Heme Binding Biguanides Target Cytochrome P450-Dependent Cancer Cell Mitochondria.

The mechanisms by which cancer cell-intrinsic CYP monooxygenases promote tumor progression are largely unknown. CYP3A4 was unexpectedly associated with breast cancer mitochondria and synthesized arachidonic acid (AA)-derived epoxyeicosatrienoic acids (EETs), which promoted the electron transport chain/respiration and inhibited AMPKα. CYP3A4 knockdown activated AMPKα, promoted autophagy, and prevented mammary tumor formation. The diabetes drug metformin inhibited CYP3A4-mediated EET biosynthesis and depleted cancer cell-intrinsic EETs. Metformin bound to the active-site heme of CYP3A4 in a co-crystal structure, establishing CYP3A4 as a biguanide target. Structure-based design led to discovery of N1-hexyl-N5-benzyl-biguanide (HBB), which bound to the CYP3A4 heme with higher affinity than metformin. HBB potently and specifically inhibited CYP3A4 AA epoxygenase activity. HBB also inhibited growth of established ER+ mammary tumors and suppressed intratumoral mTOR. CYP3A4 AA epoxygenase inhibition by biguanides thus demonstrates convergence between eicosanoid activity in mitochondria and biguanide action in cancer, opening a new avenue for cancer drug discovery.

An Innovative High-Throughput Screening Approach for Discovery of Small Molecules That Inhibit TNF Receptors.

Tumor necrosis factor receptor 1 (TNFR1) is a transmembrane receptor that binds tumor necrosis factor or lymphotoxin-alpha and plays a critical role in regulating the inflammatory response. Upregulation of these ligands is associated with inflammatory and autoimmune diseases. Current treatments reduce symptoms by sequestering free ligands, but this can cause adverse side effects by unintentionally inhibiting ligand binding to off-target receptors. Hence, there is a need for new small molecules that specifically target the receptors, rather than the ligands. Here, we developed a TNFR1 FRET biosensor expressed in living cells to screen compounds from the NIH Clinical Collection. We used an innovative high-throughput fluorescence lifetime screening platform that has exquisite spatial and temporal resolution to identify two small-molecule compounds, zafirlukast and triclabendazole, that inhibit the TNFR1-induced IκBα degradation and NF-κB activation. Biochemical and computational docking methods were used to show that zafirlukast disrupts the interactions between TNFR1 pre-ligand assembly domain (PLAD), whereas triclabendazole acts allosterically. Importantly, neither compound inhibits ligand binding, proving for the first time that it is possible to inhibit receptor activation by targeting TNF receptor-receptor interactions. This strategy should be generally applicable to other members of the TNFR superfamily, as well as to oligomeric receptors in general.

Methemoglobin Formation and Characterization of Hemoglobin Adducts of Carcinogenic Aromatic Amines and Heterocyclic Aromatic Amines.

Arylamines (AAs) and heterocyclic aromatic amines (HAAs) are structurally related carcinogens formed during the combustion of tobacco or cooking of meat. They undergo cytochrome P450 mediated N-hydroxylation to form metabolites which bind to DNA and lead to mutations. The N-hydroxylated metabolites of many AAs also can undergo a co-oxidation reaction with oxy-hemolgobin (HbO2) to form methemoglobin (met-Hb) and the arylnitroso intermediates, which react with the ß-Cys(93) chain of Hb to form Hb-arylsulfinamide adducts. The biochemistry of arylamine metabolism has been exploited to biomonitor certain AAs through their Hb arylsulfinamide adducts in humans. We examined the reactivity of HbO2 with the N-hydroxylated metabolites of 4-aminobiphenyl (ABP, HONH-ABP), aniline (ANL, HONH-ANL), and the HAAs 2-amino-9H-pyrido[2,3-b]indole (AαC, HONH-AαC), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP, HONH-PhIP), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx, HONH-MeIQx). HONH-ABP, HO-ANL, and HONH-AαC induced methemoglobinemia and formed Hb sulfinamide adducts. However, HONH-MeIQx and HONH-PhIP did not react with the oxy-heme complex, and met-Hb formation and chemical modification of the ß-Cys(93) residue were negligible. Molecular modeling studies showed that the distances between the H-ON-AA or H-ON-HAA substrates and the oxy-heme complex of HbO2 were too far away to induce methemoglobinemia. Different conformational changes in flexible helical and loop regions around the heme pocket induced by the H-ON-AA or H-ON-HAAs may explain the different proclivities of these chemicals to induce methemoglobinemia. Hb-Cys(93ß) sulfinamide and sulfonamide adducts of ABP, ANL, and AαC were identified, by Orbitrap MS, following the proteolysis of Hb with trypsin, Glu-C, or Lys-C. Hb sulfinamide and sulfonamide adducts of ABP were identified in the blood of mice exposed to ABP, by Orbitrap MS. This is the first report of the identification of intact Hb sulfinamide adducts of carcinogenic AAs in vivo. The high reactivity of HONH-AαC with HbO2 suggests that the Hb sulfinamide adduct of AαC may be a promising biomarker of exposure to this HAA in humans.

