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Biomedical subjects

Kit S Lam

Publications and source records attributed to Kit S Lam.

At least 19 recordsLinked to original sources
PubMed2007

Microwave-mediated heterocyclization to benzimidazo[2,1-b]quinazolin-12(5H)-ones.

An effective route to benzimidazo[2,1-b]quinazolin-12(5H)-ones from commercially available o-aryl isothiocyanate esters and o-phenylenediamines is reported. This method accommodates a variety of substituents on either starting material and proceeds under microwave irradiation in the presence of barium hydroxide, conditions that do not hydrolyze methyl ester substituents. The pharmacologically pertinent benzimidazoquinazolinone heterocycle is delivered in excellent yield and purity via both solution- and solid-phase protocols, the latter involving traceless release from the resin.

Benzimidazoles↗
PubMed2006

Aminodeoxychorismate synthase inhibitors from one-bead one-compound combinatorial libraries: "staged" inhibitor design.

4-Amino-4-deoxychorismate synthase (ADCS) catalyzes the first step in the conversion of chorismate into p-aminobenzoate, which is incorporated into folic acid. We aim to discover compounds that inhibit ADCS and serve as leads for a new class of antimicrobial compounds. This report presents (1) synthesis of a mass-tag encoded library based on a "staged" design, (2) massively parallel fluorescence-based on-bead screening, (3) rapid structural identification of hits, and (4) full kinetic analysis of ADCS. All inhibitors are competitive against chorismate and Mg(2+). The most potent ADCS inhibitor identified has a K(i) of 360 microM. We show that the combinatorial diversity elements add substantial binding affinity by interacting with residues outside of but proximal to the active site. The methods presented here constitute a paradigm for inhibitor discovery through active site targeting, enabled by rapid library synthesis, facile massively parallel screening, and straightforward hit identification.

Acetates↗
PubMed2006

Combining the rapid MTT formazan exocytosis assay and the MC65 protection assay led to the discovery of carbazole analogs as small molecule inhibitors of Abeta oligomer-induced cytotoxicity.

The discovery of small molecule inhibitors of cytotoxicity induced by amyloid-beta (Abeta) oligomers, either applied extracellularly or accumulated intraneuronally, is an important goal of drug development for Alzheimer's disease (AD), but has been limited by the lack of efficient screening methods. Here we describe our approach using two cell-based methods. The first method takes advantage of the unique ability of extracellularly applied Abeta oligomers to rapidly induce the exocytosis of formazan formed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). We employed a short protocol to quantify this toxicity, and quickly identified two novel inhibitors, code-named CP2 and A5, from two compound libraries. A second independent screen of the same libraries using our previously published MC65 protection assay, which identifies inhibitors of toxicity related to intracellular Abeta oligomers, also selected the same two leads, suggesting that both assays select for the same anti-Abeta oligomer properties displayed by these compounds. We further demonstrated that A5 attenuated the progressive aggregation of existing Abeta oligomers, reduced the level of intracellular Abeta oligomers, and prevented the Abeta oligomer-induced death of primary cortical neurons, effects similar to those demonstrated by CP2. Our results suggest that, when combined, the two methods would generate fewer false results and give a high likelihood of identifying leads that show promises in ameliorating Abeta oligomer-induced toxicities within both intraneuronal and extracellular sites. Both assays are simple, suitable for rapid screening of a large number of medicinal libraries, and amenable for automation.

Amyloid beta-Peptides↗
PubMed2006

The minimum element of a synthetic peptide required to block prostate tumor cell migration.

