Substrate specificity of the ileal and the hepatic Na(+)/bile acid cotransporters of the rabbit. II. A reliable 3D QSAR pharmacophore model for the ileal Na(+)/bile acid cotransporter

To design a reliable 3D QSAR model of the intestinal Na(+)/bile acid cotransporter, we have used a training set of 17 inhibitors of the rabbit ileal Na(+)/bile acid cotransporter. The IC(50) values of the training set of compounds covered a range of four orders of magnitude for inhibition of [(3)H]cholyltaurine uptake by CHO cells expressing the rabbit ileal Na(+)/bile acid cotransporter allowing the generation of a pharmacophore using the CATALYST algorithm. After thorough conformational analysis of each molecule, CATALYST generated a pharmacophore model characterized by five chemical features: one hydrogen bond donor, one hydrogen bond acceptor, and three hydrophobic features. The 3D pharmacophore was enantiospecific and correctly estimated the activities of the members of the training set. The predicted interactions of natural bile acids with the pharmacophore model of the ileal Na(+)/bile acid cotransporter explain exactly the experimentally found structure;-activity relationships for the interaction of bile acids with the ileal Na(+)/bile acid cotransporter (Kramer et al. 1999. J. Lipid. Res. 40: 1604;-1617). The natural bile acid analogues cholyltaurine, chenodeoxycholyltaurine, or deoxycholyltaurine were able to map four of the five features of the pharmacophore model: a) the five-membered ring D and the methyl group at position 18 map one hydrophobic site and the 21-methyl group of the side chain maps a second hydrophobic site; b) one of the alpha-oriented hydroxyl groups at position 7 or 12 fits the hydrogen bond donor feature; c) the negatively charged side chain acts as hydrogen bond acceptor; and d) the hydroxy group at position 3 does not specifically map any of the five binding features of the pharmacophore model. The 3-hydroxy group of natural bile acids is not essential for interactions with ileal or hepatic Na(+)/bile acid cotransporters. A modification of the 3-position of a natural bile acid molecule is therefore the preferred position for drug targeting strategies using bile acid transport pathways.     

Synthesis and anti-HIV activity of cosalane analogues incorporating two dichlorodisalicylmethane pharmacophore fragments

A new series of cosalane analogues incorporating two fragments of the dichlorodisalicylmethane pharmacophore has been synthesized. In order to identify the position for the attachment of the pharmacophore fragments to the steroid ring that results in the most potent analogues, two types of compounds were designed. In the first type, the two pharmacophore fragments were attached at C-3 and C-17 of the steroid ring by using appropriate linker units. In the second type, both pharmacophore groups were connected to C-3 of the steroid through an alkenyl chain containing an amide moiety. All of the new compounds displayed antiviral activity versus HIV-1(RF), HIV-1(IIIB), and HIV-2(ROD) in cell culture. The relative potencies of the compounds resulting from the two attachment strategies were found to depend on the viral strain as well as the cell type. Overall, the attachment of the second pharmacophore did not result in either a large gain or a large loss in anti-HIV activity, and the results are therefore consistent with the hypothesis that the two pharmacophores act independently, and one at a time, with positively charged amino acid side chains present on the surface of gp120 and CD4.     

Discovery of potential pancreatic cholesterol esterase inhibitors using pharmacophore modelling, virtual screening, and optimization studies

Pancreatic cholesterol esterase (CEase) is a serine hydrolase involved in the hydrolysis of variety of lipids and transport of free cholesterol. In this study, pharmacophore hypotheses based on known inhibitors were generated using common feature pharmacophore generation protocol available in Discovery Studio program. The best pharmacophore model containing two hydrogen bond acceptor and three hydrophobic features was selected and validated. It was further used in screening three diverse chemical databases. Hit compounds were subjected to drug-likeness and molecular docking studies. Four hits, namely SEW00846, NCI0040784, GK03167, and CD10645, were selected based on the GOLD fitness score and interaction with active site amino acids. All hit compounds were further optimized to improve their binding in the active site. The optimized compounds were found to have improved binding at the active site. Strongly binding optimized hits at the active site can act as virtual leads in potent CEase inhibitor designing.     

