ß-1 tubulin R307H SNP alters microtubule dynamics and affects severity of a hereditary thrombocytopenia.

BACKGROUND: Single nucleotide polymorphisms (SNPs) in platelet-associated genes partly explain inherent variability in platelet counts. Patients with monoallelic Bernard Soulier syndrome due to the Bolzano mutation (GPIBA A156V) have variable platelet counts despite a common mutation for unknown reasons. OBJECTIVES: We investigated the effect of the most common SNP (R307H) in the hematopoietic-specific tubulin isotype ß-1 in these Bernard Soulier patients and potential microtubule-based mechanisms of worsened thrombocytopenia. PATIENTS/METHODS: Ninety-four monoallelic Bolzano mutation patients were evaluated for the R307H ß-1 SNP and had platelet counts measured by three methods; the Q43P SNP was also evaluated. To investigate possible mechanisms underlying this association, we used molecular modeling of ß-1 tubulin with and without the R307H SNP. We transfected SNP or non-SNP ß-1 tubulin into MCF-7 and CMK cell lines and measured microtubule regrowth after nocodazole-induced depolymerization. RESULTS: We found that patients with at least one R307H SNP allele had significantly worse thrombocytopenia; manual platelet counting revealed a median platelet count of 124 in non-SNP patients and 76 in SNP patients (both ×10(9)  L(-1) ; P < 0.01). The Q43P SNP had no significant association with platelet count. Molecular modeling suggested a structural relationship between the R307H SNP and microtubule stability via alterations in the M-loop of ß tubulin; in vitro microtubule recovery assays revealed that cells transfected with R307H SNP ß-1 had significantly impaired microtubule recovery. CONCLUSIONS: Our data show that the R307H SNP is significantly associated with the degree of thrombocytopenia in congenital and acquired platelet disorders, and may affect platelets by altering microtubule behavior.

Transport spectroscopy of single Pt impurities in silicon using Schottky barrier MOSFETs.

We investigate low temperature electron transport in silicon Schottky barrier metal-oxide-semiconductor field-effect transistors (MOSFETs), which consist of PtSi metallic source/drain electrodes. Measurements are made on approximately 23 inversion layers and resonances attributed to single impurities close to the metal/semiconductor interface are observed. We ascribe these impurities to Pt atoms that have diffused into the semiconductor channel from the contacts.

Ciprofloxacin microprecipitates and macroprecipitates in the human corneal epithelium.

In 4 corneal transplantation patients treated preoperatively with ciprofloxacin ophthalmic drops, microprecipitates associated with damaged corneal epithelium were noted in 2 patients. Another patient developed a large macroprecipitate in a corneal ulcer. All specimens were examined by electron microscopy and high-pressure liquid chromatography. The crystalline precipitates were pure ciprofloxacin. The macroprecipitate demonstrated a large zone of inhibition on agar plates seeded with a susceptible organism at 24 and 48 hours. It was bioactive and bioavailable in vitro.

Synthesis and biological evaluation of novel macrocyclic paclitaxel analogues.

[reaction: see text] This work describes the synthesis of two novel macrocyclic taxoid constructs by ring-closing olefin metathesis (RCM) and their biological evaluation. Computational studies examine conformational profiles of 1 and 2 for their fit to the beta-tubulin binding site determined by electron crystallography. The results support the hypothesis that paclitaxel binds to microtubules in a "T" conformation.

Polyoxometalate HIV-1 protease inhibitors. A new mode of protease inhibition.

