciatus, a carrier of West Nile virus, one of its two SLC1 EAAT-like genes encodes a transporter that displays an unusual selectivity for dicarboxylic acids over acidic amino acids. In eukaryotes, dicarboxylic acid uptake has been previously thought to be mediated exclusively by transporters outside the SLC1 family. The dicarboxylate selectivity was found to be associated with two residues in transmembrane domain 8, near the presumed substrate binding site. These residues appear to be conserved in all eukaryotic SLC1 transporters (Asp444 and Thr448, human EAAT3 numbering) with the exception of this novel C. quinquefasciatus transporter and an ortholog from the yellow fever mosquito Aedes aegypti, in which they are changed to Asn and Ala. In the prokaryotic EAAT-like SLC1 transporter DctA, a dicarboxylate transporter which was lost in the lineage leading to eukaryotes, the corresponding TMD8 residues are Ser and Ala. Functional analysis of engineered mutant mosquito and human transporters expressed in Xenopus laevis oocytes provide support for a model defining interactions of charged and polar transporter residues in TMD8 with α-amino acids and ions. Together with the phylogenetic evidence, the functional data suggest that a novel route of dicarboxylic acid uptake evolved in these mosquitos by mutations in an ancestral glutamate transporter gene.

In this study we characterized the pharmacological selectivity and physiological actions of a new arylaspartate glutamate transporter blocker, L-threo-ß-benzylaspartate (L-TBA). At concentrations up to 100 µM, L-TBA did not act as an AMPA receptor (AMPAR) or NMDA receptor (NMDAR) agonist or antagonist when applied to outside-out patches from mouse hippocampal CA1 pyramidal neurons. L-TBA had no effect on the amplitude of field excitatory postsynaptic potentials (fEPSPs) recorded at the Schaffer collateral-CA1 pyramidal cell synapse. Excitatory postsynaptic currents (EPSCs) in CA1 pyramidal neurons were unaffected by L-TBA in the presence of physiological extracellular Mg(2+) concentrations, but in Mg(2+)-free solution, EPSCs were significantly prolonged as a consequence of increased NMDAR activity. Although L-TBA exhibited approximately four-fold selectivity for neuronal EAAT3 over glial EAAT1/EAAT2 transporter subtypes expressed in Xenopus oocytes, the L-TBA concentration-dependence of the EPSC charge transfer increase in the absence of Mg(2+) was the same in hippocampal slices from EAAT3 +/+ and EAAT3 -/- mice, suggesting that TBA effects were primarily due to block of glial transporters. Consistent with this, L-TBA blocked synaptically evoked transporter currents in CA1 astrocytes with a potency in accord with its block of heterologously expressed glial transporters. Extracellular recording in the presence of physiological Mg(2+) revealed that L-TBA prolonged fEPSPs in a frequency-dependent manner by selectively increasing the NMDAR-mediated component of the fEPSP during short bursts of activity. The data indicate that glial glutamate transporters play a dominant role in limiting extrasynaptic transmitter diffusion and binding to NMDARs. Furthermore, NMDAR signaling is primarily limited by voltage-dependent Mg(2+) block during low-frequency activity, while the relative contribution of transport increases during short bursts of higher frequency signaling.

A prominent aqueous cavity is formed by the junction of three identical subunits in the excitatory amino acid transporter (EAAT) family. To investigate the effect of this structure on the interaction of ligands with the transporter, we recorded currents in voltage-clamped Xenopus oocytes expressing EAATs and used concentration jumps to measure binding and unbinding rates of a high-affinity aspartate analog that competitively blocks transport (β-2-fluorenyl-aspartylamide; 2-FAA). The binding rates of the blocker were approximately one order of magnitude slower than l-Glu and were not significantly different for EAAT1, EAAT2, or EAAT3, but 2-FAA exhibited higher affinity for the neuronal transporter EAAT3 as a result of a slower dissociation rate. Unexpectedly, the rate of recovery from block was increased by l-Glu in a saturable and concentration-dependent manner, ruling out a first-order mechanism and suggesting that following unbinding, there is a significant probability of ligand rebinding to the same or neighboring subunits within a trimer. Consistent with such a mechanism, coexpression of wild-type subunits with mutant (R447C) subunits that do not bind glutamate or 2-FAA also increased the unblocking rate. The data suggest that electrostatic and steric factors result in an effective dissociation rate that is approximately sevenfold slower than the microscopic subunit unbinding rate. The quaternary structure, which has been conserved through evolution, is expected to increase the transporters' capture efficiency by increasing the probability that following unbinding, a ligand will rebind as opposed to being lost to diffusion.

