Biomedical subjects

R Kiplin Guy

Publications and source records attributed to R Kiplin Guy.

At least 19 recordsLinked to original sources

PubMed2006

Inactivation of the p53 pathway in retinoblastoma.

Most human tumours have genetic mutations in their Rb and p53 pathways, but retinoblastoma is thought to be an exception. Studies suggest that retinoblastomas, which initiate with mutations in the gene retinoblastoma 1 (RB1), bypass the p53 pathway because they arise from intrinsically death-resistant cells during retinal development. In contrast to this prevailing theory, here we show that the tumour surveillance pathway mediated by Arf, MDM2, MDMX and p53 is activated after loss of RB1 during retinogenesis. RB1-deficient retinoblasts undergo p53-mediated apoptosis and exit the cell cycle. Subsequently, amplification of the MDMX gene and increased expression of MDMX protein are strongly selected for during tumour progression as a mechanism to suppress the p53 response in RB1-deficient retinal cells. Our data provide evidence that the p53 pathway is inactivated in retinoblastoma and that this cancer does not originate from intrinsically death-resistant cells as previously thought. In addition, they support the idea that MDMX is a specific chemotherapeutic target for treating retinoblastoma.

Animals↗

PubMed2006

Design of a selective chemical probe for class I PDZ domains.

Covalent labeling has been widely used for structural and functional analyses of proteins. To target a wide range of PDZ domains, we designed a chemical scaffold mimicking the E/D-T/S-XV peptide, which is a PDZ domain that binds ligands in higher occurrence. A chemical probe (2) that contained this moiety alkylated diverse PDZ domains, including NHERF-1 PDZ2, and differentially visualized the cellular proteome.

Alkylation↗

PubMed2006

Rational design of a nonpeptide general chemical scaffold for reversible inhibition of PDZ domain interactions.

Novel small molecules were designed to specifically target the ligand-binding pocket of a PDZ domain. Iterative molecular docking and modeling allowed the design of an indole scaffold 10a as a reversible inhibitor of ligand binding. The 10a scaffold inhibited the interaction between MAGI-3 and PTEN and showed cellular activities that are consistent with the inhibition of NHERF-1 function.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Synthesis of highly substituted dibenzo[b,f]azocines and their evaluation as protein kinase inhibitors.

Synthetic routes towards highly substituted eight membered ring heterocycles fused to aryl rings such as the dibenzo[b,f]azocine system are still lacking. Herein, we present a convenient convergent synthetic route towards this heterocyclic class of compounds with possible variations at positions 4, 7, and 11. One member of a library of dibenzo[b,f]azocines with different substituents at position 11 was identified to inhibit protein kinase A activity (IC(50)=122microM) but not protein kinase C.

Azocines↗

PubMed2006

Incorporation of an intramolecular hydrogen-bonding motif in the side chain of 4-aminoquinolines enhances activity against drug-resistant P. falciparum.

Previous data showing that several chloroquine analogues containing an intramolecular hydrogen-bonding motif were potent against multidrug-resistant P. falciparum led to the exploration of the importance of this motif. A series of 116 compounds containing four different alkyl linkers and various aromatic substitutions with hydrogen bond accepting capability was synthesized. The series showed broad potency against the drug-resistant W2 strain of P. falciparum. In particular, a novel series containing variations of the alpha-aminocresol motif gave eight compounds with IC50 values more potent than 5 nM against the W2 strain. Such simple modifications, significantly altering the pKa and sterics of the basic side chain in chloroquine analogues, may prove to be part of a strategy for overcoming the problem of worldwide resistance to affordable antimalarial drugs.

Aminoquinolines↗

PubMed2006

Structure-activity relationship study of 9-aminoacridine compounds in scrapie-infected neuroblastoma cells.

A focused library of variously substituted 9-aminoacridine compounds was screened for bioactivity against accumulation of the infectious prion protein isoform, denoted PrP(Sc), in a cell model of prion replication. The efficacy of compounds against PrP(Sc) accumulation was influenced by both substituents of the distal tertiary amine and acridine heterocycle, while cellular cytotoxicity was encoded in the acridine heterocycle substituents.

Aminacrine↗

PubMed2006

A high-throughput screening method to identify small molecule inhibitors of thyroid hormone receptor coactivator binding.

To provide alternative methods for regulation of gene transcription initiated by the binding of thyroid hormone (T3) to the thyroid receptor (TR), we have developed a high-throughput method for discovering inhibitors of the interaction of TR with its transcriptional coactivators. The screening method is based on fluorescence polarization (FP), one of the most sensitive and robust high-throughput methods for the study of protein-protein interactions. A fluorescently labeled coactivator is excited by polarized light. The emitted polarized light is a function of the molecular properties of the labeled coactivator, especially Brownian molecular rotation, which is very sensitive to changes in the molecular mass of the labeled complex. Dissociation of hormone receptor from fluorescently labeled coactivator peptide in the presence of small molecules can be detected by this competition method, and the assay can be performed in a high-throughput screening format. Hit compounds identified by this method are evaluated by several secondary assay methods, including a dose-response analysis, a semiquantitative glutathione-S-transferase assay, and a hormone displacement assay. Subsequent in vitro transcription assays can detect inhibition of thyroid signaling at low micromolar concentrations of small molecules in the presence of T3.

