Modifications in the aminoether side chain of clomiphene influence affinity for a specific antiestrogen binding site in MCF 7 cell cytosol

Studies on the binding of a series of clomiphene derivatives to a specific high affinity antiestrogen binding site in MCF 7 cell cytosol revealed that substitutions in the aminoether side chain influenced affinity for this site. Removal of the aminoether side chain completely eliminated binding while changes in length of the C chain, and changes in the number and size of the substituents on the terminal amino group modified binding affinity. Conversion of the ether linkage to an amine also markedly reduced affinity. It is concluded that the aminoether side chain which is essential for antiestrogenic activity is a major structural determinant of the binding of synthetic triphenylethylene antiestrogens to the specific antiestrogen binding site.     

The discovery of fluoropyridine-based inhibitors of the factor VIIa/TF complex--Part 2

The activated factor VII/tissue factor complex (FVIIa/TF) is known to play a key role in the formation of blood clots. Inhibition of this complex may lead to new antithrombotic drugs. A fluoropyridine-based series of FVIIa/TF inhibitors was discovered which utilized a diisopropylamino group for binding in the S2 and S3 binding pockets of the active site of the enzyme complex. In this series, an enhancement in binding affinity was observed by substitution at the 5-position of the hydroxybenzoic acid sidechain. An X-ray crystal structure indicates that amides at this position may increase inhibitor binding affinity through interactions with the S1'/S2' pocket.     

Development of substituted N-[3-(3-methoxylphenyl)propyl] amides as MT2-selective melatonin agonists: Improving metabolic stability

A series of novel and selective N-[3-(6-benzyloxy-3-methoxyphenyl)propyl] amides has recently been shown to possess sub-nanomolar range binding affinity to the type 2 melatonin receptor (MT₂). Pharmacokinetics studies suggested that these compounds were subject to vigorous CYP450-mediated metabolism, resulting in a series of metabolites with significantly decreased or diminished binding affinities toward MT₂ receptor. The ether bonds were found to be the major positions susceptible to metabolism. In this study, the benzyl ether bond was either removed or replaced with a carbon–carbon bond in an attempt to improve metabolic stability and enhance their resistance towards phase I oxidation. The synthesis, receptor binding affinity, intrinsic potency and metabolic stability of modified structures are reported in this article. By removal or replacement of metabolic labile ether linkerage with carbon linkers, a novel compound was identified with good potency and MT₂ selectivity, and with increased metabolic stability.     

Uncoupling the Folding and Binding of an Intrinsically Disordered Protein

The relationship between helical stability and binding affinity was examined for the intrinsically disordered transactivation domain of the myeloblastosis oncoprotein, c-Myb, and its ordered binding partner, KIX. A series of c-Myb mutants was designed to either increase or decrease helical stability without changing the binding interface with KIX. This included a complimentary series of A, G, P, and V mutants at three non-interacting sites. We were able to use the glycine mutants as a reference state and show a strong correlation between binding affinity and helical stability. The intrinsic helicity of c-Myb is 21%, and helicity values of the mutants ranged from 8% to 28%. The c-Myb helix is divided into two conformationally distinct segments. The N-terminal segment, from K291–L301, has an average helicity greater than 60% and the C-terminal segment, from S304–L315, has an average helicity less than 10%. We observed different effects on binding when these two segments were mutated. Mutants in the N-terminal segment that increased helicity had no effect on the binding affinity to KIX, while helix destabilizing glycine and proline mutants reduced binding affinity by more than 1 kcal/mol. Mutants that either increased or decreased helical stability in the C-terminal segment had almost no effect on binding. However, several of the mutants reveal the presence of multiple conformations accessible in the bound state based on changes in enthalpy and linkage analysis of binding free energies. These results may explain the high level of sequence identity (> 90%), even at non-interacting sites, for c-Myb homologues.     

NMDA-NR2B subtype selectivity of stereoisomeric 2-(1,2,3,4-tetrahydro-1-isoquinolyl)ethanol derivatives

Enantiopure 2-(1,2,3,4-tetrahydro-1-isoquinolyl)ethanol derivatives were tested for their affinity to the ifenprodil binding site of the NMDA receptor, their potency to inhibit [3H]MK801 binding and their NMDA-NR2B subtype selectivity. The (1S,1'S)-configurated series displayed the highest affinity to the ifenprodil binding site. A reasonable potency and NMDA-NR2B subtype selectivity was found for (1S,1'S)-4c (R1=Me, R2=OMe). A high affinity to HERG K+ channels, however, suggests that (1S,1'S)-4c may involve an increased risk of cardiovascular side effects.     

