Biomedical subjects

Joseph J Barchi

Publications and source records attributed to Joseph J Barchi.

15 recordsLinked to original sources

PubMed2006

Conformational analysis of an alpha3beta1 integrin-binding peptide from thrombospondin-1: implications for antiangiogenic drug design.

The integrin alpha3beta1 plays important roles in development, angiogenesis, and the pathogenesis of cancer, suggesting potential therapeutic uses for antagonists of this receptor. Recently, an alpha3beta1 integrin-binding site was mapped to residues 190-201 (FQGVLQNVRFVF) of the N-terminal domain of the secreted protein thrombospondin-1 (TSP1). This sequence displays diverse biological activities in vitro and inhibits angiogenesis in vivo. Herein we describe the NMR solution conformation of this segment in both water and dodecylphosphocholine micelles. While essentially unstructured in water, a more well-defined conformation is populated in micelles, particularly in the C-terminal half of the peptide and correlated with increased biological activity of the micellar peptide. The data suggested that the residues that are critical for biological activity are contained in a structurally well-defined segment of the peptide. These data support the role of the NVR motif as a required element of full-length TSP1 for specific molecular recognition by the alpha3beta1 integrin.

Angiogenesis Inhibitors↗

PubMed2006

Mining the NCI antiviral compounds for HIV-1 integrase inhibitors.

HIV-1 integrase (IN) is an essential enzyme for effective viral replication and is a validated target for the development of antiretroviral drugs. Currently, there are no approved drugs targeting this enzyme. In this study, we have identified 11 structurally diverse small-molecule inhibitors of IN. These compounds have been selected by mining the moderately active antiviral molecules from a collection of 90,000 compounds screened by the National Cancer Institute (NCI) Antiviral Program. These compounds, which were screened at the NCI during the past 20 years, resulted in approximately 4000 compounds labeled as 'moderately active.' In our study, chalcone 11 shows the most potent activity with an IC(50) of 2+/-1 microM against purified IN in the presence of both Mn(2+) and Mg(2+) as cofactors. Docking simulations using the 11 identified inhibitors as a training set have elucidated two unique binding areas within the active site: the first encompasses the conserved D64-D116-E152 motif, while the other involves the flexible loop region formed by amino acid residues 140-149. The tested inhibitors exhibit favorable interactions with important amino acid residues through van der Waals and H-bonding contacts.

Anti-HIV Agents↗

PubMed2006

Reaction of geldanamycin and C17-substituted analogues with glutathione: product identifications and pharmacological implications.

17-Dimethylaminoethylamino-17-demethoxygeldanamycin (DMAG) and 17-allylamino-17-demethoxygeldanamycin (17-AAG) are two derivatives of geldanamycin (GA) that are currently undergoing clinical evaluation as anticancer agents. These agents bind to heat shock protein 90 (hsp90), resulting in the destabilization of client proteins and inhibition of tumor growth. In a search for the mechanism of hepatotoxicity, which is a dose-limiting toxicity for these agents, we found that GA and its derivatives, 17-AAG and 17-DMAG, react chemically (i.e., nonenzymatically) with glutathione (GSH). A combination of liquid chromatography/electrospray ionization/mass spectrometry and nuclear magnetic resonance analyses were used to identify the product of this reaction as a GSH adduct in which the thiol group of GSH is substituted in the 19-position of the benzoquinone ring. The reaction proceeds rapidly with GA and 17-DMAG (half-lives of approximately 1.5 and 36 min, respectively) and less rapidly with 17-AAG and its major metabolite, 17-AG (half-lives of approximately 9.8 and 16.7 h). The reaction occurs at pH 7.0, 37 degrees C, and a physiological concentration of GSH, indicating that cellular GSH could play a role in modulating the cellular toxicity of these agents and therefore be a factor in their mechanism of differential toxicity. Moreover, reactions with thiol groups of critical cellular proteins could be important to the mechanism of toxicity with this class of anticancer agents.

Antibiotics, Antineoplastic↗

PubMed2005

Diazeniumdiolate ions as leaving groups in anomeric displacement reactions: a protection-deprotection strategy for ionic diazeniumdiolates.