Acetyltransferases↗

PubMed2006

An alpha-helical peptidomimetic inhibitor of the HIV-1 Rev-RRE interaction.

The interaction between the HIV-1 Rev protein and the Rev-Responsive Element (RRE) RNA is an attractive target for anti-viral therapy. We have designed alpha-helical peptidomimetics of Rev-like peptides using side chain-side chain macrolactam formation between positions i and i+4. One peptidomimetic having an appropriate location, orientation, and length of the macrolactam exhibited both significant helical character and specific RRE binding. This molecule displays 2-fold greater RNA specificity than the wild-type Rev peptide and more than 20-fold greater specificity than an uncyclized control peptide. Thus, specific, high affinity recognition of the RRE is feasible utilizing a small, relatively rigid peptidomimetic scaffold.

Amino Acid Sequence↗

PubMed2006

Synthesis and testing of a focused phenothiazine library for binding to HIV-1 TAR RNA.

We have synthesized a series of phenothiazine derivatives, which were used to test the structure-activity relationship of binding to HIV-1 TAR RNA. Variations from our initial compound, 2-acetylphenothiazine, focused on two moieties: ring substitutions and n-alkyl substitutions. Binding characteristics were ascertained via NMR, principally by saturation transfer difference spectra of the ligand and imino proton resonance shifts of the RNA. Both ring and alkyl substitutions manifested NMR changes upon binding. In general, the active site, while somewhat flexible, has regions that can be capitalized for increased binding through van der Waals interactions and others that can be optimized for solubility in subsequent stages of development. However, binding can be nontrivially enhanced several-fold through optimization of van der Waals and hydrophilic sites of the scaffold.

Binding Sites↗

PubMed2006

Searching for new antimalarial therapeutics amongst known drugs.

The need to discover and develop new antimalarial therapeutics is overwhelming. The annual mortality attributed to malaria, currently approximately 2.5 million, is increasing due primarily to widespread resistance to currently used drugs. One strategy to identify new treatment alternatives for malaria is to examine libraries of diverse compounds for the possible identification of novel scaffolds. Beginning with libraries of drug or drug-like compounds is an ideal starting point because, in the case of approved drugs, substantial pharmacokinetic and toxicologic data should be available for each compound series. We have employed a high-throughput screen of the MicroSource Spectrum and Killer Collections, a library of known drugs, bioactive compounds, and natural products. Our screening assay identifies compounds that inhibit growth of Plasmodium falciparum cultured in human erythrocytes. We have identified 36 novel inhibitors of P. falciparum, of which 19 are therapeutics, and five of these drugs exhibit effective 50% inhibitory concentrations within similar ranges to therapeutic serum concentrations for their recently indicated uses: propafenone, thioridazine, chlorprothixene, perhexiline, and azlocillin. The findings we report here indicate that this is an effective strategy to identify novel scaffolds and therefore aid in antimalarial drug discovery efforts.

Animals↗

PubMed2005

Discovery of small molecule inhibitors of the interaction of the thyroid hormone receptor with transcriptional coregulators.

Thyroid hormone (3,5,3'-triiodo-L-thyronine, T3) is an endocrine hormone that exerts homeostatic regulation of basal metabolic rate, heart rate and contractility, fat deposition, and other phenomena (1, 2). T3 binds to the thyroid hormone receptors (TRs) and controls their regulation of transcription of target genes. The binding of TRs to thyroid hormone induces a conformational change in TRs that regulates the composition of the transcriptional regulatory complex. Recruitment of the correct coregulators (CoR) is important for successful gene regulation. In principle, inhibition of the TR-CoR interaction can have a direct influence on gene transcription in the presence of thyroid hormones. Herein we report a high throughput screen for small molecules capable of inhibiting TR coactivator interactions. One class of inhibitors identified in this screen was aromatic beta-aminoketones, which exhibited IC50 values of approximately 2 microm. These compounds can undergo a deamination, generating unsaturated ketones capable of reacting with nucleophilic amino acids. Several experiments confirm the hypothesis that these inhibitors are covalently bound to TR. Optimization of these compounds produced leads that inhibited the TR-CoR interaction in vitro with potency of approximately 0.6 microm and thyroid signaling in cellular systems. These are the first small molecules irreversibly inhibiting the coactivator binding of a nuclear receptor and suppressing its transcriptional activity.

Binding, Competitive↗

PubMed2005

Parallel synthesis of 9-aminoacridines and their evaluation against chloroquine-resistant Plasmodium falciparum.

