Bifunctional inhibition of HIV-1 reverse transcriptase: a first step in designing a bifunctional triphosphate.

The onset of resistance to approved anti-AIDS drugs by HIV necessitates the search for novel inhibitors of HIV-1 reverse transcriptase (RT). Developing single molecular agents concurrently occupying the nucleoside and nonnucleoside binding sites in RT is an intriguing idea but the proof of concept has so far been elusive. As a first step, we describe molecular modeling to guide focused chemical syntheses of conjugates having nucleoside (d4T) and nonnucleoside (TIBO) moieties tethered by a flexible polyethylene glycol (PEG) linker. A triphosphate of d4T-6PEG-TIBO conjugate was successfully synthesized that is recognized as a substrate by HIV-1 RT and incorporated into a double-stranded DNA.

Novel compounds with antiproliferative activity against imatinib-resistant cell lines.

Chronic myelogenous leukemia is caused by the Bcr-Abl hybrid gene that encodes the p210Bcr-Abl chimeric oncoprotein. Although it reduces the total body burden of leukemia cells, the use of imatinib mesylate as a single agent may be accompanied by the evolution of resistance due mainly to the acquisition of point mutations. Imatinib has been combined with drugs that inhibit both the active and the inactive states of the p210Bcr-Abl kinase. These combinations have reduced but not completely eliminated the rate at which point mutations are acquired in the p210Bcr-Abl kinase. Thus, it is important to identify additional new inhibitors of the p210Bcr-Abl kinase. One possible method to prevent evolution of resistance is to simultaneously use multiple kinase inhibitors each with a different mechanism of action. To identify such a new class of inhibitors that could suppress the growth of chronic myelogenous leukemia cells and prevent the evolution of cells that are resistant to imatinib, we screened two low-complexity libraries of compounds based on planar and linear scaffolds. These libraries were screened using a cell-based assay for molecules that suppress p210Bcr-Abl-dependent cell growth. The application of this method resulted in the isolation of two new classes of drugs, both of which inhibited imatinib-resistant cells in the low micromolar range. Some of these drugs were potent inhibitors not only of Abl tyrosine kinase but also of the Src, Lyn, and Fyn tyrosine kinases.

EBP, a program for protein identification using multiple tandem mass spectrometry datasets.

MS/MS combined with database search methods can identify the proteins present in complex mixtures. High throughput methods that infer probable peptide sequences from enzymatically digested protein samples create a challenge in how best to aggregate the evidence for candidate proteins. Typically the results of multiple technical and/or biological replicate experiments must be combined to maximize sensitivity. We present a statistical method for estimating probabilities of protein expression that integrates peptide sequence identifications from multiple search algorithms and replicate experimental runs. The method was applied to create a repository of 797 non-homologous zebrafish (Danio rerio) proteins, at an empirically validated false identification rate under 1%, as a resource for the development of targeted quantitative proteomics assays. We have implemented this statistical method as an analytic module that can be integrated with an existing suite of open-source proteomics software.

Paclitaxel induces calcium oscillations via an inositol 1,4,5-trisphosphate receptor and neuronal calcium sensor 1-dependent mechanism.

Taxol (Paclitaxel) is an important natural product for the treatment of solid tumors. Despite a well documented tubulin-stabilizing effect, many side effects of taxol therapy cannot be explained by cytoskeletal mechanisms. In the present study submicromolar concentrations of taxol, mimicking concentrations found in patients, induced cytosolic calcium (Ca(2+)) oscillations in a human neuronal cell line. These oscillations were independent of extracellular and mitochondrial Ca(2+) but dependent on intact signaling via the phosphoinositide signaling pathway. We identified a taxol binding protein, neuronal Ca(2+) sensor 1 (NCS-1), a Ca(2+) binding protein that interacts with the inositol 1,4,5-trisphosphate receptor from a human brain cDNA phage display library. Taxol increased binding of NCS-1 to the inositol 1,4,5-trisphosphate receptor. Short hairpin RNA-mediated knockdown of NCS-1 in the same cell line abrogated the response to taxol but not to other agonists stimulating the phosphoinositide signaling pathway. These findings are important for studies involving taxol as a research tool in cell biology and may help to devise new strategies for the management of side effects induced by taxol therapy.

Identification of a molecular recognition role for the activation loop phosphotyrosine of the SRC tyrosine kinase.

A human cDNA phage display library screen, using a phosphopeptide designed to mimic the activation loop phosphotyrosine of the Src tyrosine kinase, has identified the N-terminal SH2 domain of the p85 regulatory subunit of phosphatidyl inositol-3 kinase (PI3K) as an interacting recognition domain. Activation loop phosphorylation is known to play a conformational role in kinase activation, but is largely not thought to play a role in protein/protein recognition. Affinity chromatography and biochemical evaluation in mouse fibroblast cells has confirmed the dependence of this interaction on both the Src activation loop phosphotyrosine and the N-terminal SH2 domain of PI3K.