Influence of solvent and configuration of residues at positions 2 and 3 on distance and mobility of pharmacophore groups at positions 1 and 4 in cyclic enkephalin analogues

The analgesic activity of opioid peptides is mainly connected with their affinity and selectivity for the mu-receptors. The biological activity of cyclic opioid analogues depends on mutual orientation and conformational freedom of aromatic pharmacophore groups at positions 1 and 4. The distance and distance distributions between chromophores at positions 1 [Phe(p-NO(2)), p-nitrophenylalanine] and 4 [Nal, beta-(2-naphthyl)alanine], which constitute an energy donor-acceptor pair, were calculated based on measured fluorescence intensity decays of a donor (Nal). The influence of the solvent and configuration of the residues at position 2 and 3 on donor-acceptor distance distribution and mobility of pharmacophore groups at position 1 and 4 in cyclic enkephalin analogues are discussed.     

3-O-arylmethylgalangin, a novel isostere for anti-HCV 1,3-diketoacids (DKAs)

Through chelation of the metal ions at the enzyme active site, 1,3-diketoacids (DKAs) show potent inhibition of viral enzymes such as HIV integrase and HCV NS5B. In order to optimize the antiviral activity of the DKAs, structural modification of their metal-binding units, keto-enol acids or monoketo acids, have been actively performed. In this study, we proposed 3-O-arylmethylgalangin 3 as an alternative to ortho-substituted aromatic DKA, a potent inhibitor of HCV NS5B. As a proof-of-concept study, a series of 3-O-arylmethylgalangin derivatives (3a-3r) were prepared and their inhibitory activity against HCV NS5B was estimated. Structure-activity relationship of the 3-O-arylmethylgalangin derivatives was in good accordance with that of the ortho-substituted aromatic DKA series. In particular, two galangin ethers (3g and 3i) completely superimposable with the most potent ortho-substituted aromatic DKA analogue (2) in atom-by-atom fashion showed equipotent inhibitory activity to that of 2. Taken together, this result provides convincing evidence that the 3-O-arylmethylgalangin can successfully mimic the chelating function of the DKA pharmacophore to show potent inhibitory activity against the target enzyme, HCV NS5B.     

Analysis of stereoelectronic properties, mechanism of action and pharmacophore of synthetic indolo[2,1-b]quinazoline-6,12-dione derivatives in relation to antileishmanial activity using quantum chemical, cyclic voltammetry and 3-D-QSAR CATALYST procedures

Several indolo[2,1-b]quinazoline-6,12-dione (tryptanthrin) derivatives exhibited remarkable activity at concentrations below 100 ng/mL when tested against in vitro Leishmania donovani amastigotes. The in vitro toxicity studies indicate that the compounds are fairly well tolerated in both macrophage and neuronal lines. An analysis based on qualitative and quantitative structure-activity relationship studies between in vitro antileishmanial activity and molecular electronic structure of 27 analogues of indolo[2,1-b]quinazoline-6,12-dione is presented here by using a combination of semi-empirical AM1 quantum chemical, cyclic voltammetry and a pharmacophore generation (CATALYST) methods. A modest to good correlation is observed between activity and a few calculated molecular properties such as molecular density, octanol-water partition coefficient, molecular orbital energies, and redox potentials. Electron transfer seems to be a plausible path in the mechanism of action of the compounds. A pharmacophore generated by using the 3-D QSAR of CATALYST produced a fairly accurate predictive model of antileishmanial activity of the tryptanthrins. The validity of the pharmacophore model extends to structurally different class of compounds that could open new frontiers for study. The carbonyl group of the five- and six-membered rings in the indolo[2,1-b]quinazoline-6,12-dione skeleton and the electron transfer ability to the carbonyl atom appear to be crucial for activity.     