Probing the S2' Subsite of the Anthrax Toxin Lethal Factor Using Novel N-Alkylated Hydroxamates.

The lethal factor (LF) enzyme secreted by Bacillus anthracis is a zinc hydrolase that is chiefly responsible for anthrax-related cell death. Although many studies of the design of small molecule LF inhibitors have been conducted, no LF inhibitor is yet available as a therapeutic agent. Inhibitors with considerable chemical diversity have been developed and investigated; however, the LF S2' subsite has not yet been systematically explored as a potential target for lead optimization. Here we present synthesis, experimental evaluation, modeling, and structural biology for a novel series of sulfonamide hydroxamate LF inhibitor analogues specifically designed to extend into, and probe chemical preferences of, this S2' subsite. We discovered that this region accommodates a wide variety of chemical functionalities and that a broad selection of ligand structural modifications directed to this area can be incorporated without significant deleterious alterations in biological activity. We also identified key residues in this subsite that can potentially be targeted to improve inhibitor binding.

Identification of novel anthrax toxin countermeasures using in silico methods.

Anthrax is an acute infectious disease caused by the spore-forming, gram-positive, rod-shaped bacterium Bacillus anthracis. The anthrax toxin lethal factor (LF) is the primary anthrax toxin component responsible for cytotoxicity and host death and has been a heavily researched target for design of postexposure therapeutics in the event of a bioterror attack. Various computer-aided drug design methodologies have proven useful for pinpointing new antianthrax drug scaffolds, optimizing existing leads and probes, and elucidating key mechanisms of action. We present a selection of in silico virtual screening protocols incorporating docking and scoring, shape-based searching, and pharmacophore mapping techniques to identify and prioritize small molecules with potential biological activity against LF. We also recommend screening parameters that have been shown to increase the accuracy and reliability of these computational results.

Development of a comprehensive, validated pharmacophore hypothesis for anthrax toxin lethal factor (LF) inhibitors using genetic algorithms, Pareto scoring, and structural biology.

Anthrax is an acute infectious disease caused by the spore-forming bacterium Bacillus anthracis. The anthrax toxin lethal factor (LF), an 89-kDa zinc hydrolase secreted by the bacilli, is the toxin component chiefly responsible for pathogenesis and has been a popular target for rational and structure-based drug design. Although hundreds of small-molecule compounds have been designed to target the LF active site, relatively few reported inhibitors have exhibited activity in cell-based assays, and no LF inhibitor is currently available to treat or prevent anthrax. This study presents a new pharmacophore map assembly, validated by experiment, designed to rapidly identify and prioritize promising LF inhibitor scaffolds from virtual compound libraries. The new hypothesis incorporates structural information from all five available LF enzyme-inhibitor complexes deposited in the Protein Data Bank (PDB) and is the first LF pharmacophore map reported to date that includes features representing interactions involving all three key subsites of the LF catalytic binding region. In a wide-ranging validation study on all 546 compounds for which published LF biological activity data exist, this model displayed strong selectivity toward nanomolar-level LF inhibitors, successfully identifying 72.1% of existing nanomolar-level compounds in an unbiased test set, while rejecting 100% of weakly active (>100 µM) compounds. In addition to its capabilities as a database searching tool, this comprehensive model points to a number of key design principles and previously unidentified ligand-receptor interactions that are likely to influence compound potency.