Identification of neutral 4-O-alkyl quinolone nonpeptide GnRH receptor antagonists

A series of neutral, nonbasic quinolone GnRH antagonists were prepared via Mitsunobu alkylation of protected and unprotected 4-hydroxy quinolone intermediates. The synthetic route was improved by utilization of unique reactivity and convergency afforded by the use of mono and bis-trimethylsilylethyl protected quinolones. Potent neutral GnRH antagonists were identified, including ether and lactam derivatives, that show similar in vitro binding affinity and functional activity as compared to the earlier basic 4-aminoalkyl quinolone series of nonpeptide GnRH antagonists.     

Transformation of Streptococcus sanguis to intrinsic penicillin resistance

A series of step-level penicillin-resistant derivatives of Streptococcus sanguis V288 (Challis) were obtained through successive genetic transformations. The DNA donor used was a laboratory-derived, penicillin-resistant multistep mutant of the recipient strain. Detection of the penicillin-binding proteins (PBPs) of wild-type and transformants revealed five major PBPs. While it was found that S. sanguis can acquire intrinsic resistance in a stepwise manner and the mechanism was similar to those of some other organisms (changes in penicillin-binding protein affinity and/or in extent of penicillin binding), multiple-PBP changes accompanied a single step-level of resistance. All of the PBPs showed varying degrees of decreased affinity for [3H]benzylpenicillin with increasing penicillin resistance. Of these, the consistent, dramatic and progressive decrease of PBP 4 binding was most notable. After an initial decrease at the first step-level of resistance, PBP 5 was restored to wild-type levels, indicating a possible important role in survival. Genetic linkage of the first two step-levels of resistance was demonstrated by examination of transformation frequencies and by hit-kinetics experiments. A convenient method is described for the quantitative comparison of fluorographs containing PBPs with a wide range of affinities for penicillin.     

Estrogen pyrazoles: defining the pyrazole core structure and the orientation of substituents in the ligand binding pocket of the estrogen receptor

Previously, we reported that certain tetrasubstituted 1,3,5-triaryl-4-alkyl-pyrazoles bind to the estrogen receptor (ER) with high affinity (Fink, B. E.; Mortenson, D. S.; Stauffer, S. R.; Aron, Z. D.; Katzenellenbogen, J. A. Chem. Biol. 1999, 6, 205-219; Stauffer, S. R.; Katzenellenbogen, J. A. J. Comb/. Chem. 2000, 2. 318 329; Stauffer, S. R.: Coletta, C. J.: Sun, J.; Tedesco, R., Katzenellenbogen, B. S.; Katzenellenbogen, J. A. J. Med. Chem. 2000, submitted). To investigate how cyclic permutation of the two nitrogen atoms of a pyrazole might affect ER binding affinity, we prepared a new pyrazole core isomer, namely a 1,3,4-triaryl-5-alkyl-pyrazole (2), to compare it with our original pyrazole (1). We also prepared several peripherally matched core pyrazole isomer sets to investigate whether the two pyrazole series share a common binding orientation. Our efficient, regioselective synthetic route to these pyrazoles relies on the acylation of a hydrazone anion, followed by cyclization, halogenation, and Suzuki coupling. We found that the ER accommodates 1,3,4-triaryl-pyrazoles of the isomeric series only somewhat less well than the original 1,3,5-triaryl series, and it appears that both series share a common binding mode. This preferred orientation for the 1,3,5-triaryl-4-alkyl-pyrazoles is supported by binding affinity measurements of analogues in which the phenolic hydroxyl groups were systematically removed from each of the three aryl groups, and the orientation is consistent, as well, with molecular modeling studies. These studies provide additional insight into the design of heterocyclic core structures for the development of high affinity ER ligands by combinatorial methods.     

Modulation of the Benzodiazepine/γ-Aminobutyric Acid Receptor Chloride Channel Complex by Inhalation Anesthetics

Inhalation anesthetics, such as diethyl ether, halothane, and enflurane, increase 36Cl- uptake into rat cerebral cortical synaptoneurosomes in a concentration-dependent, picrotoxin-sensitive fashion. At concentrations consistent with those that stimulate 36Cl- uptake, inhalation anesthetics also inhibit the binding of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) to well-washed cortical membranes. Scatchard analysis of [35S]TBPS binding indicates that these agents reduce the apparent affinity of this radioligand and have little effect on the Bmax. The ability of inhalation anesthetics to directly stimulate 36Cl- uptake and inhibit [35S]TBPS binding is a property shared by nonvolatile anesthetics. Nonetheless, there are differences between nonvolatile agents (such as barbiturates and alcohols) and inhalation anesthetics, because the former compounds augment muscimol (a GABAmimetic) stimulated 36Cl- uptake, whereas the latter group (such as ether and enflurane) inhibit this effect. These findings demonstrate that therapeutically relevant concentrations of inhalation anesthetics perturb the benzodiazepine/gamma-aminobutyric acid receptor chloride channel complex, and suggest this oligomeric protein may be a common mediator of some aspects of anesthetic action.     