........................................................................................................................ ii Acknowledgments ......................................................................................................... iii List of Figures ................................................................................................................ v Chapter 1: Background and Significance ...................................................................... 1 Chapter 2: Interactions of alkali cations with glutamate transporters ............................ 19 Chapter 3: A model for sequential binding in glutamate transporters ........................... 26 Chapter 4: Further computational work ......................................................................... 47 Chapter 5: Discussion and future directions .................................................................. 82

The transport of glutamate is coupled to the co-transport of three Na + ions and the countertransport of one K + ion. In addition to this carrier-type exchange behaviour, glutamate transporters also behave as chloride channels. The chloride channel activity is strongly influenced by the cations that are involved in coupled flux, making glutamate transporters representative of the ambiguous interface between carriers and channels. In this paper, we review the interaction of alkali cations with glutamate transporters in terms of these diverse functions. We also present a model derived from electrostatic mapping of the predicted cation-binding sites in the X-ray crystal structure of the Pyrococcus horikoshii transporter Glt Ph and in its human glutamate transporter homologue EAAT3. Two predicted Na + -binding sites were found to overlap precisely with the Tl + densities observed in the aspartate-bound complex. A novel third site predicted to favourably bind Na + (but not Tl + ) is formed by interaction with the substrate and the occluding HP2 loop. A fourth predicted site in the apo state exhibits selectivity for K + over both Na + and Tl + . Notably, this K + site partially overlaps the glutamate-binding site, and their binding is mutually exclusive. These results are consistent with kinetic and structural data and suggest a plausible mechanism for the flux coupling of glutamate with Na + and K + ions.

A 1H69 crystal structure-based in silico model of the NAD(P)H:quinone oxidoreductase 1 (NQO1) active site has been developed to facilitate NQO1-directed lavendamycin antitumor agent development. Lavendamycin analogues were designed as NQO1 substrates utilizing structure-based design criteria. Computational docking studies were performed using the model to predict NQO1 substrate specificity. Designed N-acyllavendamycin esters and amides were synthesized by Pictet-Spengler condensation. Metabolism and cytotoxicity studies were performed on the analogues with recombinant human NQO1 and human colon adenocarcinoma cells (NQO1-deficient BE and NQO1-rich BE-NQ). Docking and biological data were found to be correlated where analogues 12, 13, 14, 15, and 16 were categorized as good, poor, poor, poor, and good NQO1 substrates, respectively. Our results demonstrated that the ligand design criteria were valid, resulting in the discovery of two good NQO1 substrates. The observed consistency between the docking and biological data suggests that the model possesses practical predictive power.

Glutamate transporters have a homotrimeric subunit structure with a large central water-filled cavity that extends partially into the plane of the lipid bilayer (Yernool et al., 2004). In addition to uptake of glutamate, the transporters also mediate a chloride conductance that is increased in the presence of substrate. Whether the chloride channel is located in the central pore of the trimer or within the individual subunits has been controversial. We find that coexpression of wild-type neuronal glutamate transporter EAAT3 subunits with subunits mutated at R447, a residue governing substrate selectivity (Bendahan et al., 2000), results in transport activity consistent with two distinct noninteracting populations of transporters, in agreement with previous work suggesting that each subunit operates independently to transport substrate (Awes et al., 2004; Grewer et al., 2005; Koch and Larsson, 2005). In wild-type homotrimeric transporters, the glutamate concentration dependence of the anion conductance and the kinetics of glutamate flux were isolated and measured, and the anion channel activation was fitted to analytical expressions corresponding to (1) a central pore gated by binding to one or more subunits and (2) a channel pore in each subunit. The data indicate that glutamate-binding sites, transport pathways, and chloride channels reside in individual subunits in a trimer and function independently.

Factors that restrict a virus from establishing productive infection in a new host species are important to understand because cross-species transmission events are often associated with emergent viral diseases. To determine the evolutionary pressures on viruses in new host species, we evaluated the molecular evolution of a feline immunodeficiency virus derived from a wild cougar, Puma concolor, during infection of domestic cats. Analyses were based on the coding portion of genome sequences recovered at intervals over 37 weeks of infection of six cats inoculated by either intravenous or oral-nasal routes. All cats inoculated intravenously, but only one inoculated orally-nasally, became persistently viremic. There were notable accumulations of lethal errors and predominance of G-to-A alterations throughout the genome, which were marked in the viral polymerase gene, pol. Viral structural (env and gag) and accessory (vif and orfA) genes evolved neutrally or were under purifying selection. However, sites under positive selection were identified in reverse transcriptase that involved residues in the nucleotide binding pocket or those contacting the RNA-DNA duplex. The findings of extensive G-to-A alterations in this cross-species infection are consistent with the recently described editing of host cytidine deaminase on lentivirus genomes. Additionally, we demonstrate that the primary site of hypermutation is the viral pol gene and the dominant selective force acting on this feline immunodeficiency virus as it replicates in a new host species is on key residues of the virus polymerase.