Design, synthesis and evaluation of analogs of initiation factor 4E (eIF4E) cap-binding antagonist Bn7-GMP.

Aberrant regulation of cap-dependent translation has been frequently observed in the development of cancer. Association of the cap-binding protein eIF4E with N(7)-methylated guanosine capped mRNA is the rate limiting step governing translation initiation; and therefore represents an attractive process for cancer drug discovery. Previously, replacement of the 7-Me group of the Me(7)-guanosine monophosphate with a benzyl group has been found to increase binding affinity to eIF4E. Recent X-ray crystallographic studies have revealed that the cap-binding pocket undergoes a unique structural change in order to accommodate the benzyl group. To explore the structure-activity relationships governing the affinity of N(7)-benzylated guanosine monophosphate (Bn(7)-GMP) for eIF4E, we virtually screened a library of 80 Bn(7)-GMP analogs utilizing CombiGlide as implemented in Schrodinger. A subset library of substituted Bn(7)-GMP analogs was synthesized and their dissociation constants (K(d)) were determined. Due to the poor correlation between docking/scoring results and experimental binding affinities, three-dimensional quantitative structure-activity relationship (3D-QSAR) calculations were performed. Two highly predictive and self-consistent CoMFA (comparative molecular field analysis) and CoMSIA (comparative molecular similarity indices analysis) models were derived and optimized. These models may be useful for the future design of eIF4E cap-binding antagonists.

Total synthesis and evaluation of C26-hydroxyepothilone D derivatives for photoaffinity labeling of beta-tubulin.

Three photoaffinity labeled derivatives of epothilone D were prepared by total synthesis, using efficient novel asymmetric synthesis methods for the preparation of two important synthetic building blocks. The key step for the asymmetric synthesis of (S,E)-3-(tert-butyldimethylsilyloxy)-4-methyl-5-(2-methylthiazol-4-yl)pent-4-enal involved a ketone reduction with (R)-Me-CBS-oxazaborolidine. For the synthesis of (5S)-5,7-di[(tert-butyldimethylsilyl)oxy]-4,4-dimethylheptan-3-one an asymmetric Noyori reduction of a beta-ketoester was employed. The C26 hydroxyepothilone D derivative was constructed following a well-established total synthesis strategy and the photoaffinity labels were attached to the C26 hydroxyl group. The photoaffinity analogues were tested in a tubulin assembly assay and for cytotoxicity against MCF-7 and HCT-116 cancer cell lines. The 3- and 4-azidobenzoic acid analogues were found to be as active as epothilone B in a tubulin assembly assay, but demonstrated significantly reduced cellular cytotoxicity compared to epothilone B. The benzophenone analogue was inactive in both assays. Docking and scoring studies were conducted that suggested that the azide analogues can bind to the epothilone binding site, but that the benzophenone analogue undergoes a sterically driven ligand rearrangement that interrupts all hydrogen bonding and therefore protein binding. Photoaffinity labeling studies with the 3-azidobenzoic acid derivative did not identify any covalently labeled peptide fragments, suggesting that the phenylazido side chain was predominantly solvent-exposed in the bound conformation.

Identification of novel non-hydroxamate anthrax toxin lethal factor inhibitors by topomeric searching, docking and scoring, and in vitro screening.

