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.

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.

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.

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.

Conformationally restricted inhibitors of angiotensin converting enzyme: synthesis and computations.

A series of inhibitors of angiotensin converting enzyme (ACE, dipeptidyl carboxypeptidase, EC 3.4.15.1) is described which addresses certain conformational aspects of the enzyme-inhibitor interaction. In this study the alanylproline portion of the potent ACE inhibitor enalaprilat (2) is replaced by a series of monocyclic lactams containing the required recognition and binding elements. In order to more fully assess the lactam ring conformations and the key backbone angle psi as defined in 3 with respect to possible enzyme-bound conformations, a series of model lactams was investigated with use of molecular mechanics. The results point to a correlation between inhibitor potency (IC50) and the computed psi angle for the lowest energy conformation of the model compounds. Thus the psi angle as defined in 3 is an important determinant in the binding of inhibitors to ACE. The inhibition data in conjunction with the computational data have served to define a window of psi angles from 130 degrees to 170 degrees which seems to be acceptable to the ACE active site.

Unsuspected syphilitic hepatitis in a patient with low-grade proteinuria and abnormal liver function.

A 25-year-old patient was found to have cholestatic liver enzyme abnormalities during assessment for asymptomatic low-grade proteinuria at the US Naval Hospital in Portsmouth, Virginia. These abnormalities persisted for a 6-month period, and an extensive workup, including viral serologic studies, rapid plasma reagin test, iron studies, ceruloplasmin, antimitochondrial, antinuclear, and anti-human immunodeficiency virus antibodies, endoscopic retrograde cholangiopancreatography, and liver biopsy, was unrevealing until serologic tests for syphilis were repeated to evaluate a new onset of urethral discharge. The patient had none of the more characteristic signs of secondary syphilis. The liver enzyme abnormalities rapidly resolved after treatment with penicillin. Syphilis remains the great impostor and still must be considered in the differential diagnosis of unexplained liver enzyme abnormalities, even in a patient with no symptoms or signs of early syphilis.

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.

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.

Characterization of D-3,4-cyclopropylglutamates as N-methyl-D-aspartate receptor agonists.

The 4 configurational isomers of D-3,4-cyclopropylglutamate (D-CGA) have been synthesized and analyzed for their interactions as excitatory amino acid recognition sites. Additionally, functional assessment of the action of these compounds at the N-methyl-D-aspartate (NMDA) receptor was performed. All 4 analogs function as agonists at the NMDA receptor as evidenced by their ability to stimulate [3H]MK-801 binding to the coupled PCP recognition site. Furthermore, the rank order of potency of these compounds in stimulating [3H]MK-801 binding corresponds with their Ki values for the displacement of NMDA-selective L-[3H]glutamate and [3H]CGS-19755 binding (D-CGA-C greater than D-CGA-B greater than D-CGA-D greater than D-CGA-A). The D-CGA-C isomer has affinity and potency at the NMDA receptor similar to the endogenous agonist, L-glutamate. This high potency coupled with greater specificity than L-glutamate, makes D-CGA-C a potentially useful pharmacological tool for the study of this receptor.

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, 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.

A novel comprehensive opiate-receptor model.

A new, comprehensive opiate-receptor model based on calculations of possible events at the molecular level is proposed. Agonist versus antagonist action, ultra long-lasting activity and N-dealkylation accompanying receptor binding are included.

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.

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.

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.

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.

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.