Diazeniumdiolate ions [R2N-N(O)=N-O-] are of growing interest pharmacologically for their ability to generate up to two molar equivalents of bioactive nitric oxide (NO) spontaneously on protonating the amino nitrogen. Accordingly, their stability increases as the pH is raised. Here we show that the corresponding beta-glucosides [R2N-N(O)=N-O-Glc] decreased in stability with pH; when R2N was diethylamino, the rate equation was kobs = ko + kOH- [OH-], where ko = 7.8 x 10-7 s-1 and kOH- = 5.3 x 10-3 M-1 s-1. The primary products were 1,6-anhydroglucose and the regenerated R2N-N(O)=N-O- ion. The results were qualitatively similar to those of beta-glucosyl fluoride and p-nitrophenoxide, whose hydrolyses have been rationalized as proceeding via a glycal oxide intermediate. This chemistry offers a convenient strategy for protecting heat- and acid-sensitive diazeniumdiolate ions during manipulations that would otherwise destroy them. As an example, a poly(urethane) film that generated NO in physiological buffer at a surface flux comparable to that of the mammalian vascular endothelium was prepared by glucosylating the ionic diazeniumdiolate group attached to the diol monomer before reacting it with the bis-isocyanate, then removing the saccharide with base when the protecting group was no longer needed.

Azo Compounds↗

PubMed2005

Evaluation of macrocyclic Grb2 SH2 domain-binding peptide mimetics prepared by ring-closing metathesis of C-terminal allylglycines with an N-terminal beta-vinyl-substituted phosphotyrosyl mimetic.

Preferential binding of ligands to Grb2 SH2 domains in beta-bend conformations has made peptide cyclization a logical means of effecting affinity enhancement. This is based on the concept that constraint of open-chain sequences to bend geometries may reduce entropy penalties of binding. The current study extends this approach by undertaking ring-closing metathesis (RCM) macrocyclization between i and i+3 residues through a process involving allylglycines and beta-vinyl-functionalized residues. Ring closure in this fashion results in minimal macrocyclic tetrapeptide mimetics. The predominant effects of such macrocyclization on Grb2 SH2 domain binding affinity were increases in rates of association (from 7- to 16-fold) relative to an open-chain congener, while decreases in dissociation rates were less pronounced (approximately 2-fold). The significant increases in association rates were consistent with pre-ordering of solution conformations to near those required for binding. Data from NMR experiments and molecular modeling simulations were used to interpret the binding results. An understanding of the conformational consequences of such i to i+3 ring closure may facilitate its application to other systems where bend geometries are desired.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Zebularine: a unique molecule for an epigenetically based strategy in cancer chemotherapy. The magic of its chemistry and biology.

1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine) is structurally 4-deamino cytidine. The increased electrophilic character of this simple aglycon endows the molecule with unique chemical and biological properties, making zebularine a versatile starting material for the synthesis of complex nucleosides and an effective inhibitor of cytidine deaminase and DNA cytosine methyltransferase. Zebularine is a stable, antitumor agent that preferentially targets cancer cells and shows activity both in vitro and in experimental animals, even after oral administration.

Administration, Oral↗

PubMed2005

Engineering DNA topology with locked nucleosides: a structural study.

DNA dodecamers modified with nucleotide building blocks based on a bicyclo[3. 1.0]hexane system that effectively locks the ribose template into an RNA-like or North (N) conformation were analyzed by various biophysical techniques including high field nuclear magnetic resonance (NMR). Replacement of either one or both of the center thymidines in the Dickerson Drew dodecamer (CGCGAAT*T*CGCG) caused a progressive shift in the bending propensity of the double helix as shown by a newly developed rapid technique that compares the residual dipolar coupling (RDC) values of the modified duplexes with those previously determined for the native DNA.

Biophysics↗

PubMed2004

Changes in DNA bending induced by restricting nucleotide ring pucker studied by weak alignment NMR spectroscopy.

Changes in bending of the DNA helix axis caused by the introduction of conformationally locked nucleotide analogs into the center region of the palindromic Dickerson dodecamer, d(CGCGAATTCGCG)(2), have been studied by NMR measurement of residual one-bond (13)C-(1)H dipolar couplings. Thymidine analogs, in which the deoxyribose was substituted by bicyclo[3.1.0]hexane, were incorporated in the T7, T8, and T7T8 positions. These nucleotide analogs restrict the ring pucker to the C2'-exo or "north" conformation, instead of C2'-endo or "south," which dominates in regular B-form DNA. For all three oligomers, bending toward the major groove is found relative to the native molecule. The effects are additive with bending of 5 +/- 1 degrees per locked nucleotide. Measurement of the change in bending is more accurate than measurement of the bending angle itself and requires far fewer experimental data.