Influence of ring- and side-chain substituents on the transamination of aromatic amino acids by two multispecific aspartate-aromatic aminotransferase isozymes purified from bushbean shoots

The breadth of substrate specificity shown by the multispecific aspartate-aromatic aminotransferase of bushbean (Phaseolus vulgaris) has been investigated by testing the ability of two cytosolic isozymes (I and II), purified from shoot tissue, to catalyse transamination reactions between a range of ring- and sidechain-substituted aromatic amino acids and 2-oxoglutarate. Ring-substituted phenylalanines were the most reactive substrates whereas ring-substitution in tyrosine or tryptophan resulted in transamination rates lower than those observed with the parent amino acids. All side chain-substituted analogues were found to be totally inactive. The highest activity shown by any ring-substituted phenylalanine was observed with the 4-amino- compound, followed closely by the 4-hydroxy- and 4-halogen-compounds. In contrast, 4-nitrophenylalanine was completely inactive. These trends were consistent for both isozymes I and II, but only isozyme II showed greatly enhanced activity over that found with the parent amino acid when certain ring-substituted analogues were tested. The varying capacity of the bushbean isozymes to utilize the present range of substituted amino acids is compared with previous reports on the substrate specificity shown by aspartate and aromatic aminotransferases isolated from mammalian and microbial systems. A model for the mechanism of activation observed with bushbean isozyme II in the presence of certain 4-substituted aromatic amino acids is proposed, based on current understanding of the nature of the active site of animal aspartate aminotransferases.     

Synthesis of new bile acid analogues and their metabolism in the hamster: 3 alpha, 6 alpha-dihydroxy-6 beta-methyl-5 beta-cholanoic acid and 3 alpha, 6 beta-dihydroxy-6 alpha-methyl-5 beta-cholanoic acid

This paper reports the chemical synthesis of two new bile acid analogues, namely, 3 alpha, 6 beta-dihydroxy-6 alpha-methyl-5 beta-cholanoic acid from 3 alpha-hydroxy-6-oxo-5 beta-cholanoic acid and describes their metabolism in the hamster. A Grignard reaction of the oxo acid with methyl magnesium iodide in tetrahydrofuran gave two epimeric dihydroxy-6-methyl-cholanoic acids which were separated as the methyl esters by silica gel column chromatography. The configuration of the 6-methyl groups was assigned by proton nuclear magnetic resonance spectroscopy and was supported by the chromatographic properties of the new compounds. The metabolism of the two new bile acid analogues was studied in the hamster. After intraduodenal administration of the 14C-labeled analogues into bile fistula hamsters, both compounds were absorbed rapidly from the intestine and secreted into bile. Intravenous infusion studies revealed that these compounds were efficiently extracted by the liver; the administered analogues became major biliary bile acids, present as either the glycine or taurine conjugates. These compounds are useful to study the effect of methyl-substituted bile acids on cholesterol and bile acid metabolism and may possibly possess cholelitholytic properties.     

The influence of diphosphonic analogues of inorganic pyrophosphate on activity of RNA-polymerases from the calf thymus

Diphosphonic analogues of inorganic pyrophosphate (PPi): methylene-, oxyethylidene-, aminomethylenediphosphonic acids as well as phosphonacetic, imidodiphosphoric bis- (phosphonomethyl)-phosphonic acids and methylenediphosphonic and phosphonic acid monoanhydrides were studied for their effect on the RNA-synthesizing activity of thymocytes. DNA-dependent RNA-polymerases I and II from the calf thymus nuclei were used for these studies. The analogues and PPi under study are shown to be inhibitors of both RNA-polymerases in nuclei from calf thymus and of purified RNA-polymerase II, which is more sensitive to the effect of diphosphonates. Methylenediphosphonic acid is the strongest inhibitor among the studied analogues, and imidodiphosphoric and phosphonacetic acids are the weakest inhibitors. Inhibition of purified RNA-polymerase II by diphosphonates has a complex character and includes both interaction of the PPi analogues with enzymes and chelating by them of Mn ions which are cofactors for RNA polymerase.     

