John G. Wise

Catalytic transitions in the human MDR1 P-glycoprotein drug binding sites.

Multidrug resistance proteins that belong to the ATP-binding cassette family like the human P-glycoprotein (ABCB1 or Pgp) are responsible for many failed cancer and antiviral chemotherapies because these membrane transporters remove the chemotherapeutics from the targeted cells. Understanding the details of the catalytic mechanism of Pgp is therefore critical to the development of inhibitors that might overcome these resistances. In this work, targeted molecular dynamics techniques were used to elucidate catalytically relevant structures of Pgp. Crystal structures of homologues in four different conformations were used as intermediate targets in the dynamics simulations. Transitions from conformations that were wide open to the cytoplasm to transition state conformations that were wide open to the extracellular space were studied. Twenty-six nonredundant transitional protein structures were identified from these targeted molecular dynamics simulations using evolutionary structure analyses. Coupled movement of nucleotide binding domains (NBDs) and transmembrane domains (TMDs) that form the drug binding cavities were observed. Pronounced twisting of the NBDs as they approached each other as well as the quantification of a dramatic opening of the TMDs to the extracellular space as the ATP hydrolysis transition state was reached were observed. Docking interactions of 21 known transport ligands or inhibitors were analyzed with each of the 26 transitional structures. Many of the docking results obtained here were validated by previously published biochemical determinations. As the ATP hydrolysis transition state was approached, drug docking in the extracellular half of the transmembrane domains seemed to be destabilized as transport ligand exit gates opened to the extracellular space.

The cell cycle as a therapeutic target against Trypanosoma brucei: Hesperadin inhibits Aurora kinase-1 and blocks mitotic progression in bloodstream forms

Aurora kinase family members co‐ordinate a range of events associated with mitosis and cytokinesis. Anti‐cancer therapies are currently being developed against them. Here, we evaluate whether Aurora kinase‐1 (TbAUK1) from pathogenic Trypanosoma brucei might be targeted in anti‐parasitic therapies as well. Conditional knockdown of TbAUK1 within infected mice demonstrated its essential contribution to infection. An in vitro kinase assay was developed which used recombinant trypanosome histone H3 as a substrate. Tandem mass spectroscopy identified a novel phosphorylation site in the carboxyl‐tail of recombinant trypanosome histone H3. Hesperadin, an inhibitor of human Aurora B, prevented the phosphorylation of substrate with IC50 of 40 nM. Growth of cultured bloodstream forms was also sensitive to Hesperadin (IC50 of 50 nM). Hesperadin blocked nuclear division and cytokinesis but not other aspects of the cell cycle. Consequently, growth arrested cells accumulated multiple kinetoplasts, flagella and nucleoli, similar to the effects of RNAi‐dependent knockdown of TbAUK1 in cultured bloodstream forms cells. Molecular models predicted high‐affinity binding of Hesperadin to both conserved and novel sites in TbAUK1. Collectively, these data demonstrate that cell cycle progression is essential for infections with T. brucei and that parasite Aurora kinases can be targeted with small‐molecule inhibitors.

Multiple Drug Transport Pathways through human P-Glycoprotein

P-Glycoprotein (P-gp) is a plasma membrane efflux pump that is commonly associated with therapy resistances in cancers and infectious diseases. P-gp can lower the intracellular concentrations of many drugs to subtherapeutic levels by translocating them out of the cell. Because of the broad range of substrates transported by P-gp, overexpression of P-gp causes multidrug resistance. We reported previously on dynamic transitions of P-gp as it moved through conformations based on crystal structures of homologous ABCB1 proteins using in silico targeted molecular dynamics techniques. We expanded these studies here by docking transport substrates to drug binding sites of P-gp in conformations open to the cytoplasm, followed by cycling the pump through conformations that opened to the extracellular space. We observed reproducible transport of two substrates, daunorubicin and verapamil, by an average of 11-12 Å through the plane of the membrane as P-gp progressed through a catalytic cycle. Methylpyrophosphate, a ligand that should not be transported by P-gp, did not show this movement through P-gp. Drug binding to either of two subsites on P-gp appeared to determine the initial pathway used for drug movement through the membrane. The specific side-chain interactions with drugs within each pathway seemed to be, at least in part, stochastic. The docking and transport properties of a P-gp inhibitor, tariquidar, were also studied. A mechanism of inhibition by tariquidar that involves stabilization of an outward open conformation with tariquidar bound in intracellular loops or at the drug binding domain of P-gp is presented.

