Orexin Antagonists: A Wake-Up Call for Medicinal Chemists?

Orexin receptors are G protein coupled receptors that may be useful targets for sleep disorders, eating disorders, or addictive behavior. Recent work shows that binding of antagonists to these receptors is complex, with strong dependence on hydrophobic hot spots and networks of water-mediated hydrogen bonds. Despite the minimal structural differences between receptor types, selectivity can be achieved in a number of different ways.

Potent HIV-1 protease inhibitors incorporating squaramide-derived P2 ligands: Design, synthesis, and biological evaluation.

We describe the design, synthesis, and biological evaluation of novel HIV-1 protease inhibitors containing a squaramide-derived scaffold as the P2 ligand in combination with a (R)-hydroxyethylamine sulfonamide isostere. Inhibitor 3h with an N-methyl-3-(R)-aminotetrahydrofuranyl squaramide P2-ligand displayed an HIV-1 protease inhibitory Ki value of 0.51 nM. An energy minimized model of 3h revealed the major molecular interactions between HIV-1 protease active site and the tetrahydrofuranyl squaramide scaffold that may be responsible for its potent activity.

Variation of the aryl substituent on the piperazine ring within the 4-(piperazin-1-yl)-2,6-di(pyrrolidin-1-yl)pyrimidine scaffold unveils potent, non-competitive inhibitors of the inflammatory caspases.

The inflammatory caspases (caspase-1, -4 and -5) are potential therapeutic targets for autoimmune and inflammatory diseases due to their involvement in the immune response upon inflammasome formation. A series of small molecules based on the 4-(piperazin-1-yl)-2,6-di(pyrrolidin-1-yl)pyrimidine scaffold were synthesized with varying substituents on the piperazine ring. Several compounds were pan-selective inhibitors of the inflammatory caspases, caspase-1, -4 and -5, with the ethylbenzene derivative CK-1-41 displaying low nanomolar Ki values across this family of caspases. Three analogs were nearly 10 fold selective for caspase-5 over caspase-1 and -4. The compounds display non-competitive, time dependent inhibition profiles. To our knowledge, this series is the first example of small molecule inhibitors of all three inflammatory caspases.

Antipsychotics inhibit glucose transport: Determination of olanzapine binding site in Staphylococcus epidermidis glucose/H(+) symporter.

The antipsychotic drug olanzapine is widely prescribed to treat schizophrenia and other psychotic disorders. However, it often causes unwanted side effects, including diabetes, due to disruption of insulin-dependant glucose metabolism through a mechanism yet to be elucidated. To determine if olanzapine can affect the first step in glucose metabolism - glucose transport inside cells - we investigated the effect of this drug on the transport activity of a model glucose transporter. The glucose transporter from Staphylococcus epidermidis (GlcPSe) is specific for glucose, inhibited by various human glucose transporter (GLUT) inhibitors, has high sequence and structure homology to GLUTs, and is readily amenable to transport assay, mutagenesis, and computational modeling. We found that olanzapine inhibits glucose transport of GlcPSe with an IC50 0.9 ± 0.1 mM. Computational docking of olanzapine to the GlcPSe structure revealed potential binding sites that were further examined through mutagenesis and transport assay to identify residues important for olanzapine inhibition. These investigations suggest that olanzapine binds in a polar region of the cytosolic part of the transporter, and interacts with residues R129, strictly conserved in all GLUTs, and N136, conserved in only a few GLUTs, including the insulin-responsive GLUT4. We propose that olanzapine inhibits GlcPSe by impeding the alternating opening and closing of the substrate cavity necessary for glucose transport. It accomplishes this by disrupting a key salt bridge formed by conserved residues R129 and E362, that stabilizes the outward-facing conformation of the transporter.

Design and synthesis of potent macrocyclic HIV-1 protease inhibitors involving P1-P2 ligands.

A series of potent macrocyclic HIV-1 protease inhibitors have been designed and synthesized. The compounds incorporated 16- to 19-membered macrocyclic rings between a nelfinavir-like P2 ligand and a tyrosine side chain containing a hydroxyethylamine sulfonamide isostere. All cyclic inhibitors are more potent than their corresponding acyclic counterparts. Saturated derivatives showed slight reduction of potency compared to the respective unsaturated derivatives. Compound containing a 16-membered ring as the P1-P2 ligand showed the most potent enzyme inhibitory and antiviral activity.

Properties of two cataract-associated mutations located in the NH2 terminus of connexin 46.