Nb-containing polyoxometalates (POMs) of the Wells-Dawson class inhibit HIV-1 protease (HIV-1P) by a new mode based on kinetics, binding, and molecular modeling studies. Reaction of alpha(1)-K(9)Li[P(2)W(17)O(61)] or alpha(2)-K(10)[P(2)W(17)O(61)] with aqueous H(2)O(2) solutions of K(7)H[Nb(6)O(19)] followed by treatment with HCl and KCl and then crystallization affords the complexes alpha(1)-K(7)[P(2)W(17)(NbO(2))O(61)] (alpha(1)()1) and alpha(2)-K(7)[P(2)W(17)(NbO(2))O(61)] (alpha(2)()1) in 63 and 86% isolated yields, respectively. Thermolysis of the crude peroxoniobium compounds (72-96 h in refluxing H(2)O) prior to treatment with KCl converts the peroxoniobium compounds to the corresponding polyoxometalates (POMs), alpha(1)-K(7)[P(2)W(17)NbO(62)] (alpha(1)()2) and alpha(2)-K(7)[P(2)W(17)NbO(62)] (alpha(2)()2), in moderate yields (66 and 52%, respectively). The identity and high purity of all four compounds were confirmed by (31)P NMR and (183)W NMR. The acid-induced dimerization of the oxo complexes differentiates sterically between the cap (alpha(2)) site and the belt (alpha(1)) site in the Wells-Dawson structure (alpha(2)()2 dimerizes in high yield; alpha(1)()2 does not). All four POMs exhibit high activity in cell culture against HIV-1 (EC(50) values of 0.17-0.83 microM), are minimally toxic (IC(50) values of 50 to >100 microM), and selectively inhibit purified HIV-1 protease (HIV-1P) (IC(50) values for alpha(1)()1, alpha(2)()1, alpha(1)()2, and alpha(2)()2 of 2.0, 1.2, 1.5, and 1.8 microM, respectively). Thus, theoretical, binding, and kinetics studies of the POM/HIV-1P interaction(s) were conducted. Parameters for [P(2)W(17)NbO(62)](7)(-) were determined for the Kollman all-atom (KAA) force field in Sybyl 6.2. Charges for the POM were obtained from natural population analysis (NPA) at the HF/LANL2DZ level of theory. AutoDock 2.2 was used to explore possible binding locations for the POM with HIV-1P. These computational studies strongly suggest that the POMs function not by binding to the active site of HIV-1P, the mode of inhibition of all other HIV-1P protease inhibitors, but by binding to a cationic pocket on the "hinge" region of the flaps covering the active site (2 POMs and cationic pockets per active homodimer of HIV-1P). The kinetics and binding studies, conducted after the molecular modeling, are both in remarkable agreement with the modeling results: 2 POMs bind per HIV-1P homodimer with high affinities (K(i) = 1.1 +/- 0.5 and 4.1 +/- 1.8 nM in 0.1 and 1.0 M NaCl, respectively) and inhibition is noncompetitive (k(cat) but not K(m) is affected by the POM concentration).

Novel d-seco paclitaxel analogues: synthesis, biological evaluation, and model testing.

Four new D-secopaclitaxel analogues were synthesized from paclitaxel. The key step of the synthesis involved the opening of the D-ring by Jones oxidation. Two of the compounds had been predicted to be nearly as active as paclitaxel in a minireceptor model of the binding site on tubulin, but all were biologically inactive in an in vitro cytotoxic assay and a tubulin assembly assay. The biological results identify a weakness in our predictive minireceptor model and suggest a corrective remedy in which additional amino acids are needed to accommodate ligand-protein steric effects around the oxetane ring. These changes to the model lead to correct predictions of the bioactivity. Conformational analysis and dynamics simulations of the compounds showed that the 4-acetyl substituent is as important as the oxetane in determining the A ring conformation.

Synthesis and NMR-driven conformational analysis of taxol analogues conformationally constrained on the C13 side chain.