Structure of the Cytosolic Part of the Subunit b-Dimer of Escherichia coli F0F1-ATP Synthase ☆ ☆☆

The structure of the external stalk and its function in the catalytic mechanism of the F(0)F(1)-ATP synthase remains one of the important questions in bioenergetics. The external stalk has been proposed to be either a rigid stator that binds F(1) or an elastic structural element that transmits energy from the small rotational steps of subunits c to the F(1) sector during catalysis. We employed proteomics, sequence-based structure prediction, molecular modeling, and electron spin resonance spectroscopy using site-directed spin labeling to understand the structure and interfacial packing of the Escherichia coli b-subunit homodimer external stalk. Comparisons of bacterial, cyanobacterial, and plant b-subunits demonstrated little sequence similarity. Supersecondary structure predictions, however, show that all compared b-sequences have extensive heptad repeats, suggesting that the proteins all are capable of packing as left-handed coiled-coils. Molecular modeling subsequently indicated that b(2) from the E. coli ATP synthase could pack into stable left-handed coiled-coils. Thirty-eight substitutions to cysteine in soluble b-constructs allowed the introduction of spin labels and the determination of intersubunit distances by ESR. These distances correlated well with molecular modeling results and strongly suggest that the E. coli subunit b-dimer can stably exist as a left-handed coiled-coil.

Subunit b-Dimer of the Escherichia coli ATP Synthase Can Form Left-Handed Coiled-Coils

One remaining challenge to our understanding of the ATP synthase concerns the dimeric coiled-coil stator subunit b of bacterial synthases. The subunit b-dimer has been implicated in important protein interactions that appear necessary for energy conservation and that may be instrumental in energy conservation during rotary catalysis by the synthase. Understanding the stator structure and its interactions with the rest of the enzyme is crucial to the understanding of the overall catalytic mechanism. Controversy exists on whether subunit b adopts a classic left-handed or a presumed right-handed dimeric coiled-coil and whether or not staggered pairing between nonhomologous residues in the homodimer is required for intersubunit packing. In this study we generated molecular models of the Escherichia coli subunit b-dimer that were based on the well-established heptad-repeat packing exhibited by left-handed, dimeric coiled-coils by employing simulated annealing protocols with structural restraints collected from known structures. In addition, we attempted to create hypothetical right-handed coiled-coil models and left- and right-handed models with staggered packing in the coiled-coil domains. Our analyses suggest that the available structural and biochemical evidence for subunit b can be accommodated by classic left-handed, dimeric coiled-coil quaternary structures.

Investigating the structure of nucleotide binding sites on the chloroplast F1-ATPase using electron spin resonance spectroscopy

Abstract The relative structure and binding properties of nucleotide binding sites of the latent, nonactivated chloroplast F1(CF1)ATPase have been investigated by employing ESR spectroscopy using 2-N3-2′,3′-SL-ATP (2-N3-SL-ATP), a spin-labeled photoaffinity analog of ATP. These results are compared to data obtained in analogous experiments using CF1 that was either depleted of its e-subunit or activated by different methods. Nonactivated (na) CF1 in complex with 2-N3-SL-ATP exhibits ESR spectra typical for enzyme-bound spin labels. At increased 2-N3-SL-ATP concentration, a second spectral component for enzyme-bound spin label is observed. The line-shape of the second signal indicates an environment of the enzyme-bound radical that differs from the spin-labeled nucleotide bound first. It can be explained by an enzyme-bound radical bound in a way that allows for higher mobility, e.g. a nucleotide binding site in an “open” or “loose” conformation. Maximal binding of about 5 mol 2-N3-SL-ANP per mol of enzyme has been reached. Similar results are obtained when using enzyme that has been either previously depleted of the e-subunit or treated with the reducing agent dithiothreitol (DTT) in the cold. Upon heat-activation of CF1 in the presence of ATP and the presence or absence of the reducing agent DTT, the line-shape of the ESR spectra is observed to be quite different from the non-heat-treated enzyme forms. The “loose” or “open” nucleotide binding site described above (or at least an environment of the enzyme similar to this site) is observed to be accessible to 2-N3-SL-ATP even at substoichiometric concentrations of the nucleotide analog. The results presented indicate that the enzyme form of CF1 generated after heat treatment in the presence of ATP with or without DTT exhibits altered binding specificities mainly with respect to the sequence of occupation of two different types of nucleotide binding sites.

In Silico Screening for Inhibitors of P-Glycoprotein That Target the Nucleotide Binding Domains

Multidrug resistances and the failure of chemotherapies are often caused by the expression or overexpression of ATP-binding cassette transporter proteins such as the multidrug resistance protein, P-glycoprotein (P-gp). P-gp is expressed in the plasma membrane of many cell types and protects cells from accumulation of toxins. P-gp uses ATP hydrolysis to catalyze the transport of a broad range of mostly hydrophobic compounds across the plasma membrane and out of the cell. During cancer chemotherapy, the administration of therapeutics often selects for cells which overexpress P-gp, thereby creating populations of cancer cells resistant to a variety of chemically unrelated chemotherapeutics. The present study describes extremely high-throughput, massively parallel in silico ligand docking studies aimed at identifying reversible inhibitors of ATP hydrolysis that target the nucleotide-binding domains of P-gp. We used a structural model of human P-gp that we obtained from molecular dynamics experiments as the protein target for ligand docking. We employed a novel approach of subtractive docking experiments that identified ligands that bound predominantly to the nucleotide-binding domains but not the drug-binding domains of P-gp. Four compounds were found that inhibit ATP hydrolysis by P-gp. Using electron spin resonance spectroscopy, we showed that at least three of these compounds affected nucleotide binding to the transporter. These studies represent a successful proof of principle demonstrating the potential of targeted approaches for identifying specific inhibitors of P-gp.