Evidence for allosteric coupling between the ribosome and repressor binding sites of a translationally regulated mRNA

Escherichia coli ribosomal protein S4 is a translational repressor regulating the expression of four ribosomal genes in the alpha operon. In vitro studies have shown that the protein specifically recognizes an unusual mRNA pseudoknot secondary structure which links sequences upstream and downstream of the ribosome binding site for rpsM (S13) [Tang, C. K., & Draper, D. E. (1989) Cell 57, 531]. We have prepared fusions of the rpsM translational initiation site and lacZ that allows us to detect repression in cells in which overproduction of S4 repressor can be induced. Twenty-five mRNA sequence variants have been introduced into the S13-lacZ fusions and the levels of translational repression measured. Sets of compensating base changes confirm the importance of the pseudoknot secondary structure for translational repression. An A residue in a looped, single-stranded sequence is also required for S4 recognition and may contact S4 directly. Comparison of translational repression levels and S4 binding constants for the set of mRNA mutations show that nine mutants are repressed much more weakly than predicted from their affinity for S4; in extreme cases no repression can be detected for variants with unchanged S4 binding. We suggest that the mRNA contains functionally distinct ribosome and repressor binding sites that are allosterically coupled. Mutations can relieve translational repression by disrupting the linkage between the two sites without altering S4 binding. This proposal assigns to the mRNA a more active role in mediating translational repression than found in other translational repression systems.     

Characterization of the carbohydrate binding specificity of the leukoagglutinating lectin from Maackia amurensis. Comparison with other sialic acid-specific lectins

The sialic acid-specific leukoagglutinating lectin from the seeds of Maackia amurensis (MAL) has been studied by the techniques of quantitative precipitin formation, hapten inhibition of precipitation, hapten inhibition using an enzyme-linked immunosorbent assay, and lectin affinity chromatography. The ability of the immobilized lectin to fractionate oligosaccharides based on their content of sialic acid has also been investigated. Our results indicate that MAL reacts with greatest affinity with the trisaccharide sequence Neu5Ac/Gc alpha 2,3Gal beta 1,4GlcNAc/Glc. The lectin requires three intact sugar units for binding and does not interact when the beta 1,4-linkage is replaced by a beta 1,3-linkage nor when the "reducing sugar" of the trisaccharide is reduced. Results from enzyme-linked immunosorbent assays show that an N-acetyllactosamine repeating sequence is not required; however, the N-acetyllactosamine repeating sequence does appear to enhance the binding of MAL to a series of glycolipids. In addition, the sialic acid may be substituted with either N-acetyl or N-glycolyl groups without reduction in binding. The C-8 and C-9 hydroxyl groups of sialic acid do not play a role in binding as shown by the strong reaction of periodate-treated glycoproteins. Comparison of the specificity of the three sialic acid-binding lectins indicates that Limax flavus agglutinin binds to Neu5Ac in any linkage and in any position in a glycoconjugate, Sambucus nigra lectin requires a disaccharide of the structure Neu5Ac alpha 2,6Gal/GalNAc, and MAL has a binding site complimentary to the trisaccharide Neu5Ac alpha 2,3Gal beta 1,4GlcNAc/Glc, to which sialic acid contributes less to the total binding affinity than for either S. nigra lectin or L. flavus agglutinin.     

Novel, potent THC/anandamide (hybrid) analogs

The structure–activity relationship (SAR) of the end pentyl chain in anandamide (AEA) has been established to be very similar to that of Δ⁹-tetrahydrocannabinol (Δ⁹-THC). In order to broaden our understanding of the structural similarities between AEA and THC, hybrid structures 1–3 were designed. In these hybrids the aromatic ring of THC–DMH was linked to the AEA moiety through an ether linkage with the oxygen of the phenol of THC. Hybrid 1 (O-2220) was found to have very high binding affinity to CB1 receptors (K_i = 8.5 nM), and it is interesting to note that the orientation of the side chain with respect to the oxygen in the phenol is the same as in THCs. To further explore the SAR in this series the terminal carbon of the side chain was modified by adding different substituents. Several such analogs were synthesized and tested for their CB1 and CB2 binding affinities and in vivo activity (tetrad tests). The details of the synthesis and the biological activity of these compounds are described.     