Anthrax is an infectious disease caused by Bacillus anthracis, a Gram-positive, rod-shaped, anaerobic bacterium. The lethal factor (LF) enzyme is secreted by B. anthracis as part of a tripartite exotoxin and is chiefly responsible for anthrax-related cytotoxicity. As LF can remain in the system long after antibiotics have eradicated B. anthracis from the body, the preferred therapeutic modality would be the administration of antibiotics together with an effective LF inhibitor. Although LF has garnered a great deal of attention as an attractive target for rational drug design, relatively few published inhibitors have demonstrated activity in cell-based assays and, to date, no LF inhibitor is available as a therapeutic or preventive agent. Here we present a novel in silico high-throughput virtual screening protocol that successfully identified 5 non-hydroxamic acid small molecules as new, preliminary LF inhibitor scaffolds with low micromolar inhibition against that target, resulting in a 12.8% experimental hit rate. This protocol screened approximately 35 million nonredundant compounds for potential activity against LF and comprised topomeric searching, docking and scoring, and drug-like filtering. Among these 5 hit compounds, none of which has previously been identified as a LF inhibitor, three exhibited experimental IC(50) values less than 100 microM. These three preliminary hits may potentially serve as scaffolds for lead optimization as well as templates for probe compounds to be used in mechanistic studies. Notably, our docking simulations predicted that these novel hits are likely to engage in critical ligand-receptor interactions with nearby residues in at least two of the three (S1', S1-S2, and S2') subsites in the LF substrate binding area. Further experimental characterization of these compounds is in process. We found that micromolar-level LF inhibition can be attained by compounds with non-hydroxamate zinc-binding groups that exhibit monodentate zinc chelation as long as key hydrophobic interactions with at least two LF subsites are retained.

Total synthesis and evaluation of 22-(3-azidobenzoyloxy)methyl epothilone C for photoaffinity labeling of beta-tubulin.

The total synthesis of 22-(3-azidobenzoyloxy)methyl epothilone C is described as a potential photoaffinity probe to elucidate the beta-tubulin binding site. A sequential Suzuki-aldol-Yamaguchi macrolactonization strategy was utilized employing a novel derivatized C1-C6 fragment. The C22-functionalized analog exhibited good activity in microtubule assembly assays, but cytotoxicity was significantly reduced. Molecular modeling simulations indicated that excessive steric bulk in the C22 position is accommodated by the large hydrophobic pocket of the binding site. Photoaffinity labeling studies were inconclusive suggesting non-specific labeling.

Development of a new pharmacophore model that discriminates active compstatin analogs.

Compstatin and its active peptide analogs can potentially be used for therapeutic purposes because their binding to the third component of complement prohibits its conversion into the proteolytically activated form of the third component of complement, thus inhibiting complement cascades in all three complement pathways. Mallik and Morikis built three quasi-dynamic pharmacophore models for compstatin peptide analogs before, but only nine compstatin peptide analogs were incorporated in their study and the most active compstatin analog had only medium inhibitory activity. Since then, many more compstatin analogs have been synthesized and their inhibitory activities tested. Furthermore, the X-ray structure of AcCompNH2-V4W-H9A bound to the third component of complement has become available (PDB ID: 2QKI). In this paper, we utilized all the new information and built a new pharmacophore model using a distinct approach. Our model demonstrated good performance in a separate test set of 82 compstatin analogs: it accurately identified 70% of the analogs of medium or high inhibitory activities and misclassified only 8.5% of the analogs of low or no inhibitory activities. The results proved our pharmacophore model to be a filter of great sensitivity and specificity.

Total synthesis and evaluation of C25-benzyloxyepothilone C for tubulin assembly and cytotoxicity against MCF-7 breast cancer cells.

The total synthesis of C25-benzyloxy epothilone C is described. A sequential Suzuki-Aldol-Yamaguchi macrolactonization strategy was utilized employing a novel derivatized C8-C12 fragment. The C25-benzyloxy analog exhibited significantly reduced biological activity in microtubule assembly and cytotoxicity assays. Molecular modeling simulations indicated that excessive steric bulk in the C25 position may reduce activity by disrupting key hydrogen bonds that are crucial for epothilone binding to beta-tubulin.

Comparative molecular modeling of Anopheles gambiae CYP6Z1, a mosquito P450 capable of metabolizing DDT.