Mutations in connexin 46 are associated with congenital cataracts. The purpose of this project was to characterize cellular and functional properties of two congenital cataract-associated mutations located in the NH2 terminus of connexin 46: Cx46D3Y and Cx46L11S, which we found localized to gap junctional plaques like wild-type Cx46 in transfected HeLa cells. Dual two-microelectrode-voltage-clamp studies of Xenopus oocyte pairs injected with wild-type or mutant rat Cx46 showed that oocyte pairs injected with D3Y or L11S cRNA failed to induce gap junctional coupling, whereas oocyte pairs injected with Cx46 showed high levels of coupling. D3Y, but not L11S, functionally paired with wild-type Cx46. To determine whether coexpression of D3Y or L11S affected the junctional conductance produced by wild-type lens connexins, we studied pairs of oocytes coinjected with equal amounts of mutant and wild-type connexin cRNA. Expression of D3Y or L11S almost completely abolished gap junctional coupling induced by Cx46. In contrast, expression of D3Y or L11S failed to inhibit junctional conductance induced by Cx50. To examine effects of the D3Y and L11S mutations on hemichannel activity, hemichannel currents were measured in connexin cRNA-injected oocytes. Oocytes expressing D3Y exhibited reduced hemichannel activity as well as alterations in voltage gating and charge selectivity while oocytes expressing L11S showed no hemichannel activity. Moreover, coexpression of mutant with wild-type Cx50 or Cx46 gave rise to hemichannels with distinct electrophysiological properties, suggesting that the mutant connexins were forming heteromeric channels with wild-type connexins. These data suggest D3Y and L11S cause cataracts by similar but not identical mechanisms.

Oligomycin frames a common drug-binding site in the ATP synthase.

We report the high-resolution (1.9 Å) crystal structure of oligomycin bound to the subunit c(10) ring of the yeast mitochondrial ATP synthase. Oligomycin binds to the surface of the c(10) ring making contact with two neighboring molecules at a position that explains the inhibitory effect on ATP synthesis. The carboxyl side chain of Glu59, which is essential for proton translocation, forms an H-bond with oligomycin via a bridging water molecule but is otherwise shielded from the aqueous environment. The remaining contacts between oligomycin and subunit c are primarily hydrophobic. The amino acid residues that form the oligomycin-binding site are 100% conserved between human and yeast but are widely different from those in bacterial homologs, thus explaining the differential sensitivity to oligomycin. Prior genetics studies suggest that the oligomycin-binding site overlaps with the binding site of other antibiotics, including those effective against Mycobacterium tuberculosis, and thereby frames a common "drug-binding site." We anticipate that this drug-binding site will serve as an effective target for new antibiotics developed by rational design.

Design and synthesis of potent HIV-1 protease inhibitors incorporating hexahydrofuropyranol-derived high affinity P(2) ligands: structure-activity studies and biological evaluation.

The design, synthesis, and evaluation of a new series of hexahydrofuropyranol-derived HIV-1 protease inhibitors are described. We have designed a stereochemically defined hexahydrofuropyranol-derived urethane as the P2-ligand. The current ligand is designed based upon the X-ray structure of 1a-bound HIV-1 protease. The synthesis of (3aS,4S,7aR)-hexahydro-2H-furo[2,3-b]pyran-4-ol, (-)-7, was carried out in optically active form. Incorporation of this ligand provided inhibitor 35a, which has shown excellent enzyme inhibitory activity and antiviral potency. Our structure-activity studies have indicated that the stereochemistry and the position of oxygens in the ligand are important to the observed potency of the inhibitor. Inhibitor 35a has maintained excellent potency against multidrug-resistant HIV-1 variants. An active site model of 35a was created based upon the X-ray structure of 1b-bound HIV-1 protease. The model offers molecular insights regarding ligand-binding site interactions of the hexahydrofuropyranol-derived novel P2-ligand.

Binding of glutamate to the umami receptor.

The umami taste receptor is a heterodimer composed of two members of the T1R taste receptor family: T1R1 and T1R3. It detects glutamate in humans, and is a more general amino acid detector in other species. We have constructed homology models of the ligand binding domains of the human umami receptor (based on crystallographic structures of the metabotropic glutamate receptor of the central nervous system). We have carried out molecular dynamics simulations of the ligand binding domains, and we find that the likely conformation is that T1R1 receptor protein exists in the closed conformation, and T1R3 receptor in the open conformation in the heterodimer. Further, we have identified the important binding interactions and have made an estimate of the relative free energies associated with the two glutamate binding sites.