Binding of selected iodothyronine analogues to receptor sites of isolated rat hepatic nuclei. High correlation between structural requirements for nuclear binding and biological activity.

The limited capacity, high affinity binding of 35 iodothyronine analogues by rat liver nuclei has been examined in an in vitro system. The in vitro nuclear binding of all the analogues tested was highly correlated with their published thyromimetic potencies in the intact animals. Binding and biological activity are greater for 3'-mono-than 3',5'-di-substituted iodothyronines. A 4'-hydroxyl group is essential, but the 3' substituent can be several halogen or non-halogen groups for which the distal conformation is preferred. The ether linkage can be replaced equally well by a methylene or sulfur group. The presence of both 3 and 5 groups which are limited to halogens or small alkyl groups are necessary for the maintenance of significant activity. Halogen-free iodothyronines have very low, but significant activity both in vitro and in vivo. The data provide information on the structural requirements for thyroid hormone action and further support the physiological relevance of the nuclear sites.     

Sequence recognition in the minor groove of DNA by covalently linked formamido imidazole-pyrrole-imidazole polyamides: effect of H-pin linkage and linker length on selectivity and affinity

The polyamide N-formamido imidazole-pyrrole-imidazole (f-ImPyIm) binds with an exceptionally high affinity for its cognate site 5'-ACGCGT-3' as a stacked, staggered, and noncovalent cooperative dimer. Investigations are presented into its sequence specificity and binding affinity when linked covalently as an H-pin "dimer". Five f-ImPyIm cross-linked analogues with six to nine methylene linkers and an eight-linked ethylene glycol linker were examined to investigate the effect of linkage and linker length on DNA binding. Thermal denaturation studies on short DNA hairpins showed preferential binding by both f-ImPyIm (DeltaTm = 7.8 degrees C) and its cross-linked derivatives (DeltaTm > 30 degrees C) at 5'-ACGCGT-3', indicating sequence specificity was retained on linkage. DNase I footprinting confirmed strict cognate site selectivity and demonstrated that affinity increased with linker length (f-ImPyIm-9 = f-ImPyIm-8 = f-ImPyIm-EG-8 > f-ImPyIm-7 > f-ImPyIm-6). The eight- and nine-linked derivatives bound at 100-fold lower concentrations at the cognate site relative to f-ImPyIm-6, and with 10-fold higher affinity than unlinked f-ImPyIm. Use of an ethylene glycol linkage in f-ImPyIm-EG-8 to improve solubility slightly increased the cognate site affinity relative to those of f-ImPyIm-8 and f-ImPyIm-9, although some selectivity was lost at high ligand concentration. CD demonstrated that cognate site binding by eight and nine-linked compounds occurred in the minor groove. SPR analysis gave a binding affinity (K) for f-ImPyIm-EG-8 at the cognate site of 2 x 10(10) M-1, representing a 100-fold increase relative to that of f-ImPyIm. This study demonstrates that the high-affinity cooperative binding of f-ImPyIm can be enhanced significantly by suitable covalent linkage, while maintaining its strict cognate site selectivity.     

Effect of the position of the phenolic group in morphinans on their affinity for opiate receptor binding

Homologous series of N-methyl, N-allyl and N-cyclopropylmethylmorphinans, differing only in the position of the plenolic hydroxy group, were examined with respect to their binding affinities for the opiate receptor. IC₅₀'s were determined for competition with ³H-naltrexone in the presence and absence of 100 mM NaCl. While the compounds with the hydroxy in the 3-position had, as expected, by far the highest affinity, the corresponding molecules with the hydroxy in the 2- or 4- position had significant binding affinity ranging from 30 nM in the cyclopropyl- methyl series to 400 nM for the 2-hydroxy N-methyl morphinan. The sodium indices were also very similar to those of the corresponding 3-hydroxy compounds. The only 1-hydroxy derivative available was about 5-fold weaker than the corresponding 2- and 4-hydroxy compounds. Covering or removing the hydroxy group greatly weakened the binding but did not totally destroy it. There was good correlation between binding affinity and pharmacological potency for all except the methoxy compounds. Their high potency is consonant with $\text{in vivo}$ hydrolysis of the methyl ether.     

