The cyclin-dependent kinase inhibitor roscovitine and the nucleoside analog sangivamycin induce apoptosis in caspase-3 deficient breast cancer cells independent of caspase mediated P-glycoprotein cleavage: Implications for therapy of drug resistant breast cancers

Resistance to multiple chemotherapeutic agents is a common clinical problem which can arise during cancer treatment. Drug resistance often involves overexpression of the multidrug resistance MDR1 gene, encoding P-glycoprotein (P-gp), a 170-kDa glycoprotein belonging to the ATP-binding cassette superfamily of membrane transporters. We have recently demonstrated apoptosis-induced, caspase-3-dependent P-gp cleavage in human T-lymphoblastoid CEM-R VBL100 cells. However, P-gp contain many aspartate residues which could be targeted by caspases other than caspase-3. To test whether other caspases could cleave P-gp in vivo, we investigated the fate of P-gp during roscovitine- and sangivamycin- induced apoptosis in MCF7 human breast cancer cells, as they lack functional caspase-3. MCF7 cells were stably transfected with human cDNA encoding P-gp. P-gp was cleaved in vitro by purified recombinant caspase-3, -6 and -7. However, P-gp cleavage was not detected in vivo in MCF7 cells induced to undergoing apoptosis by either roscovitine or sangivamycin, despite activation of both caspase-6 and -7. Interestingly, P-gp overexpressing MCF7 cells were more sensitive to either roscovitine or sangivamycin than wild-type cells, suggesting a novel potential therapeutic strategy against P-gp overexpressing cells. Taken together, our results support the concept that caspase-3 is the only caspase responsible for in vivo cleavage of P-gp and also highlight small molecules which could be effective in treating P-gp overexpressing cancers.     

Crystal structures of a high-affinity macrocyclic peptide mimetic in complex with the Grb2 SH2 domain

The high-affinity binding of the growth factor receptor-bound protein 2 (Grb2) SH2 domain to tyrosine-phosphorylated cytosolic domains of receptor tyrosine kinases (RTKs) is an attractive target for therapeutic intervention in many types of cancer. We report here two crystal forms of a complex between the Grb2 SH2 domain and a potent non-phosphorus-containing macrocyclic peptide mimetic that exhibits significant anti-proliferative effects against erbB-2-dependent breast cancers. This agent represents a "second generation" inhibitor with greatly improved binding affinity and bio-availability compared to its open-chain counterpart. The structures were determined at 2.0A and 1.8A with one and two domain-swapped dimers per asymmetric unit, respectively. The mode of binding and specific interactions between the protein and the inhibitor provide insight into the high potency of this class of macrocylic compounds and may aid in further optimization as part of the iterative rational drug design process.     

Crystal structures of beryllium fluoride-free and beryllium fluoride-bound CheY in complex with the conserved C-terminal peptide of CheZ reveal dual binding modes specific to CheY conformation

Chemotaxis, the environment-specific swimming behavior of a bacterial cell is controlled by flagellar rotation. The steady-state level of the phosphorylated or activated form of the response regulator CheY dictates the direction of flagellar rotation. CheY phosphorylation is regulated by a fine equilibrium of three phosphotransfer activities: phosphorylation by the kinase CheA, its auto-dephosphorylation and dephosphorylation by its phosphatase CheZ. Efficient dephosphorylation of CheY by CheZ requires two spatially distinct protein-protein contacts: tethering of the two proteins to each other and formation of an active site for dephosphorylation. The former involves interaction of phosphorylated CheY with the small highly conserved C-terminal helix of CheZ (CheZ(C)), an indispensable structural component of the functional CheZ protein. To understand how the CheZ(C) helix, representing less than 10% of the full-length protein, ascertains molecular specificity of binding to CheY, we have determined crystal structures of CheY in complex with a synthetic peptide corresponding to 15 C-terminal residues of CheZ (CheZ(200-214)) at resolutions ranging from 2.0 A to 2.3A. These structures provide a detailed view of the CheZ(C) peptide interaction both in the presence and absence of the phosphoryl analog, BeF3-. Our studies reveal that two different modes of binding the CheZ(200-214) peptide are dictated by the conformational state of CheY in the complex. Our structures suggest that the CheZ(C) helix binds to a "meta-active" conformation of inactive CheY and it does so in an orientation that is distinct from the one in which it binds activated CheY. Our dual binding mode hypothesis provides implications for reverse information flow in CheY and extends previous observations on inherent resilience in CheY-like signaling domains.     