Analogues of Taxol (paclitaxel) with the side chain conformationally restricted by insertion of a carbon linker between the 2'-carbon and the ortho-position of the 3'-phenyl ring were synthesized. Biological evaluation of these new taxoids showed that activity was dependent on the length of the linker and the configuration at C2' and C3'. Two analogues in the homo series, 9a and 24a, showed tubulin binding and cytotoxicity comparable to that of Taxol. NAMFIS (NMR analysis of molecular flexibility in solution) deconvolution of the averaged 2-D NMR spectra for 9a yields seven conformations. Within the latter set, the hydrophobically collapsed "nonpolar" and "polar" classes are represented by one conformation each with predicted populations of 12-15%. The five remaining conformers, however, are extended, two of which correspond to the T-conformation (47% of the total population). The latter superimpose well with the recently proposed T-Taxol binding conformer in beta-tubulin. The results provide evidence for the existence of two previously unrecognized structural features that support Taxol-like activity: (1) a reduced torsion angle between C2' and C3' and (2) an orthogonal arrangement of the mean plane through C1', C2' and the 2'-hydroxyl and the 3'-phenyl plane, the latter ring bisected by the former plane. By contrast, epimerization at 2',3' and homologation of the tether to CH2-CH2 were both detrimental for activity. The decreased activity of these analogues is apparently due to configurational and steric factors, respectively.

Total synthesis of SR 121463 A, a highly potent and selective vasopressin v(2) receptor antagonist.

SR 121463 A, 1, is a promising nonpeptide prototype for potent and selective antagonism of the vasopressin V(2) receptor subtype and, thus, a candidate for control of the clinically debilitating condition of hyponatremia and its associated syndromes. In the present work, we present a novel and stereoselective synthesis that stems from the preparation of three key intermediates: the substituted benzenesulfonyl chloride 2, the N-protected oxindole 3, and protected dibromide 4. The synthesis of 1 has been achieved in good overall yield, each step proceeding in greater than 80% yield. In addition, intermediate 2 and the syn isomer of 1 were prepared with complete control of stereochemistry. The latter reduction appears to proceed by lithium cation mediated chelation control. Molecular mechanics calculations with the MM3* and MMFF force fields underscore geometric and energetic aspects of the reaction.

The binding conformation of Taxol in beta-tubulin: a model based on electron crystallographic density.

The chemotherapeutic drug Taxol is known to interact within a specific site on beta-tubulin. Although the general location of the site has been defined by photoaffinity labeling and electron crystallography, the original data were insufficient to make an absolute determination of the bound conformation. We have now correlated the crystallographic density with analysis of Taxol conformations and have found the unique solution to be a T-shaped Taxol structure. This T-shaped or butterfly structure is optimized within the beta-tubulin site and exhibits functional similarity to a portion of the B9-B10 loop in the alpha-tubulin subunit. The model provides structural rationalization for a sizeable body of Taxol structure-activity relationship data, including binding affinity, photoaffinity labeling, and acquired mutation in human cancer cells.

The oxetane ring in taxol.

Numerous structure-activity studies combining synthesis and bioassay have been performed for the anti-cancer drug Taxol. The four-membered D-ring, an oxetane, is one of four structural features regarded to be essential for biological activity. This proposition is examined by application of a Taxol-epothilone minireceptor, K(i) estimation for microtubule binding and docking of Taxol analogues into a model of the Taxol-tubulin complex. In this way, we evaluate the two characteristics considered responsible for oxetane function: (1) rigidification of the tetracyclic Taxol core to provide an appropriate framework for presenting the C-2, C-4, C-13 side chains to the microtubule protein and (2) service as a hydrogen-bond acceptor. An energy decomposition analysis for a series of Taxol analogues demonstrates that the oxetane ring clearly operates by both mechanisms. However, a broader analysis of four-membered ring containing compounds, C- and D-seco derivatives, and structures with no oxetane equivalent underscores that the four-membered ring is not necessary for Taxol analogue bioactivity. Other functional groups and ligand-protein binding characteristics are fully capable of delivering Taxol biobehavior as effectively as the oxetane D-ring. This insight may contribute to the design and development of novel anticancer drugs.

A unified and quantitative receptor model for the microtubule binding of paclitaxel and epothilone.