Congeneric but still distinct: how closely related trypsin ligands exhibit different thermodynamic and structural properties

A congeneric series of benzamidine-type ligands with a central proline moiety and a terminal cycloalkyl group—linked by a secondary amine, ether, or methylene bridge—was synthesized as trypsin inhibitors. This series of inhibitors was investigated by isothermal titration calorimetry, crystal structure analysis in two crystal forms, and molecular dynamics simulations. Even though all of these congeneric ligands exhibited essentially the same affinity for trypsin, their binding profiles at the structural, dynamic, and thermodynamic levels are very distinct. The ligands display a pronounced enthalpy/entropy compensation that results in a nearly unchanged free energy of binding, even though individual enthalpy and entropy terms change significantly across the series. Crystal structures revealed that the secondary amine-linked analogs scatter over two distinct conformational families of binding modes that occupy either the inside or of the outside the protein's S3/S4 specificity pocket. In contrast, the ether-linked and methylene-linked ligands preferentially occupy the hydrophobic specificity pocket. This also explains why the latter ligands could only be crystallized in the conformationally restricting closed crystal form whereas the derivative with the highest residual mobility in the series escaped our attempts to crystallize it in the closed form; instead, a well-resolved structure could only be achieved in the open form with the ligand in disordered orientation. These distinct binding modes are supported by molecular dynamics simulations and correlate with the shifting enthalpic/entropic signatures of ligand binding. The examples demonstrate that, at the molecular level, binding modes and thermodynamic binding signatures can be very different even for closely related ligands. However, deviating binding profiles provide the opportunity to optimally address a given target.     

Two neonatal diabetes mutations on transmembrane helix 15 of SUR1 increase affinity for ATP and ADP at nucleotide binding domain 2

K(ATP) channels, (SUR1/Kir6.2)(4) (sulfonylurea receptor type 1/potassium inward rectifier type 6.2) respond to the metabolic state of pancreatic β-cells, modulating membrane potential and insulin exocytosis. Mutations in both subunits cause neonatal diabetes by overactivating the pore. Hyperactive channels fail to close appropriately with increased glucose metabolism; thus, β-cell hyperpolarization limits insulin release. K(ATP) channels are inhibited by ATP binding to the Kir6.2 pore and stimulated, via an uncertain mechanism, by magnesium nucleotides at SUR1. Glibenclamide (GBC), a sulfonylurea, was used as a conformational probe to compare nucleotide action on wild type versus Q1178R and R1182Q SUR1 mutants. GBC binds with high affinity to aporeceptors, presumably in the inward facing ATP-binding cassette configuration; MgATP reduces binding affinity via a shift to the outward facing conformation. To determine nucleotide affinities under equilibrium, non-hydrolytic conditions, Mg(2+) was eliminated. A four-state equilibrium model describes the allosteric linkage. The K(D) for ATP(4-) is ~1 versus 12 mM, Q1178R versus wild type, respectively. The linkage constant is ~10, implying that outward facing conformations bind GBC with a lower affinity, 9-10 nM for Q1178R. Thus, nucleotides cannot completely inhibit GBC binding. Binding of channel openers is reported to require ATP hydrolysis, but diazoxide, a SUR1-selective agonist, concentration-dependently augments ATP(4-) action. An eight-state model describes linkage between diazoxide and ATP(4-) binding; diazoxide markedly increases the affinity of Q1178R for ATP(4-) and ATP(4-) augments diazoxide binding. NBD2, but not NBD1, has a higher affinity for ATP (and ADP) in mutant versus wild type (with or without Mg(2+)). Thus, the mutants spend more time in nucleotide-bound conformations, with reduced affinity for GBC, that activate the pore.     

Incorporation of (2S,3S) and (2S,3R) beta-methyl aspartic acid into RGD-containing peptides

We report the synthesis and biological activity of a series of side-chain-constrained RGD peptides containing the (2S,3R) or (2S,3S) beta-methyl aspartic acid within the RGD sequence. These compounds have been assayed for binding to the integrin receptors alpha(IIb)beta3 and alpha(v)beta3 and the results demonstrate the importance of the side-chain orientation of this particular residue within the RGD sequence. Based on our findings, the (2S,3S) beta-methylated analogues of our RGD sequences maintain their binding potency to the integrin receptors while the (2S,3R) beta-methylated analogues exhibit a drastically reduced binding affinity. Our studies demonstrate that the three-dimensional orientation of the aspartyl side chain is a very important parameter for integrin binding and that small changes that affect the side-chain orientations give rise to drastic changes in binding affinity. These results provide important information for the design of more potent RGD mimetics.