4823 NAD(P)H:quinone oxidoreductase 1 (NQO1) is a homodimeric flavoenzyme containing 273 residues and one molecule of FAD per monomer. This reductase catalyzes a NAD(P)H-dependent two-electron reduction and the bioactivation of many antitumor bioreductive agents. NQO1, expressed at high levels in many human solid tumors, can be used as a unique target for enzyme directed bioreductive cancer drug development. Lavendamycin, a quinolinedione antibiotic with significant antitumor activity, is structurally related to streptonigrin, a good substrate for and efficiently bioactivated by NQO1. Computational molecular modeling studies were performed on two lavendamycin analogues, MB-353 and -323, very good and poor substrates for NQO1, respectively. The molecular docking was performed using the FlexX module of SYBYL6.9.1. The minimized coordinates of the crystal structure of the NQO1 complex with ARH019, an indolequinone, obtained from the Protein Data Bank (ID code: 1H69) was used as a reference structure for docking experiments. The docked conformations of ligands were evaluated using the FlexX and CSCORE modules in SYBYL. CSCORE is the consensus score computed from FlexX, ChemScore, D-Score, G-Score and PMF-Score scoring functions where docked poses are ranked from 1 to 5; where 5 is the best fit to the model. Geometric post-analysis strategies that determine hydrogen-bonding interactions of the ligands with the key residues of the active site (Tyr 126, 128 and His 161), hydride ion transfer from the flavin to the ligands, and the angle between the planes of the ligands and the flavin were performed. Of 30 possible docked conformations of MB-353, 24 poses had orientations similar to the reference ligand (ARH019) and 19 had CSCORE ≥ 4 (4 with CSCORE = 5 and 15 with CSCORE = 4). All of the 19 poses were capable of forming the hydrogen-bonding interactions and hydride ion transfer, and the departure of their planes from an exact aromatic parallel stacking with the flavin closely resembled that of ARH019. However, of 30 possible docked poses of MB-323, no conformation had a CSCORE = 5 and only 4 poses had CSCORE = 4. The remaining poses with CSCOREs of 0 and 1 did not merit further considerations. Also, none of the 30 poses had orientations similar to the reference ligand. Therefore, an increased number of possible conformations of ligand MB-353 with acceptable orientations compared to ligand MB-323 can be considered as a potential contributing factor to substrate specificity. Supported by NIH grants R15 CA78232 and NCRR P20 RR15583.

The virus population transmitted by a human immunodeficiency virus type 1 (HIV-1) infected individual undergoes restriction and subsequent diversification in the new host. However, in contrast to men, who have limited virus diversity at seroconversion, there is measurable diversity in viral envelope gene sequences in women infected with clade A HIV-1. In this study, virus sequence diversity in three unrelated, clade A infected women preceding and shortly after seroconversion was evaluated. It was demonstrated that there is measurable evolution of envelope gene sequences over this time interval. Furthermore, in each of the three individuals, amino acid substitutions arose at five or six positions in sequences derived at or shortly after seroconversion relative to sequences obtained from the seronegative sample. Presented here is a model of clade A gp120 to determine the location of substitutions that appeared as the virus population became established in three clade A HIV-1 infected women.

ABSTRACT Within the large body of research on retroviruses, the distribution and evolution of endemic retroviruses in natural host populations have so far received little attention. In this study, the epidemiology, genetic diversity, and molecular evolution of feline immunodeficiency virus specific to cougars (FIVpco) was examined using blood samples collected over several years from a free-ranging cougar population in the western United States. The virus prevalence was 58% in this population ( n = 52) and increased significantly with host age. Based on phylogenetic analysis of fragments of envelope ( env ) and polymerase ( pol ) genes, two genetically distinct lineages of FIVpco were found to cooccur in the population but not in the same individuals. Within each of the virus lineages, geographically nearby isolates formed monophyletic clusters of closely related viruses. Sequence diversity for env within a host rarely exceeded 1%, and the evolution of this gene was dominated by purifying selection. For both pol and env , our data indicate mean rates of molecular evolution of 1 to 3% per 10 years. These results support the premise that FIVpco is well adapted to its cougar host and provide a basis for comparing lentivirus evolution in endemic and epidemic infections in natural hosts.

ABSTRACT Women infected with clade A human immunodeficiency virus type 1 harbor a virus population that is genetically diverse in the envelope gene, a fact that contrasts with the homogeneous virus population identified in newly infected men. It is not known whether viral genetic diversity at this early stage of infection is manifested as phenotypic diversity. This is a significant question because phenotypic diversity in the viral population that establishes infection in women may have important implications for pathogenesis and therapeutic intervention. Thus, in this study we compared the biological properties of three pairs of chimeric viruses that contained envelope genes representative of variant groups in each of three infected women—Q23, Q45, and Q47. Envelope chimeras were evaluated for replication in stimulated and resting peripheral blood mononuclear cells alone and in competition, for coreceptor use, and for neutralization sensitivity. All viruses utilized CCR5 exclusively and had a non-syncytium-inducing phenotype on MT-2 cells and in primary culture. There were no significant differences in replication parameters between paired variants in individual cultures. However, in competition experiments, one chimera of each variant pair always dominated. The dominant virus from Q23 and Q47, but not from Q45, infected a significantly higher number of CCR5- and CD4-expressing GHOST cells than the weaker chimeras. Significantly, chimeric viruses from Q47 and Q45 showed markedly different neutralization sensitivity to antibodies to CCR5 and gp120, respectively. These data indicate that distinct envelope genotypes identified in clade A-infected women near seroconversion confer unique phenotypes that affect viral fitness and that may be due, in part, to different requirements for relative configuration of CD4 and CCR5 on infected cells.