Structure of antibody F425-B4e8 in complex with a V3 peptide reveals a new binding mode for HIV-1 neutralization

F425-B4e8 (B4e8) is a monoclonal antibody isolated from a human immunodeficiency virus type 1 (HIV-1)-infected individual that recognizes the V3 variable loop on the gp120 subunit of the viral envelope spike. B4e8 neutralizes a subset of HIV-1 primary isolates from subtypes B, C and D, which places this antibody among the very few human anti-V3 antibodies with notable cross-neutralizing activity. Here, the crystal structure of the B4e8 Fab′ fragment in complex with a 24-mer V3 peptide (RP142) at 2.8 Å resolution is described. The complex structure reveals that the antibody recognizes a novel V3 loop conformation, featuring a five-residue α-turn around the conserved GPGRA apex of the β-hairpin loop. In agreement with previous mutagenesis analyses, the Fab′ interacts primarily with V3 through side-chain contacts with just two residues, Ile^P309 and Arg^P315, while the remaining contacts are to the main chain. The structure helps explain how B4e8 can tolerate a certain degree of sequence variation within V3 and, hence, is able to neutralize an appreciable number of different HIV-1 isolates.     

Solution structure of the phosphotyrosine binding (PTB) domain of human tensin2 protein in complex with deleted in liver cancer 1 (DLC1) peptide reveals a novel peptide binding mode

The protein deleted in liver cancer 1 (DLC1) interacts with the tensin family of focal adhesion proteins to play a role as a tumor suppressor in a wide spectrum of human cancers. This interaction has been proven to be crucial to the oncogenic inhibitory capacity and focal adhesion localization of DLC1. The phosphotyrosine binding (PTB) domain of tensin2 predominantly interacts with a novel site on DLC1, not the canonical NPXY motif. In this study, we characterized this interaction biochemically and determined the complex structure of tensin2 PTB domain with DLC1 peptide by NMR spectroscopy. Our HADDOCK-derived complex structure model elucidates the molecular mechanism by which tensin2 PTB domain recognizes DLC1 peptide and reveals a PTB-peptide binding mode that is unique in that peptide occupies the binding site opposite to the canonical NPXY motif interaction site with the peptide utilizing a non-canonical binding motif to bind in an extended conformation and that the N-terminal helix, which is unique to some Shc- and Dab-like PTB domains, is required for binding. Mutations of crucial residues defined for the PTB-DLC1 interaction affected the co-localization of DLC1 and tensin2 in cells and abolished DLC1-mediated growth suppression of hepatocellular carcinoma cells. This tensin2 PTB-DLC1 peptide complex with a novel binding mode extends the versatile binding repertoire of the PTB domains in mediating diverse cellular signaling pathways as well as provides a molecular and structural basis for better understanding the tumor-suppressive activity of DLC1 and tensin2.     

Molecular modeling of human acidic mammalian chitinase in complex with the natural-product cyclopentapeptide chitinase inhibitor argifin

Human acidic mammalian chitinase (hAMCase) is an attractive target for developing anti-asthma medications. We used a variety of computational methods to investigate the interaction between hAMCase and the natural-product cyclopentapeptide chitinase inhibitor argifin. The three-dimensional structure of hAMCase was first constructed using homology modeling. The interaction mode and binding free energy between argifin and hAMCase were then examined by the molecular-docking calculation and the molecular mechanics Poisson–Boltzmann surface area method combined with molecular dynamics simulation, respectively. The results suggested that argifin binds to hAMCase in a similar fashion to the interaction mode observed in the crystal structure of argifin-human chitotriosidase complex, and possesses inhibitory activity against hAMCase in the micromolar range. We further designed argifin derivatives expected to be selective for hAMCase.     

Structure of a benzylidene derivative of 9(10H)-anthracenone in complex with tubulin provides a rationale for drug design

Microtubules are composed of αβ-tubulin heterodimers and have been treated as highly attractive targets for antitumor drugs. A broad range of agents bind to tubulin and interfere with microtubule assembly, including colchicine binding site inhibitors (CBSIs). Tubulin Polymerization Inhibitor I (TPI1), a benzylidene derivative of 9(10H)-anthracenone, is a CBSI that inhibits the assembly of microtubules. However, for a long time, the design and development of anthracenone family drugs have been hindered by the lack of structural information of the tubulin-agent complex. Here we report a 2.3 Å crystal structure of tubulin complexed with TPI1, the first structure of anthracenone family agents. This complex structure reveals the interactions between TPI1 and tubulin, and thus provides insights into the development of new anthracenone derivatives targeting the colchicine binding site.     