Synthesis and antiviral activity of helioxanthin analogues.

A series of natural product analogues based on helioxanthin (2), with particular attention to modification of the lactone ring and methylenedioxy group, were synthesized and evaluated for their antiviral activities. Among them, lactam derivative 18 and helioxanthin cyclic hydrazide 28 exhibited significant in vitro antiviral activity against hepatitis B virus (EC(50) = 0.08 and 0.03 microM, respectively). Compound 18 showed the most potent antiviral activity against hepatitis C virus (55% inhibition at 1.0 microM). Compound 12, an acid-hydrolyzed product of helioxanthin cyclic imide derivative 9, was found to exhibit broad-spectrum antiviral activity against hepatitis B virus (EC(50) = 0.8 microM), herpes simplex virus type 1 (EC(50) = 0.15 microM) and type 2 (EC(50) < 0.1 microM), Epstein-Barr virus (EC(50) = 9.0 microM), and cytomegalovirus (EC(50) = 0.45 microM). Helioxanthin lactam derivative 18 also showed marked inhibition of herpes simplex virus type 1 (EC(50) = 0.29 microM) and type 2 (EC(50) = 0.16 microM). The cyclic hydrazide derivative of helioxanthin 28 and its brominated product 42 exhibited moderately potent activities against human immunodeficiency virus (EC(50) = 2.7 and 2.5 microM, respectively). Collectively, these molecules represent a novel set of antiviral compounds with unique structural features.

Hypervalent iodine-mediated vicinal syn diazidation: application to the total synthesis of (+/-)-dibromophakellstatin.

[reaction: see text] The total synthesis of (+/-)-dibromophakellstatin is described. The molecule is constructed from a key syn-diazide, formed by a hypervalent iodine-mediated diazidation of a dihydrodipyrrolopyrazinone ring structure.

Increased anti-P-glycoprotein activity of baicalein by alkylation on the A ring.

The aqueous extract of Scutellariae baicalensis Georgi has inhibitory activity against P-gp 170, a multiple drug resistant gene product. Baicalein, one of the major flavones, was found to be responsible for this activity. The hydroxyl groups of the A ring of baicalein were systematically alkylated in order to assess the effect of such modifications on the activity against P-gp 170. The impact of the baicalein modifications on activity against the growth of a human nasopharyngeal cancer cell line KB and its P-gp 170 overexpressing cell line KB/MDR were also examined. The results indicate that alkylation of R5 of baicalein does not have a major impact on the interaction with P-gp 170, whereas alkylation of R6 or R7 alone or both, could enhance the interaction of baicalein with P-gp 170 as well as the amount of intracellular accumulation of vinblastine, a surrogate marker for the activity of P-gp 170 pump of KB/MDR cells. In this case, the optimal linear alkyl functionality is a propyl side chain. These modifications could also alter the activity of compounds inhibiting cell growth. Among the different compounds synthesized, the most potent molecule against P-gp 170 is 5-methoxy-6,7-dipropyloxyflavone (23). Its inhibitory activity against P-gp 170 is approximately 40 times better, based on EC50 (concentration of the compound enhancing 50% of the intracellular vinblastine accumulation in the KB/MDR cells) and 3 times higher, based on Amax (the intracellular vinblastine accumulation of the KB/MDR cells caused by the compound) as compared to baicalein. Compound 23 is also a more selective inhibitor than baicalein against P-gp 170, because its cytotoxicity is less than that observed for baicalein. The growth inhibitory IC50 of compound 23 against KB and KB/MDR cells are about the same, suggesting that compound 23 is unlikely to be a substrate of P-gp 170 pump. Acetylation of R6, R7 or both could also decrease EC50 and increase Amax. Acetylated compounds are more toxic than baicalein, and their potency against cell growth is compromised by the presence of P-gp 170, suggesting that these compounds are substrates of P-gp 170. Benzylation of R6 or R7 but not both also enhanced anti-P-gp170 activity and potency against cell growth; however, the presence of P-gp 170 in cells did not have an impact on their sensitivity to these molecules, suggesting that the benzylated compounds are inhibitors but not substrates of P-gp 170, and perhaps have a different mechanism of action. In conclusion, the substitutions of R6 and R7 hydroxyl groups by alkoxy groups, acetoxy groups, or benzyloxy groups could yield compounds with different modes of action against P-gp 170 with different mechanisms of action against cell growth.

Two-dimensional diversity: screening human cDNA phage display libraries with a random diversity probe for the display cloning of phosphotyrosine binding domains.