One of the challenges faced in malarial control is the acquisition of insecticide resistance that has developed in mosquitoes that are vectors for this disease. Anopheles gambiae, which has been the major mosquito vector of the malaria parasite Plasmodium falciparum in Africa, has over the years developed resistance to insecticides including dieldrin, 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), and pyrethroids. Previous microarray studies using fragments of 230 An. gambiae genes identified five P450 loci, including CYP4C27, CYP4H15, CYP6Z1, CYP6Z2, and CYP12F1, that showed significantly higher expression in the DDT-resistant ZAN/U strain compared with the DDT-susceptible Kisumu strain. To predict whether either of the CYP6Z1 and CYP6Z2 proteins might potentially metabolize DDT, we generated and compared molecular models of these two proteins with and without DDT docked in their catalytic sites. This comparison indicated that, although these two CYP6Z proteins share high sequence identity, their metabolic profiles were likely to differ dramatically from the larger catalytic site of CYP6Z1, potentially involved in DDT metabolism, and the more constrained catalytic site of CYP6Z2, not likely to metabolize DDT. Heterologous expressions of these proteins have corroborated these predictions: only CYP6Z1 is capable of metabolizing DDT. Overlays of these models indicate that slight differences in the backbone of SRS1 and variations of side chains in SRS2 and SRS4 account for the significant differences in their catalytic site volumes and DDT-metabolic capacities. These data identify CYP6Z1 as one important target for inhibitor design aimed at inactivating insecticide-metabolizing P450s in natural populations of this malarial mosquito.

Helicoverpa zea CYP6B8 and CYP321A1: different molecular solutions to the problem of metabolizing plant toxins and insecticides.

Under continual exposure to naturally occurring plant toxins and synthetic insecticides, insects have evolved cytochrome P450 monooxygenases (P450s) capable of metabolizing a wide range of structurally different compounds. Two such P450s, CYP6B8 and CYP321A1, expressed in Helicoverpa zea (a lepidopteran) in response to plant allelochemicals and plant signaling molecules metabolize these compounds with varying efficiencies. While sequence alignments of these proteins indicate highly divergent substrate recognition sites (SRSs), homology models developed for them indicate that the two active site cavities have essentially the same volume with distinct shapes dictated by side-chain differences in SRS1 and SRS5. CYP6B8 has a narrower active site cavity extending from substrate access channel pw2a with a very narrow access to the ferryl oxygen atom. This predicted shape suggests that bulkier molecules bind further from the ferryl oxygen at positions that are not as effectively metabolized. In contrast, CYP321A1 is predicted to have a more spacious cavity allowing larger molecules to access the heme-bound oxygen. The metabolic profiles for several plant toxins (xanthotoxin, angelicin) and insecticides (cypermethrin, aldrin and diazinon) correlate well with these predictive models. The absence of Thr in the I helix of CYP321A1 and hydroxyl groups on many of its substrates suggests that this insect P450 mediates oxygen activation by a mechanism different from that employed by CYP107A1 and CYP158A1, which are two bacterial P450s also lacking Thr in their I helix, and most other P450s that contain Thr in their I helix.

Development of neural network QSPR models for Hansch substituent constants. 1. Method and validations.

In an attempt to develop predictive models for Hansch substituent constants for less common substituents, neural network QSPR (Quantitative Structure-Property Relationship) studies were conducted to correlate Hansch substituent constants for hundreds of chemically diverse functional groups with two different molecular descriptor sets. The Hansch substituent constants under study were pi, MR, F and R, describing the hydrophobic, steric/polarizability, and electronic (field and resonance) characteristics of the substituents, respectively. E-state descriptors were used for pi and MR, while the molecular descriptor set based upon the approach of Kvasnicka, Sklenak, and Pospichal (J. Am. Chem. Soc. 1993, 115, 1495-1500) was adopted for F and R. Both QSPR models demonstrated good predictivity in test sets.

Development of neural network QSPR models for Hansch substituent constants. 2. Applications in QSAR studies of HIV-1 reverse transcriptase and dihydrofolate reductase inhibitors.

In this paper, the applications of a Hansch substituent constant predictor(1) to Quantitative Structure-Activity Relationships (QSAR) studies of E. coli dihydrofolate reductase (DHFR) inhibitors 2,4-diamino-5-(substituted-benzyl)pyrimidines as well as HIV-1 reverse transcriptase (RT) inhibitors 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) derivatives are demonstrated. Both data sets contain functional groups for which the substituent constants (pi, MR, F and R) could not be found in standard substituent constant tables. The substituent constant predictor allowed us to derive predicted pi, MR, F and R values for all substituents in both data sets, thus enabling the generation of easily interpretable QSAR models of comparable or better predictivity than previous models.