Computational analysis of BACE1-ligand complex crystal structures and linear discriminant analysis for identification of BACE1 inhibitors with anti P-glycoprotein binding property

More than 100 years of research on Alzheimer’s disease didn’t yield a potential cure for this dreadful disease. Poor Blood Brain Barrier (BBB) permeability and P-glycoprotein binding of BACE1 inhibitors are the major causes for the failure of these molecules during clinical trials. The design of BACE1 inhibitors with a balance of sufficient affinity to the binding site and little or no interaction with P-glycoproteins is indispensable. Identification and understanding of protein–ligand interactions are essential for ligand optimization process. Structure-based drug design (SBDD) efforts led to a steady accumulation of BACE1-ligand crystal complexes in the PDB. This study focuses on analyses of 153 BACE1-ligand complexes for the direct contacts (hydrogen bonds and weak interactions) observed between protein and ligand and indirect contacts (water-mediated hydrogen bonds), observed in BACE1-ligand complex crystal structures. Intraligand hydrogen bonds were analyzed, with focus on ligand P-glycoprotein efflux. The interactions are dissected specific to subsites in the active site and discussed. The observed protein-ligand and intraligand interactions were used to develop the linear discriminant model for the identification of BACE1 inhibitors with less or no P-glycoprotein binding property. Excellent statistical results and model’s ability to correctly predict a new data-set with an accuracy of 92% is achieved. The results are retrospectively analyzed to give input for the design of potential BACE1 inhibitors.     

Crystal structures of Tritrichomonasfoetus inosine monophosphate dehydrogenase in complex with substrate,cofactor and analogs: a structural basis for the random-in ordered-out kinetic mechanism

The enzyme inosine monophosphate dehydrogenase (IMPDH) is responsible for the rate-limiting step in guanine nucleotide biosynthesis. Because it is up-regulated in rapidly proliferating cells, human type II IMPDH is actively targeted for immunosuppressive, anticancer, and antiviral chemotherapy. The enzyme employs a random-in ordered-out kinetic mechanism where substrate or cofactor can bind first but product is only released after the cofactor leaves. Due to structural and kinetic differences between mammalian and microbial enzymes, most drugs that are successful in the inhibition of mammalian IMPDH are far less effective against the microbial forms of the enzyme. It is possible that with greater knowledge of the structural mechanism of the microbial enzymes, an effective and selective inhibitor of microbial IMPDH will be developed for use as a drug against multi-drug resistant bacteria and protists. The high-resolution crystal structures of four different complexes of IMPDH from the protozoan parasite Tritrichomonas foetus have been solved: with its substrate IMP, IMP and the inhibitor mycophenolic acid (MPA), the product XMP with MPA, and XMP with the cofactor NAD(+). In addition, a potassium ion has been located at the dimer interface. A structural model for the kinetic mechanism is proposed.     

A metallo-beta-lactamase enzyme in action: crystal structures of the monozinc carbapenemase CphA and its complex with biapenem

One strategy developed by bacteria to resist the action of beta-lactam antibiotics is the expression of metallo-beta-lactamases. CphA from Aeromonas hydrophila is a member of a clinically important subclass of metallo-beta-lactamases that have only one zinc ion in their active site and for which no structure is available. The crystal structures of wild-type CphA and its N220G mutant show the structural features of the active site of this enzyme, which is modeled specifically for carbapenem hydrolysis. The structure of CphA after reaction with a carbapenem substrate, biapenem, reveals that the enzyme traps a reaction intermediate in the active site. These three X-ray structures have allowed us to propose how the enzyme recognizes carbapenems and suggest a mechanistic pathway for hydrolysis of the beta-lactam. This will be relevant for the design of metallo-beta-lactamase inhibitors as well as of antibiotics that escape their hydrolytic activity.     

X-ray structure based evaluation of analogs of citalopram: Compounds with increased affinity and selectivity compared with R-citalopram for the allosteric site (S2) on hSERT

The recent publication of X-ray structures of SERT includes structures with the potent antidepressant S-Citalopram (S-Cit). Earlier predictions of ligand binding at both a primary (S1) and an allosteric modulator site (S2), were confirmed. We provide herein examples of a series of Citalopram analogs, showing distinct structure-activity relationship (SAR) at both sites that is independent of the SAR at the other site. Analogs with a higher affinity and selectivity than benchmark R-Citalopram (R-Cit) for the S2 versus the S1 site were identified. We deploy structural and computational analyses to explain this SAR and demonstrate the potential utility of the newly emerging X-ray structures within the neurotransmitter:sodium Symporter family for drug design.     