Bridged Bicyclo Compounds↗

PubMed2004

Facile photochemical synthesis of mixed siloxyacetal glycosides as potential pH-sensitized prodrugs for selective treatment of solid tumors.

Photochemical reactions of a variety of acylsilanes with peracetylated free glycosides in anhydrous benzene at ambient temperature yielded novel, highly acid-sensitive siloxyacetal glycosides in 75-90% yields with complete retention of configuration at the anomeric center. Subsequent deacetylation of triisopropylsiloxy- and tert-butyldimethylsiloxy derivatives with sodium methoxide in methanol afforded deprotected siloxyacetal glycosides in nearly quantitative yields. Acid hydrolysis of trimethylsilyl siloxyacetals proceeded with a half-life of 17.5 minutes at pH 6.2 which is vastly superior to the decomposition rate of conventional acetals under similar conditions. The structure of one of the novel siloxyacetals was confirmed by X-ray crystallography. In vitro biological studies showed that glucose-derived siloxyacetals may serve as potential pH-activated prodrugs for selective treatment of solid tumors.

Acetals↗

PubMed2004

An antiproliferative factor from interstitial cystitis patients is a frizzled 8 protein-related sialoglycopeptide.

Approximately 1 million people in the United States suffer from interstitial cystitis, a chronic painful urinary bladder disorder characterized by thinning or ulceration of the bladder epithelial lining; its etiology is unknown. We have identified a glycosylated frizzled-related peptide inhibitor of cell proliferation that is secreted specifically by bladder epithelial cells from patients with this disorder. This antiproliferative factor (APF) profoundly inhibits bladder cell proliferation by means of regulation of cell adhesion protein and growth factor production. The structure of APF was deduced by using ion trap mass spectrometry (MS), enzymatic digestion, lectin affinity chromatography, and total synthesis, and confirmed by coelution of native and synthetic APF derivatives on microcapillary reversed-phase liquid chromatography (microRPLC)/MS. APF was determined to be an acidic, heat-stable sialoglycopeptide whose peptide chain has 100% homology to the putative sixth transmembrane domain of frizzled 8. Both synthetic and native APF had identical biological activity in normal bladder epithelial cells and T24 bladder cancer cells. Northern blot analysis indicated binding of a probe containing the sequence for the frizzled 8 segment with mRNA extracted from cells of patients with interstitial cystitis but not controls. APF is therefore a frizzled-related peptide growth inhibitor shown to contain exclusively a transmembrane segment of a frizzled protein and is a potential biomarker for interstitial cystitis.

Antineoplastic Agents↗

PubMed2004

Synthesis and characterization of oligonucleotides containing conformationally constrained bicyclo[3.1.0]hexane pseudosugar analogs.

Oligodeoxyribonucleotides containing pseudorotationally locked sites derived from bicyclo[3.1.0]hexane pseudosugars have been synthesized using adenosine, thymidine and abasic versions of North- and South-methanocarba nucleosides. The reaction conditions for coupling and oxidation steps of oligonucleotide synthesis have been investigated and optimized to allow efficient and facile solid-phase synthesis using phosphoramidite chemistry. Our studies demonstrate that the use of iodine for P(III) to P(V) oxidation leads to strand cleavage at the sites where the pseudosugar is North. In contrast, the same cleavage reaction was not observed in the case of South pseudosugars. Iodine oxidation generates a 5'-phosphate oligonucleotide fragment on the resin and releases the North pseudosugar into the solution. This side reaction, which is responsible for the extremely low yields observed for the incorporation of the North pseudosugar analogs, has been studied in detail and can be easily overcome by replacing iodine with t-butylhydroperoxide as oxidant.

Base Sequence↗

PubMed2004

Experimental and structural evidence that herpes 1 kinase and cellular DNA polymerase(s) discriminate on the basis of sugar pucker.

Two isomers of methanocarba (MC) thymidine (T), one an effective antiherpes agent with the pseudosugar moiety locked in the North (N) hemisphere of the pseudorotational cycle (1a, N-MCT) and the other an inactive isomer locked in the antipodean South (S) conformation (1b, S-MCT) were used to determine whether kinases and polymerases discriminate between their substrates on the basis of sugar conformation. A combined solid-state and solution conformational analysis of both compounds, coupled with the direct measurement of mono-, di-, and triphosphate levels in control cells, cells infected with the Herpes simplex virus, or cells transfected with the corresponding viral kinase gene (HSV-tk), suggests that kinases prefer substrates that adopt the S sugar conformation. On the other hand, the cellular DNA polymerase(s) of a murine tumor cell line transfected with HSV-tk incorporated almost exclusively the triphosphate of the locked N conformer (N-MCTTP), notwithstanding the presence of higher triphosphate levels of the S-conformer (S-MCTTP).