Synthesis of novel N1-substituted bicyclic pyrazole amino acids and evaluation of their interaction with glutamate receptors

N1-substituted bicyclic pyrazole amino acids (S)-9a-9c and (R)-9a-9c, which are conformationally constrained analogues of glutamic acid, were prepared via a strategy based on a 1,3-dipolar cycloaddition. The new amino acids were tested for activity at ionotropic and metabotropic glutamate receptors. Some of them turned out to be selective for the NMDA receptors, where they behaved as weak antagonists. The biological activity is mainly due to the interaction with the glutamate binding site, and not with the glycine co-agonist site.     

Evaluation of endogenous fatty acid amides and their synthetic analogues as potential anti-inflammatory leads

A series of endogenous fatty acid amides and their analogues (1-78) were prepared, and their inhibitory effects on pro-inflammatory mediators (NO, IL-1β, IL-6, and TNF-α) in LPS-activated RAW264.7 cells were evaluated. Their inhibitory activity on the pro-inflammatory chemokine MDC in IFN-γ-activated HaCaT cells was also examined. The results showed that the activity is strongly dependent on the nature of the fatty acid part of the molecules. As expected, the amides derived from enone fatty acids showed significant activity and were more active than those derived from other types of fatty acids. A variation of the amine headgroup also altered bioactivity profile remarkably, possibly by modulating cell permeability. Regarding the amine part of the molecules, N-acyl dopamines exhibited the most potent activity (IC(50) ～2 μM). This is the first report of the inhibitory activity of endogenous fatty acid amides and their analogues on the production of nitric oxide, cytokines (IL-1β, IL-6, and TNF-α) and the chemokine MDC. This study suggests that the enone fatty acid-derived amides (such as N-acyl ethanolamines and N-acyl amino acids) and N-acyl dopamines may be potential anti-inflammatory leads.     

Synthesis and biological activities of new side chain and backbone cyclic bradykinin analogues.

A series of conformationally constrained cyclic analogues of the peptide hormone bradykinin (BK, Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) was synthesized to check different turned structures proposed for the bioactive conformation of BK agonists and antagonists. Cycles differing in the size and direction of the lactam bridge were performed at the C- and N-terminal sequences of the molecule. Glutamic acid and lysine were introduced into the native BK sequence at different positions for cyclization through their side chains. Backbone cyclic analogues were synthesized by incorporation of N-carboxy alkylated and N-amino alkylated amino acids into the peptide chain. Although the coupling of Fmoc-glycine to the N-alkylated phenylalanine derivatives was effected with DIC/HOAt in SPPS, the dipeptide building units with more bulky amino acids were pre-built in solution. For backbone cyclization at the C-terminus an alternative building unit with an acylated reduced peptide bond was preformed in solution. Both types of building units were handled in the SPPS in the same manner as amino acids. The agonistic and antagonistic activities of the cyclic BK analogues were determined in rat uterus (RUT) and guinea-pig ileum (GPI) assays. Additionally, the potentiation of the BK-induced effects was examined. Among the series of cyclic BK agonists only compound 3 with backbone cyclization between positions 2 and 5 shows a significant agonistic activity on RUT. To study the influence of intramolecular ring closure we used an antagonistic analogue with weak activity, [D-Phe7]-BK. Side chain as well as backbone cyclization in the N-terminus of [D-Phe7]-BK resulted in analogues with moderate antagonistic activity on RUT. Also, compound 18 in which a lactam bridge between positions 6 and 9 was achieved via an acylated reduced peptide bond has moderate antagonistic activity on RUT. These results support the hypothesis of turn structures in both parts of the molecule as a requirement for BK antagonism. Certain active and inactive agonists and antagonists are able to potentiate the bradykinin-induced contraction of guinea-pig ileum.     