A combination of cation exchange and ligand-affinity chromatography for purification of two molecular species of the folate binding protein in human milk,one equipped with a hydrophobic glycosyl phosphatidylinositol tail: characterization of hydrophobicity and electrical charge

Cation exchange chromatography combined with ligand (methotrexate) affinity chromatography on a column desorbed with a pH-gradient was used for separation and large scale purification of two folate binding proteins in human milk. One of the proteins, which had a molecular size of 27 kDa on gel filtration and eluted from the affinity column at pH 5-6 was a cleavage product of a 100 kDa protein eluted at pH 3-4 as evidenced by identical N-terminal amino acid sequences and a reduction in the molecular size of the latter protein to 27 kDa after cleavage of its hydrophobic glycosylphosphatidyl-inositol tail that inserts into Triton X-100 micelles. Chromatofocusing showed that both proteins possessed multiple isoelectric points within the pH range 7-9. The 100 kDa protein exhibited a high affinity to hydrophobic interaction chromatographic gels, whereas this was only the case with unliganded forms of the 27 kDa protein indicative of a decrease in the hydrophobicity of the protein after ligand binding.     

Determination of topological structure of ARL6ip1 in cells: Identification of the essential binding region of ARL6ip1 for conophylline

Conophylline (CNP) has various biological activities, such as insulin production. A recent study identified ADP-ribosylation factor-like 6-interacting protein 1 (ARL6ip1) as a direct target protein of CNP. In this study, we revealed that ARL6ip1 is a three-spanning transmembrane protein and determined the CNP-binding domain of ARL6ip1 by deletion mutation analysis of ARL6ip1 with biotinyl-amino-CNP. These results suggest that CNP is expected to be useful for future investigation of ARL6ip1 function in cells. Because of the anti-apoptotic function of ARL6ip1, CNP may be an effective therapeutic drug and/or a novel chemosensitizer for human cancers and other diseases.     

The NDUFB6 subunit of the mitochondrial respiratory chain complex I is required for electron transfer activity: A proof of principle study on stable and controlled RNA interference in human cell lines

Molecular bases of inherited deficiencies of mitochondrial respiratory chain complex I are still unknown in a high proportion of patients. Among 45 subunits making up this large complex, more than half has unknown function(s). Understanding the function of these subunits would contribute to our knowledge on mitochondrial physiology but might also reveal that some of these subunits are not required for the catalytic activity of the complex. A direct consequence of this finding would be the reduction of the number of candidate genes to be sequenced in patients with decreased complex I activity. In this study, we tested two different methods to stably extinct complex I subunits in cultured cells. We first found that lentivirus-mediated shRNA expression frequently resulted in the unpredicted extinction of additional gene(s) beside targeted ones. This can be ascribed to uncontrolled genetic material insertions in the genome of the host cell. This approach thus appeared inappropriate to study unknown functions of a gene. Next, we found it possible to specifically extinct a CI subunit gene by direct insertion of a miR targeting CI subunits in a Flp site (HEK293 Flp-In cells). By using this strategy we unambiguously demonstrated that the NDUFB6 subunit is required for complex I activity, and defined conditions suitable to undertake a systematic and stable extinction of the different supernumerary subunits in human cells.     

Computer modelling in combination with in vitro studies reveals similar binding affinities of Drosophila Crumbs for the PDZ domains of Stardust and DmPar-6

Formation of multiprotein complexes is a common theme to pattern a cell, thereby generating spatially and functionally distinct entities at specialised regions. Central components of these complexes are scaffold proteins, which contain several protein-protein interaction domains and provide a platform to recruit a variety of additional components. There is increasing evidence that protein complexes are dynamic structures and that their components can undergo various interactions depending on the cellular context. However, little is known so far about the factors regulating this behaviour. One evolutionarily conserved protein complex, which can be found both in Drosophila and mammalian epithelial cells, is composed of the transmembrane protein Crumbs/Crb3 and the scaffolding proteins Stardust/Pals1 and DPATJ/PATJ, respectively, and localises apically to the zonula adherens. Here we show by in vitro analysis that, similar as in vertebrates, the single PDZ domain of Drosophila DmPar-6 can bind to the four C-terminal amino acids (ERLI) of the transmembrane protein Crumbs. To further evaluate the binding capability of Crumbs to DmPar-6 and the MAGUK protein Stardust, analysis of the PDZ structural database and modelling of the interactions between the C-terminus of Crumbs and the PDZ domains of these two proteins were performed. The results suggest that both PDZ domains bind Crumbs with similar affinities. These data are supported by quantitative yeast two-hybrid interactions. In vivo analysis performed in cell cultures and in the Drosophila embryo show that the cytoplasmic domain of Crumbs can recruit DmPar-6 and DaPKC to the plasma membrane. The data presented here are discussed with respect to possible dynamic interactions between these proteins.     