Mitochondrial and Plasma Membrane Citrate Transporters: Discovery of Selective Inhibitors and Application to Structure/Function Analysis.

Cytoplasmic citrate is the prime carbon source for fatty acid, triacylglycerol, and cholesterol biosyntheses, and also regulates glucose metabolism via its allosteric inhibition of phosphofructokinase. It originates either via the efflux of citrate from the mitochondrial matrix on the inner membrane citrate transport protein (CTP) or via the influx of extracellular citrate on the plasma membrane citrate transporter (PMCT). Despite their common substrate, the two transport proteins share little sequence similarity and they transport citrate via fundamentally different mechanisms. We tested the ability of a set of previously identified CTP inhibitors, to inhibit the PMCT. We found that of the top 10 CTP inhibitors only one substantially inhibited the PMCT. Conversely, we identified two other inhibitors that inhibited the PMCT but had little effect on the CTP. All three identified PMCT inhibitors displayed a noncompetitive mechanism. Furthermore, models to explain inhibitor interactions with the CTP are proposed. As part of the present studies a PMCT homology model has been developed based on the crystal structure of the leucine transporter, and a possible citrate binding site has been identified and its composition compared with the two known citrate binding sites present within the CTP. The ability to selectively inhibit the PMCT may prove key to the pharmacologic amelioration of metabolic disorders resulting from the synthesis of excess lipid, cholesterol, and glucose, including human obesity, hyperlipidemia, hyper-cholesterolemia, and Type 2 diabetes.

Conformational changes in BAK, a pore-forming proapoptotic Bcl-2 family member, upon membrane insertion and direct evidence for the existence of BH3-BH3 contact interface in BAK homo-oligomers.

During apoptosis, the pro-apoptotic Bcl-2 family proteins BAK and BAX form large oligomeric pores in the mitochondrial outer membrane. Apoptotic factors, including cytochrome c, are released through these pores from the mitochondrial intermembrane space into the cytoplasm where they initiate the cascade of events leading to cell death. To better understand this pivotal step toward apoptosis, a method was developed to induce membrane permeabilization by BAK in the membrane without using the full-length protein. Using a soluble form of BAK with a hexahistidine tag at the C terminus and a liposomal system containing the Ni(2+)-nitrilotriacetic acid lipid analog that can bind hexahistidine-tagged proteins, BAK oligomers were formed in the presence of the activator protein p7/p15Bid. In this system, we determined the conformational changes in BAK upon membrane insertion by applying the site-directed spin labeling method of EPR to 13 different amino acid locations. Upon membrane insertion, the BH3 domains were reorganized, and the alpha5-alpha6 helical hairpin structure was partially exposed to the membrane environment. The monomer-monomer interface in the oligomeric structure was also mapped by measuring the distance-dependent spin-spin interactions for each residue location. Spin labels attached in the BH3 domain were juxtaposed within 5-10 A distance in the oligomeric form in the membrane. These results are consistent with the current hypothesis that BAK or BAX forms homodimers, and these homodimers assemble into a higher order oligomeric pore. Detailed analyses of the data provide new insights into the structure of the BAX or BAK homodimer.

The yeast mitochondrial citrate transport protein: molecular determinants of its substrate specificity.

The objective of this study was to identify the role of individual amino acid residues in determining the substrate specificity of the yeast mitochondrial citrate transport protein (CTP). Previously, we showed that the CTP contains at least two substrate-binding sites. In this study, utilizing the overexpressed, single-Cys CTP-binding site variants that were functionally reconstituted in liposomes, we examined CTP specificity from both its external and internal surfaces. Upon mutation of residues comprising the more external site, the CTP becomes less selective for citrate with numerous external anions able to effectively inhibit [(14)C]citrate/citrate exchange. Thus, the site 1 variants assume the binding characteristics of a nonspecific anion carrier. Comparison of [(14)C]citrate uptake in the presence of various internal anions versus water revealed that, with the exception of the R189C mutant, the other site 1 variants showed substantial uniport activity relative to exchange. Upon mutation of residues comprising site 2, we observed two types of effects. The K37C mutant displayed a markedly enhanced selectivity for external citrate. In contrast, the other site 2 mutants displayed varying degrees of relaxed selectivity for external citrate. Examination of internal substrates revealed that, in contrast to the control transporter, the R181C variant exclusively functioned as a uniporter. This study provides the first functional information on the role of specific binding site residues in determining mitochondrial transporter substrate selectivity. We interpret our findings in the context of our homology-modeled CTP as it cycles between the outward-facing, occluded, and inward-facing states.