A parallel synthetic strategy to the 9-aminoacridine scaffold of the classical anti-malarial drug quinacrine (2) is presented. The method features a new route to 9-chloroacridines that utilizes triflates of salicylic acid derivatives, which are commercially available in a variety of substitution patterns. The route allows ready variation of the two diversity elements present in this class of molecules: the tricyclic aromatic heterocyclic core, and the disubstituted diamine sidechain. In this study, a library of 175 compounds was designed, although only 93 of the final products had purities acceptable for screening. Impurity was generally due to incomplete removal of 9-acridones (18), a degradation product of the 9-chloroacridine synthetic intermediates. The library was screened against two strains of Plasmodium falciparum, including a model of the drug-resistant parasite, and six novel compounds were found to have IC(50) values in the low nanomolar range.

Aminacrine↗

PubMed2005

A solid-phase approach to the phallotoxins: total synthesis of [Ala7]-phalloidin.

Herein we report a solid-phase synthetic approach to [Ala7]-phalloidin (1). Prior syntheses of phallotoxins were carried out using solution-phase routes that required large scale and preclude library production. The route presented here consists of solution-phase preparation of key orthogonally protected amino acid building blocks, followed by a solid-phase peptide synthesis sequence, featuring two resin-bound macro-cyclization reactions. The final product mixture was composed of two atropisomeric compounds, one designated "natural" (1) and the other designated "non-natural" (1'). The structures of these species were modeled using restrained energy minimization with NMR-derived restraints.

Amanitins↗

PubMed2005

Synthesis of ring-substituted 4-aminoquinolines and evaluation of their antimalarial activities.

A simple two-step synthesis method was used to make 51 B-ring-substituted 4-hydroxyquinolines allowing analysis of the effect of ring substitutions on inhibition of growth of chloroquine sensitive and resistant strains of Plasmodium falciparum, the dominant cause of malaria morbidity. Substituted quinoline rings other than the 7-chloroquinoline ring found in chloroquine were found to have significant activity against the drug-resistant strain of P. falciparum W2.

Aminoquinolines↗

PubMed2005

Coregulator interactions with the thyroid hormone receptor.

The thyroid hormone receptor (TR) directly regulates the transcription of thyroid hormone-responsive genes in response to changing levels of thyroid hormone. Mechanistically TR utilizes a complex set of binding interactions, with hormone, response elements, and coregulatory proteins, to provide specific local control of patterns of transcriptional response that are partially responsible for inducing the tissue-selective responses to the circulating hormone. One of the apparently dominant phenomena in the regulation of thyroid hormone responses is the protein interactions between TR and its coregulators. This review summarizes the current state of knowledge with respect to the identity of these coregulators, their interaction with TR, and the consequences of those interactions.

Amino Acid Sequence↗

PubMed2005

Discovery of potent thiosemicarbazone inhibitors of rhodesain and cruzain.

Herein we report the synthesis and evaluation of a series of thiosemicarbazones as potential inhibitors of cysteine proteases relevant to parasitic diseases. Derivatives of thiosemicarbazone 1 were discovered to be potent inhibitors of cruzain and rhodesain, crucial proteases in the life cycles of Trypanosoma cruzi and T. brucei rhodesiense, the organisms causing Chagas' disease and sleeping sickness. However, the entire series had only modest potency against falcipain-2, an essential protease for Plasmodium falciparum, the organism causing malaria. Among the active inhibitors, several potently inhibited proliferation of cultures of T. brucei. However, only modest activity was observed in inhibition of proliferation of T. cruzi or P. falciparum.

Cysteine Endopeptidases↗

PubMed2004

The molecular mechanisms of coactivator utilization in ligand-dependent transactivation by the androgen receptor.

Androgens drive sex differentiation, bone and muscle development, and promote growth of hormone-dependent cancers by binding the nuclear androgen receptor (AR), which recruits coactivators to responsive genes. Most nuclear receptors recruit steroid receptor coactivators (SRCs) to their ligand binding domain (LBD) using a leucine-rich motif (LXXLL). AR is believed to recruit unique coactivators to its LBD using an aromatic-rich motif (FXXLF) while recruiting SRCs to its N-terminal domain (NTD) through an alternate mechanism. Here, we report that the AR-LBD interacts with both FXXLF motifs and a subset of LXXLL motifs and that contacts with these LXXLL motifs are both necessary and sufficient for SRC-mediated AR regulation of transcription. Crystal structures of the activated AR in complex with both recruitment motifs reveal that side chains unique to the AR-LBD rearrange to bind either the bulky FXXLF motifs or the more compact LXXLL motifs and that AR utilizes subsidiary contacts with LXXLL flanking sequences to discriminate between LXXLL motifs.

Amino Acid Motifs↗

PubMed2004

Synthesis of medium ring heterocycles using an intramolecular Heck reaction.

Historically, general convergent syntheses of medium ring heterocycles have been difficult to develop. Herein, we describe the synthesis of five classes of heterocycles: dihydrodibenzo[b,f]azepine, -oxocine, and -thiocine and dibenzo[b,f]azepine and -oxepine using a strategy of alkylation followed by highly selective intramolecular Heck arylation reaction. The hetero-tricyclic compounds were available in only two steps starting from commercially available starting materials.

Catalysis↗