A random phosphopeptide probe (bio-pYZZZ) has been used for the isolation and identification of multiple SH2 domains from human cDNA-displaying phage libraries. In addition, on-phage analysis and quantification of binding affinities for these phage-displayed proteins has shown them to be functional domains, retaining the same characteristics as in their native state.

Right ventricle-sparing heart transplantation effective against iatrogenic pulmonary hypertension.

BACKGROUND: Right heart failure is the predominant cause of death following heart transplantation, occurring with disturbingly high frequency in patients with severe antecedent pulmonary hypertension. We have recently reported a novel technique of heart transplantation that spares the recipient right ventricle, excising only the recipient left ventricle. The resulting model has 2 right hearts and 1 left heart. The aim is to preserve the recipient's right ventricle, which is already conditioned to pulmonary hypertension. The hope is that, in this way, death due to right heart failure can be prevented in humans. Our prior report was a feasibility study in normal dogs. This study challenges this new technique by creating iatrogenic pulmonary hypertension in the recipient animals. METHODS: Iatrogenic pulmonary hypertension was created in 4 recipient canines by intravenous injection of the pulmonary toxin monocrotaline pyrrole (single bolus of 3.5 to 4.5 mg/kg intravenously [i.v.]). RESULTS: Within 6 weeks of monocrotaline administration, relative pulmonary hypertension occurred (mean pulmonary artery [PA] pressure 20 mm Hg vs 10 mm Hg for controls [p < 0.01]) (pulmonary vascular resistance [PVR] 4.2 vs 1.5 Wood units [P < 0.01]), and right ventricular (RV) hypertrophy developed (RV thickness 11 mm vs 2 mm [P < 0.04]). Histologic examination confirmed severe muscle infiltration and thickening of the media of the pulmonary arterioles. RV-sparing heart transplantation was performed successfully in all 4 animals with pulmonary hypertension. In all cases, the animals were weaned without difficulty from cardiopulmonary bypass, despite the ambient pulmonary hypertension, on low-dose epinephrine, maintaining systolic blood pressure of 104 mm Hg at right atrial pressure of 7 mm Hg. Both right hearts contracted well without dilation or strain. A single "control" traditional orthotopic transplant experiment in an animal with monocrotaline-induced pulmonary hypertension resulted in immediate death from right heart failure. CONCLUSIONS: Right ventricle-sparing heart transplantation ("one-and-one-half heart model") can handle pulmonary hypertension without difficulty. This evidence adds impetus for further pursuing of right ventricle-sparing heart transplantation to decrease the incidence of death from right heart failure in recipients with severe antecedent pulmonary hypertension.

Adenosine-anchored triphosphate subsite probing: distinguishing between HER-2 and HER-4 tyrosine protein kinases.

A strategy of full-site occupancy and stereospecific recognition in the triphosphate subsite was used to specifically inhibit two protein kinases HER-2 and HER-4 from the EGFR family. The SAR profiles of a panel of adenosine-anchored bicyclic heterocycles against HER-2 and HER-4 indicated that specificity can be derived for highly homologous protein kinases from stereospecific recognition in the triphosphate-subsite.

A transient N-O-linked Pauson-Khand strategy for the synthesis of the deschloro carbocyclic core of the palau'amines and styloguanidines.

A stereocontrolled route to the deschloro cyclopentyl core of the palau'amines and styloguanidines has been developed. This strategy makes use of the intramolecular Pauson-Khand cyclization of an enyne with a "transient N-O tether" to construct a five-membered carbocycle in a diastereoselective fashion. [reaction: see text]

Modular evolution of a chiral auxiliary for the 1,3-dipolar cycloaddition of isomünchnones with vinyl ethers.

[reaction: see text]. The 1,3-dipolar cycloaddition reaction has long been recognized as a powerful methodology in organic synthesis. More recently, this reaction has become a popular manifold for the construction of chemical diversity. Herein, we report the development of a chiral template for the facially selective cycloaddition of isomünchnones, a common class of 1,3-dipoles. The modular format of the asymmetric unit allowed a systematic optimization of selectivity. In addition, the chiral auxiliary was removed through an unusually facile ester aminolysis.

Diastereofacial solid phase synthesis and self-promoted cleavage of a [2.2.1] bicyclic diversity scaffold.

[reaction: see text]. We have previously described a diastereofacially selective 1,3-dipolar cycloaddition reaction of isomünchnones with vinyl ethers. While adapting this methodology for solid phase synthesis, we discovered a chemoselective and self-promoted linker aminolysis that provides liberated product in high purity, at a significantly enhanced rate. Herein we describe the implementation of a chiral auxiliary as a solid-phase linker, the detailed investigation of its unique aminolysis, and the utility of this cleavage within a chemical diversity format.