A conserved arginine near the filter of Kir1.1 controls Rb/K selectivity.

ROMK (Kir1.1) channels are important for K secretion and recycling in the collecting duct, connecting tubule and thick ascending limb of the mammalian nephron. We have identified a highly conserved Arg in the P loop of the channel near the selectivity filter that controls Rb/K selectivity. Mutation of this Arg to a Tyr (R128Y-Kir1.1b, R147Y-Kir1.1a) increased the macroscopic conductance ratio, G(Rb)/G(K) by 17 ± 3 fold and altered the selectivity sequence from NH(4) > K > Tl > Rb >> Cs in wt-Kir1.1 to: Rb > Cs > Tl > NH(4) >> K in R128Y, without significant change in the high K/Na permeability ratio of Kir1.1. R128M produced similar, but smaller, increases in Rb, Tl, NH(4) and Cs conductance relative to K. R128Y remained susceptible to block by both external Ba and the honeybee toxin, TPNQ, although R128Y had a reduced affinity for TPNQ, relative to wild-type. The effect of R128Y-Kir1.1b on the G(Rb)/G(K) ratio can be partly explained by a larger single-channel Rb conductance (12.4 ± 0.5 pS) than K conductance (<1.5 pS) in this mutant. The kinetics of R128Y gating at -120 mV with Rb as the permeant ion were similar to those of wt-Kir1.1 conducting Rb, but with a longer open time (129 ms vs. 6 ms for wt) and two closed states (13 ms, 905 ms), resulting in an open probability (Po) of 0.5, compared to a Po of 0.9 for wt-Kir1.1, which had a single closed state of 1 ms at -120 mV. Single-channel R128Y rectification was eliminated in excised, insideout patches with symmetrical Rb solutions. The large increase in the Rb/K conductance ratio, with no change in K/Na permeability or rectification, is consistent with R128Y-Kir1.1b causing a subtle change in the selectivity filter, perhaps by disruption of an intra-subunit salt bridge (R128-E118) near the filter.

Probing the effect of transport inhibitors on the conformation of the mitochondrial citrate transport protein via a site-directed spin labeling approach.

The present investigation utilized the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy to identify the effect of citrate, the natural ligand, and transport inhibitors on the conformation of the yeast mitochondrial citrate transport protein (CTP) reconstituted in liposomal vesicles. Spin label was placed at six different locations within the CTP in order to monitor conformational changes that occurred near each of the transporter's two substrate binding sites, as well as at more distant domains within the CTP architecture. We observed that citrate caused little change in the EPR spectra. In contrast the transport inhibitors 1,2,3-benzenetricarboxylate (BTC), pyridoxal 5'-phosphate (PLP), and compound 792949 resulted in spectral changes that indicated a decrease in the flexibility of the attached spin label at each of the six locations tested. The rank order of the immobilizing effect was compound 792949 > PLP > BTC. The four spin-label locations that report on the CTP substrate binding sites displayed the greatest changes in the EPR spectra upon addition of inhibitor. Furthermore, we found that when compound 792949 was added vectorially (i.e., extra- and/or intra-liposomally), the immobilizing effect was mediated nearly exclusively by external reagent. In contrast, upon addition of PLP vectorially, the effect was mediated to a similar extent from both the external and the internal compartments. In combination our data indicate that: i) citrate binding to the CTP substrate binding sites does not alter side-chain and/or backbone mobility in a global manner and is consistent with our expectation that both in the absence and presence of substrate the CTP displays the flexibility required of a membrane transporter; and ii) binding of each of the transport inhibitors tested locked multiple CTP domains into more rigid conformations, thereby exhibiting long-range inter-domain conformational communication. The differential vectorial effects of compound 792949 and PLP are discussed in the context of the CTP homology-modeled structure and potential mechanistic molecular explanations are given.

Ion selectivity of alpha-hemolysin with beta-cyclodextrin adapter. II. Multi-ion effects studied with grand canonical Monte Carlo/Brownian dynamics simulations.