[formula: see text] Paclitaxel and epothilone represent the two major classes of antimicrotubule agents that promote tubulin polymerization and, presumably, mitotic arrest during cell division. A common minireceptor binding site model at beta-tubulin has been constructed for these structurally divergent compounds. Utilizing 20 amino acids identified in photoaffinity labeling experiments, the 3-D model correlates measured and predicted Ki's with r = 0.99 and rms(delta Gcalc-delta Gexp) = 0.2 kcal/mol. In addition, the model predicts the affinity of compounds not used in the training set and explains much of the SAR for the paclitaxel and epothilone families.

Molecular design using the minireceptor concept.

Explicit molecular binding pockets were constructed and optimized around sets of superimposed ligands using the minireceptor concept. The resulting binding sites incorporate the properties of the different ligands and were shown to be suitable for the design of molecules presenting novel interaction patterns. Two applications of minireceptor construction and/or optimization, followed by molecular design are described. In the pursuit of new ligands mimicking the action of paclitaxel, a minireceptor was constructed using the primary amino acid sequence of the target protein as a guide. The active site extracted from a homology-based model of the serotonin 5-HT1A receptor was optimized around a set of three ligands using the same approach.

Synthesis, preliminary evaluation and molecular modeling studies of new, conformationally constrained analogues of the competitive NMDA receptor antagonist 4-(phosphonomethyl)-2-piperidinecarboxylic acid (CGS 19755).

Two new spirobicyclophosphonate isomers (19 and 20), conformationally constrained analogues of the potent competitive NMDA antagonist CGS 19755 (4), have been designed and synthetized with the aim of gaining insight into the conformational preference of the crucial distal phosphonate moiety at the antagonist NMDA binding site. The preliminary biological evaluation reveals that the activity as NMDA antagonist resides only in the (1R,5S,7R)-isomer (19), characterized by a (-)-gauche disposition around the C1-C5 bond, thus confirming previously reported pharmacophore models.

Second-generation leukotriene B4 receptor antagonists related to SC-41930: heterocyclic replacement of the methyl ketone pharmacophore.

Our previous reports have highlighted the first-generation leukotriene B4 (LTB4) receptor antagonist SC-41930 (7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxy]3,4- dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid) which has potent oral, topical, and intracolonic activity in various animal models of inflammation. Extensive structure-activity relationship studies, in which a series of heterocyclic replacements for the methyl ketone functional group of SC-41930 was explored, identified SC-50605 (7-[3-[2-(cyclopropylmethyl)-3-methoxy-4- (4-thiazolyl)phenoxy]propoxy]-3,4-dihydro-8-propyl-2H-1-benzopyran-2- carboxylic acid) as an optimized analog within a series of thiazoles. SC-50605 was found to be significantly more potent than SC-41930 in LTB4 receptor binding, chemotaxis, and degranulation assays. It also displayed very good activity in animal models of colitis and epidermal inflammation by oral, topical, intravenous, and intracolonic routes of administration. The resolved enantiomers of SC-50605 were obtained by chiral chromatography and both demonstrated good in vitro and in vivo activity. The (+)-isomer (SC-52798) is currently being evaluated as a potential clinical candidate for psoriasis and ulcerative colitis therapy.

Pseudo-receptor modeling: a new concept for the three-dimensional construction of receptor binding sites.

Molecular design of small molecules intended to target a macromolecule generally utilizes one of two computational approaches: "receptor fitting" or "receptor mapping". A comprehensive strategy for the design of potent, selective and novel ligands for cell-bound receptors combines the two by means of "pseudoreceptor modeling". Definition of a refined pharmacophore model is the first step. A subsequent step involves the construction of a pseudoreceptor--an explicit molecular binding pocket--for the bioactive conformation of a series of ligands with high affinity for a particular receptor subtype. The receptor-mapping program "Yak" allows the construction of a peptidic pseudoreceptor around any single small molecule or molecular ensemble of interest. The fidelity of the approach is exemplified by application to the active site of the enzymes human carbonic anhydrase I and thermolysin, followed by comparison with their known X-Ray crystal structures.