Structure of aldehyde reductase in ternary complex with coenzyme and the potent 20alpha-hydroxysteroid dehydrogenase inhibitor 3,5-dichlorosalicylic acid: implications for inhibitor binding and selectivity

The structure of aldehyde reductase (ALR1) in ternary complex with the coenzyme NADPH and 3,5-dichlorosalicylic acid (DCL), a potent inhibitor of human 20α-hydroxysteroid dehydrogenase (AKR1C1), was determined at a resolution of 2.41 Å. The inhibitor formed a network of hydrogen bonds with the active site residues Trp22, Tyr50, His113, Trp114 and Arg312. Molecular modelling calculations together with inhibitory activity measurements indicated that DCL was a less potent inhibitor of ALR1 (256-fold) when compared to AKR1C1. In AKR1C1, the inhibitor formed a 10-fold stronger binding interaction with the catalytic residue (Tyr55), non-conserved hydrogen bonding interaction with His222, and additional van der Waals contacts with the non-conserved C-terminal residues Leu306, Leu308 and Phe311 that contribute to the inhibitor’s selectivity advantage for AKR1C1 over ALR1.     

Metabolism Comparative Cytotoxicity Assay (MCCA) and Cytotoxic Metabolic Pathway Identification Assay (CMPIA) with cryopreserved human hepatocytes for the evaluation of metabolism-based cytotoxicity in vitro: proof-of-concept study with aflatoxin B1

Recently, we have improved the cryopreservation procedures for human hepatocytes, leading to cells that can be cultured after thawing (“plateable” cryopreserved human hepatocytes). The ability to culture cryopreserved human hepatocytes allows application of the cells for prolonged incubations such as long-term (days) metabolism studies, enzyme induction studies, and cytotoxicity studies. We report here the application of the plateable cryopreserved human hepatocytes to evaluate the relationship between xenobiotic metabolism and toxicity. Two assays were developed: The Metabolism Comparative Cytotoxicity Assay (MCCA) and the Cytotoxic Metabolic Pathway Identification Assay (CMPIA). The MCCA was designed for the initial identification of the role of metabolism in cytotoxicity by comparing the cytotoxic potential of a toxicant in a metabolically competent (primary human hepatocytes) and a metabolically incompetent (Chinese hamster ovary (CHO)) cell type, as well as the evaluation of the role of P450 metabolism by comparing the cytotoxicity of the toxicant in question in human hepatocytes in the presence and absence of a nonspecific, irreversible P450 inhibitor, 1-aminobenzotriazole (ABT). The CMPIA was designed for the identification of the P450 isoforms involved in metabolic activation via the evaluation of the cytotoxicity of the toxicant in the presence and absence of isoform-selective P450 inhibitors. Results of a proof-of-concept study with the MCCA and CMPIA with a known hepatotoxicant, aflatoxin B1 (AFB1), are reported. AFB1 is known to require P450 metabolism for its toxicity. In the MCCA, AFB1 was found to have significantly higher cytotoxicity in human hepatocytes than CHO cells, therefore confirming its requirement for biotransformation to be toxic. ABT was found to effectively attenuate AFB1 cytotoxicity, confirming that P450 metabolism was involved in its metabolic activation. In the CMPIA, AFB1 cytotoxicity was found to be attenuated by ketoconazole and diethyldithiocarbamate, but not by furafylline, quinidine, and sulfaphenazole. Results with the isoform-selective inhibitors suggest that the isoforms inhibited by ketoconazole (mainly CYP3A4) and diethyldithiocarbamate (mainly CYP2A6, and CYP2E1), but not the isoforms inhibited by furafylline (mainly CYP1A2), sulfaphenazole (mainly CYP2C9) and quinidine (mainly CYP2D6) are involved in the metabolic activation of AFB1. This proof-of-concept study suggests that MCCA and CMPIA with cryopreserved human hepatocytes are potentially useful for the evaluation of the relationship between human xenobiotic metabolism and toxicity.