In a previous study of ion selectivity of alpha-hemolysin (alphaHL) in complex with beta-cyclodextrin (betaCD) adapter, we calculated the potential of mean force (PMF) and characterized the self-diffusion coefficients of isolated K(+) and Cl(-) ions using molecular dynamics simulations (Y. Luo et al., "Ion Selectivity of alpha-Hemolysin with beta-Cyclodextrin Adapter: I. Single Ion Potential of Mean Force and Diffusion Coefficient"). In the present effort, these results pertaining to single isolated ions in the wide aqueous pore are extended to take into account multi-ion effects. The grand canonical Monte Carlo/Brownian dynamics (GCMC/BD) algorithm is used to simulate ion currents through the wild-type alphaHL ion channel, as well as two engineered alphaHL mutants, with and without the cyclic oligosaccaride betaCD lodged in the lumen of the pore. The GCMC/BD current-voltage curves agree well with experimental results and show that betaCD increases the anion selectivity of alphaHL. Comparisons between multi-ion PMFs from GCMC/BD simulations and single-ion PMFs demonstrate that multi-ion effects and pore shape are crucial for explaining this behavior. It is concluded that the narrow betaCD adapter increases the anion selectivity of alphaHL because it reduces the pore radius locally, which decreases the ionic screening and the dielectric shielding of the strong electrostatic field induced by a nearby ring of positively charged alphaHL side chains.

Ion selectivity of alpha-hemolysin with a beta-cyclodextrin adapter. I. Single ion potential of mean force and diffusion coefficient.

The alpha-hemolysin (alphaHL) is a self-assembling exotoxin that binds to the membrane of a susceptible host cell and causes its death. Experimental studies show that electrically neutral beta-cyclodextrin (betaCD) can insert into the alphaHL channel and significantly increase its anion selectivity. To understand how betaCD can affect ion selectivity, molecular dynamics simulations and potential of mean force (PMF) calculations are carried out for different alphaHL channels with and without the betaCD adapter. A multiscale approach based on the generalized solvent boundary potential is used to reduce the size of the simulated system. The PMF profiles reveal that betaCD has no anion selectivity by itself but can increase the Cl(-) selectivity of the alphaHL channel when lodged into the pore lumen. Analysis shows that betaCD causes a partial desolvation of ions and affects the orientation of nearby charged residues. The ion selectivity appears to result from increased electrostatic interaction between the ion and the channel due to a reduction in dielectric shielding by the solvent. These observations suggest a reasonable explanation of the ion selectivity and provide important information for further ion channel modification.

Inhibitors of the mitochondrial citrate transport protein: validation of the role of substrate binding residues and discovery of the first purely competitive inhibitor.

The mitochondrial citrate transport protein (CTP) is critical to energy metabolism in eukaryotic cells. We demonstrate that 1,2,3-benzenetricarboxylate (BTC), the classic and defining inhibitor of the mitochondrial CTP, is a mixed inhibitor of the reconstituted Cys-less CTP, with a strong competitive component [i.e., a competitive inhibition constant (K(ic)) of 0.12 +/- 0.02 mM and an uncompetitive inhibition constant (K(iu)) of 3.04 +/- 0.74 mM]. Based on docking calculations, a model for BTC binding has been developed. We then determined the K(ic) values for each of the eight substrate binding site cysteine substitution mutants and observed increases of 62- to 261-fold relative to the Cys-less control, thereby substantiating the importance of each of these residues in BTC binding. It is noteworthy that we observed parallel increases in the K(m) for citrate transport with each of these binding site mutants, thereby confirming that with these CTP variants, K(m) approximates the K(d) (for citrate) and is therefore a measure of substrate affinity. To further substantiate the importance of these binding site residues, in silico screening of a database of commercially available compounds has led to discovery of the first purely competitive inhibitor of the CTP. Docking calculations indicate that this inhibitor spans and binds to both substrate sites simultaneously. Finally, we propose a kinetic model for citrate transport in which the citrate molecule sequentially binds to the external and internal binding sites (per CTP monomer) before transport.

Synthesis and biological evaluation of novel allophenylnorstatine-based HIV-1 protease inhibitors incorporating high affinity P2-ligands.

A series of stereochemically defined cyclic ethers as P2-ligands were incorporated in an allophenylnorstatine-based isostere to provide a new series of HIV-1 protease inhibitors. Inhibitors 3b and 3c, containing conformationally constrained cyclic ethers, displayed impressive enzymatic and antiviral properties and represent promising lead compounds for further optimization.

Design and evaluation of new analogs of the sweet protein brazzein.

We have previously modeled the interaction of the sweet protein brazzein with the extracellular domains of the sweet taste receptor. Here, we describe the application of that model to the design of 12 new highly potent analogs of brazzein. Eight of the 12 analogs have higher sweetness potency than wild-type brazzein. Results are consistent with our brazzein-receptor interaction model. The model predicts binding of brazzein to the open form of T1R2 in the T1R2-T1R3 heterodimer.