Pharmacophore-based design, synthesis, and biological evaluation of novel chloro-pyridazine piperazines as human rhinovirus (HRV-3) inhibitors

A series of chloro-pyridazine piperazines were developed based on the structure of human rhinovirus (HRV) capsid-binding inhibitors with proven activity using a pharmacophore model. A preliminary evaluation demonstrated potent activity against HRV-3 with low cytotoxicity. A docking analysis indicated that 8a could fit into, and form tight interactions (e.g., H-bonds, σ-π effect) with the active site in VP1.     

Novel hybrids of 15-membered 8a- and 9a-azahomoerythromycin A ketolides and quinolones as potent antibacterials

A series of novel 6-O-substituted and 6,12-di-O-substituted 8a-aza-8a-homoerythromycin A and 9a-aza-9a-homoerythromycin A ketolides were synthesized and evaluated for in vitro antibacterial activity against a panel of representative erythromycin-susceptible and erythromycin-resistant test strains. Another series of ketolides based on 14-membered erythromycin oxime scaffold was also synthesized and their antibacterial activity compared to those of 15-membered azahomoerythromycin analogues. In general, structure-activity studies have shown that 14-membered ketolides displayed favorable antibacterial activity in comparison to their corresponding 15-membered analogues within 9a-azahomoerythromycin series. However, within 8a-azahomoerythromycin series, some compounds incorporating a ketolide combined with either quinoline or quinolone pharmacophore substructures showed significantly potent activity against a variety of erythromycin-susceptible and macrolide-lincosamide-streptogramin B (MLS(B))-resistant Gram-positive pathogens as well as fastidious Gram-negative pathogens. The best compounds in this series overcome all types of resistance in relevant clinical Gram-positive pathogens and display hitherto unprecedented in vitro activity against the constitutively MLS(B)-resistant strain of Staphylococcus aureus. In addition, they also represent an improvement over telithromycin (2) and cethromycin (3) against fastidious Gram-negative pathogens Haemophilus influenzae and Moraxella catarrhalis.     

Opioid peptides. Synthesis and binding properties of dermorphin related heptapeptides

C-Terminal amino acid residues of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) were replaced by N alpha-methyl- or D-amino acids in order to examine the effect on opioid activity. In binding studies based on displacement of mu, delta, and kappa opioid receptor selective radiolabels from guinea pig brain membranes, the 13 new analogues showed, like dermorphin, a negligible affinity for the kappa binding site. The introduction of N alpha-methyl- or D-amino acid residues at position 5, 6, or 7 of dermorphin, when matched with C-terminal amide function modifications, generally produced analogues with reversed mu/delta specificity.     

Protective activity of (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol analogues against diisopropylfluorophosphate neurotoxicity: Preliminary structure–activity relationship and pharmacophore modeling

Diisopropylfluorophosphate (DFP) is an organophosphorous insecticide used as a surrogate for the more toxic chemical warfare nerve agent sarin. DFP produces neurotoxicity in vivo and irreversibly decreases the area of population spikes recorded from the CA1 region of acute hippocampal slices. (1S,2E,4R,6R,7E,11E)-2,7,11-Cembratriene-4,6-diol (1) is a neuroprotective natural cembranoid that reverses DFP-induced damage both in vivo and in the hippocampal slice. Cembranoid 1 acts by noncompetitive inhibition of the α7 nicotinic acetylcholine receptor. This study aims at establishing a preliminary structure–activity relationship to define the neuroprotective cembranoid pharmacophores using the hippocampal slice assay and pharmacophore modeling. Fourteen natural, semisynthetic, or biocatalytic cembranoid analogues 2–15 related to 1 were tested for their capacity to protect the population spikes from DFP-induced damage and intrinsic toxicity. Twelve cembranoids caused significant reversal of DFP toxicity; only 3 active analogues displayed minor intrinsic toxicity at 10 μM. The C-4 epimer of 1 (2) and the 4-O-methyl ether analogue of 1 (3), were totally devoid of neuroprotective activity. The results suggested a model for cembranoid binding where the hydrophobic ring surface binds to a hydrophobic (Hbic) patch on the receptor molecule and an electronegative atom (oxygen or sulfur) in proper spatial relationship to the ring surface interacts with an electropositive group in the receptor binding site. A pharmacophore model consisting of 1 hydrogen bond acceptor (HBA), 2 Hbic, and 10 exclusion spheres was established using HipHop-REFINE and supported the above mentioned pharmacophoric hypothesis.     