Combinatorial Redesign of the DNA Binding Specificity of a Prokaryotic Helix-Turn-Helix Repressor

Redesign of the bacteriophage 434 Cro repressor was accomplished by using an in vivo genetic screening system to identify new variants that specifically bound previously unrecognized DNA sequences. Site-directed, combinatorial mutagenesis of the 434 Cro helix-turn-helix (HTH) motif generated libraries of new variants which were screened for binding to new target sequences. Multiple mutations of 434 Cro that functionally converted wild-type (wt) 434 Cro DNA binding-sequence specificity to that of a lambda bacteriophage-specific repressor were identified. The libraries contained variations within the HTH sequence at only three positions. In vivo and in vitro analysis of several of the identified 434 Cro variants showed that the relatively few changes in the recognition helix of the HTH motif of 434 Cro resulted in specific and tight binding of the target DNA sequences. For the best 434 Cro variant identified, an apparent K(d) for lambda O(R)3 of 1 nM was observed. In competition experiments, this Cro variant was observed to be highly selective. We conclude that functional 434 Cro repressor variants with new DNA binding specificities can be generated from wt 434 Cro by mutating just the recognition helix. Important characteristics of the screening system responsible for the successful identifications are discussed. Application of the techniques presented here may allow the identification of DNA binding protein variants that functionally affect DNA regulatory sequences important in disease and industrial and biotechnological processes.

Synthesis of a pH-sensitive spin-labeled cyclohexylcarbodiimide derivative for probing protonation reactions in proton-pumping enzymes

Abstract The synthesis of a pH-sensitive spin-labeled carbodiimide, N-cyclohexyl-N′-(1-oxyl-2, 2, 3, 5, 5-pentamethylimidazolidine-4-yl)-methylcarbodiimide (1) is described. The compound is an analog of dicyclohexylcarbodiimide and reacts specifically with the membrane sector of the proton-pumping F1F0-type ATP synthase. This reaction of1 with the ATP synthase (and perhaps other proton-pumping enzymes) should enable studies of protonation reactions in the enzyme using ESR spectroscopy. The preparation of another potentially useful pH-sensitive spin-label, 4-isothiocyanatomethyl-2, 2, 3, 5, 5-pentamethylimidazolidine-1-oxyl (8) is also described. The synthesis of N-cyclohexyl-N′-(1-oxyl-2, 2, 3, 5, 5-pentamethyl-imidazolidine-4-yl)-methylcarbodiimide1, a pH-sensitive affinity spin label, and the reaction of1 with the proton-pumping ATP synthase enzyme is described. Download : Download full-size image

In silico identified targeted inhibitors of P-glycoprotein overcome multidrug resistance in human cancer cells in culture.

Failure of cancer chemotherapies is often linked to the over expression of ABC efflux transporters like the multidrug resistance P‐glycoprotein (P‐gp). P‐gp expression in cells leads to the elimination of a variety of chemically unrelated, mostly cytotoxic compounds. Administration of chemotherapeutics during therapy frequently selects for cells that over express P‐gp and are therefore capable of robustly exporting diverse compounds, including chemotherapeutics, from the cells. P‐gp thus confers multidrug resistance to a majority of drugs currently available for the treatment of cancers and diseases like HIV/AIDS. The search for P‐gp inhibitors for use as co‐therapeutics to combat multidrug resistances has had little success to date. In a previous study (Brewer et al., Mol Pharmacol 86: 716–726, 2014), we described how ultrahigh throughput computational searches led to the identification of four drug‐like molecules that specifically interfere with the energy harvesting steps of substrate transport and inhibit P‐gp catalyzed ATP hydrolysis in vitro. In the present study, we demonstrate that three of these compounds reversed P‐gp‐mediated multidrug resistance of cultured prostate cancer cells to restore sensitivity comparable to naïve prostate cancer cells to the chemotherapeutic drug, paclitaxel. Potentiation concentrations of the inhibitors were <3 μmol/L. The inhibitors did not exhibit significant toxicity to noncancerous cells at concentrations where they reversed multidrug resistance in cancerous cells. Our results indicate that these compounds with novel mechanisms of P‐gp inhibition are excellent leads for the development of co‐therapeutics for the treatment of multidrug resistances.