Animals↗

PubMed2003

Highly efficient preparation of tumor antigen-containing glycopeptide building blocks from novel pentenyl glycosides.

O-Glycosylated amino acids containing the tumor-associated T(Tf)-antigen (beta-D-Gal-(1-->3)-alpha-D-GalNAc) disaccharide unit were conveniently synthesized in seven steps starting from D-galactose via an n-pentenyl glycoside (NPG) building block. Azidonitration of 3,4,6-tri-O-acetyl-D-galactal, followed by nitrate displacement with simultaneous acetate hydrolysis with sodium 4-penten-1-oxide, afforded n-pentenyl 2-deoxy-2-azidogalactoside (3) in near quantitative yield. Subsequent high-yielding transformations resulted in the synthesis of the key glycosyl donor n-pentenyl beta-disaccharide 5 that was employed for the stereospecific preparation of glycosyl amino acids via NIS-promoted glycosylations with serine or threonine acceptors. The surprising utility of the reaction of sodium 4-penten-1-oxide with anomeric nitrates encouraged the detailed exploration of the action of a variety of nucleophiles on anomeric nitrates for the synthesis of useful 2-azido glycosyl donors directly from the 'classic' Lemieux azidonitration product of protected galactals. This expedient synthesis (28% overall yield from 1 to 7a) that makes use of heretofore rarely exploited pentenyl 2'-azidoglycosides, should be a valuable entry in the armamentarium of routes to biologically relevant glycopeptides in both mono- and multivalent forms.

Antigens, Neoplasm↗

PubMed2003

A conformationally locked analogue of the anti-HIV agent stavudine. An important correlation between pseudorotation and maximum amplitude.

The synthesis and biological evaluation of a bicyclo[3.1.0]hexene nucleoside designed as a conformational mimic of the anti-HIV agent stavudine (1, D4T) is described. The unsaturated methanocarbocyclic pseudosugar of N-MCD4T (2) was constructed from an iodo-substituted precursor by a DBU-catalyzed olefination reaction. Mitsunobu coupling with N(3)-benzoylthymine afforded the desired target after deprotection. Both D4T and N-MCD4T are in the North (N) hemisphere of the pseudorotational cycle but 70 degrees away from a perfect N (P = 0 degrees ) conformation toward the East and West hemispheres, respectively. Despite this large difference, the double bond reduces the puckering amplitude (nu(max)) of N-MCD4T to 6.81 degrees, and the superposition of both structures showed a RMS deviation of only 0.039 A. The combined structural analysis of P and nu(max) shows that while the value of P may differ substantially, the low nu(max) resolves the differences and becomes the dominant pseudorotational parameter. N-MCD4T is active against HIV-1 and HIV-2 in CEM, MT-2, and MT-4 cells, and while it is somewhat less potent than D4T, it also appears to be less toxic. The triphosphate (N-MCD4TTP) inhibits HIV reverse transcriptase with a 10-fold higher IC(50) than D4TTP. By virtue of its carbocyclic nature, N-MCD4T (2) is a more robust molecule stable to conditions that would cleave D4T.

Animals↗

PubMed2002

New paradigms in drug design and discovery.

The new millennium has ushered in an era of science that will revolutionize a great majority of our daily activities. That revolution is being experienced by a growing number of the population who are pushing the average life expectancy closer to the 80-year mark. The primary reason for this increase is the changes we have made in the last 2-3 decades both in how we live our lives as well as how we treat our maladies when they arise. The advent of new techniques in diagnostics and surgery have allowed many to survive debilitating illnesses when their chances would have been slim only a few years ago. In addition, several new therapeutic agents have been developed in the latter part of the 20th century that have improved our quality of life and increased our overall survival time. New medicines to treat cardiovascular, degenerative, infectious, and neoplastic diseases are rapidly being discovered in an effort to further lengthen our lifetimes. The processes used by academic and industrial scientist to discover new drugs has recently experienced a true renaissance with many new and exciting techniques being developed in only the past 5-10 years. In this review, we will attempt to outline these latest protocols that chemists and biomedical scientist are currently employing to rapidly bring new drugs to the clinic.

Animals↗