Synthesis and biological evaluation of acylthiourea against DUSP1 inhibition

Structure based virtual screening attempts to discover DUSP1 inhibitors have yielded a scaffold featuring benzoxazole and acylthiourea pharmacophore. A series of its analogues were synthesized to explore structure activity relationship (SAR) of DUSP1 inhibition.     

Synthetic peptide derived from alpha-fetoprotein inhibits growth of human breast cancer: investigation of the pharmacophore and synthesis optimization.

Asynthetic peptide that inhibits the growth of estrogen receptor positive (ER+) human breast cancers, growing as xenografts in mice, has been reported. The cyclic 9-mer peptide, cyclo[EMTOVNOGQ], is derived from alpha-fetoprotein (AFP), a safe, naturally occurring human protein produced during pregnancy, which itself has anti-estrogenic and anti-breast cancer activity. To determine the pharmacophore of the peptide, a series of analogs was prepared using solid-phase peptide synthesis. Analogs were screened in a 1-day bioassay, which assessed their ability to inhibit the estrogen-stimulated growth of uterus in immature mice. Deletion of glutamic acid, Glu1, abolished activity of the peptide, but glutamine (Gln) or asparagine (Asn) could be substituted for Glu1 without loss of activity. Methionine (Met2) was replaced with lysine (Lys) or tyrosine (Tyr) with retention of activity. Substitution of Lys for Met2 in the cyclic molecule resulted in a compound with activity comparable with the Met2-containing cyclic molecule, but with a greater than twofold increase in purity and corresponding increase in yield. This Lys analog demonstrated anti-breast cancer activity equivalent to that of the original Met-containing peptide. Therefore, Met2 is not essential for biologic activity and substitution of Lys is synthetically advantageous. Threonine (Thr3) is a nonessential site, and can be substituted with serine (Ser), valine (Val), or alanine (Ala) without significant loss of activity. Hydroxyproline (Hyp), substituted in place of the naturally occurring prolines (Pro4, Pro7), allowed retention of activity and increased stability of the peptide during storage. Replacement of the first Pro (Pro4) with Ser maintains the activity of the peptide, but substitution of Ser for the second Pro (Pro7) abolishes the activity of the peptide. This suggests that the imino acid at residue 7 is important for conformation of the peptide, and the backbone atoms are part of the pharmacophore, but Pro4 is not essential. Valine (Val5) can be substituted only with branched-chain amino acids (isoleucine, leucine or Thr); replacement by d-valine or Ala resulted in loss of biologic activity. Thus, for this site, the bulky branched side chain is essential. Asparagine (Asn6) is essential for activity. Substitution with Gln or aspartic acid (Asp), resulted in reduction of biologic activity. Removal of glycine (Gly8) resulted in a loss of activity but nonconservative substitutions can be made at this site without a loss of activity indicating that it is not part of the pharmacophore. Cyclization of the peptide is facilitated by addition of Gln9, but this residue does not occur in AFP nor is it necessary for activity. Gln9 can be replaced with Asn, resulting in a molecule with similar activity. These data indicate that the pharmacophore of the peptide includes side chains of Val5 and Asn6 and backbone atoms contributed by Thr3, Val5, Asn6, Hyp7 and Gly8. Met2 and Gln9 can be modified or replaced. Glu1 can be replaced with charged amino acids, and is not likely to be part of the binding site of the peptide. The results of this study provide information that will be helpful in the rational modification of cyclo[EMTOVNOGQ] to yield peptide analogs and peptidomimetics with advantages in synthesis, pharmacologic properties, and biologic activity.