Human prostate tumor cell invasion and metastasis are dependent in part on cell adhesion to extracellular matrix proteins and cell migration. We previously identified a synthetic D-amino acid tumor cell adhesion peptide called HYD1 (kikmviswkg) that supported adhesion of tumor cells derived from breast, prostate, ovary and pancreas tissue. Alanine substitution analysis and a peptide deletion strategy were used to determine the minimal element of HYD1 necessary for bioactivity in a prostate cancer cell line called PC3N. Bioactivity was measured by assays of cell adhesion, migration and ERK signaling. The most potent element of HYD1 necessary to support cell adhesion was kmvixw, the block to migration required xkmviswxx and activation of ERK signaling required ikmviswxx. The shortest sequence active in all three assays was iswkg. The HYD1 peptide contains overlapping elements required for adhesion, blocking migration and the activation of ERK signaling. These linear peptide sequences provide the starting point for development of novel compounds to target cancer cell adhesion and migration.

Amino Acid Sequence↗
PubMed2006

Kaposi's sarcoma-associated herpesvirus-encoded protein kinase and its interaction with K-bZIP.

The oncogenic herpesvirus, Kaposi's sarcoma-associated herpesvirus, also identified as human herpesvirus 8, contains genes producing proteins that control transcription and influence cell signaling. Open reading frame 36 (ORF36) of this virus encodes a serine/threonine protein kinase, which is designated the viral protein kinase (vPK). Our recent efforts to elucidate the role of vPK in the viral life cycle have focused on identifying viral protein substrates and determining the effects of vPK-mediated phosphorylation on specific steps in viral replication. The vPK gene was transcribed into 4.2-kb and 3.6-kb mRNAs during the early and late phases of viral reactivation. vPK is colocalized with viral DNA replication/transcription compartments as marked by a polymerase processivity factor, and K-bZIP, a protein known to bind the viral DNA replication origin (Ori-Lyt) and to regulate viral transcription. The vPK physically associated with and strongly phosphorylated K-bZIP at threonine 111, a site also recognized by the cyclin-dependent kinase Cdk2. Both K-bZIP and vPK were corecruited to viral promoters targeted by K-bZIP as well as to the Ori-Lyt region. Phosphorylation of K-bZIP by vPK had a negative impact on K-bZIP transcription repression activity. The extent of posttranslational modification of K-bZIP by sumoylation, a process that influences its repression function, was decreased by vPK phosphorylation at threonine 111. Our data thus identify a new role of vPK as a modulator of viral transcription.

Basic-Leucine Zipper Transcription Factors↗
PubMed2006

Identification of 2-nonynoic acid, a cosmetic component, as a potential trigger of primary biliary cirrhosis.

Antimitochondrial antibodies (AMA) are unique among autoimmune serologic reactants because of their extremely high association with the index disease primary biliary cirrhosis (PBC). This autoantibody response is specifically directed only to the lipoyl domain of the mitochondrial 2-oxo-acid dehydrogenase complexes, which prompted us to search for environmental mimotopes in the form of xenobiotics and led to our identification of 2-octynoic acid as a high-affinity reactant for AMA. To focus on the chemical characteristics requisite for binding of AMA to the xenobiotic-modified self-peptide, quantitative structure-activity relationship (QSAR) studies were performed using a panel of alkynoic compounds, including examination of the length of the carbon chain and the location of the triple bond in the identified mimotope. Analyses of octynamides that varied in the position of the triple bond demonstrated that only the 2-octynamide reacted strongly with PBC sera. Furthermore, among 2-alkynamides with varying carbon chain length, 2-octyn-, 2-nonyn- (particularly) and 2-decynamide exhibited the highest reactivity. Thus, an optimal chemical structure of the xenobiotically modified epitope recognized by AMA-positive PBC sera is provided by 2-nonynoic acid. The methyl ester of this compound is ranked 2,324th out of 12,945 compounds to which there is occupational exposure, with an 80% female prevalence due to its use in cosmetic products. Our findings illustrate an unusual polyreactivity of anti-PDC-E2 and support the idea of epitope mimicry in the genesis of this autoantibody and perhaps of PBC itself.

Autoantibodies↗
PubMed2006

A serum glycomics approach to breast cancer biomarkers.

Because the glycosylation of proteins is known to change in tumor cells during the development of breast cancer, a glycomics approach is used here to find relevant biomarkers of breast cancer. These glycosylation changes are known to correlate with increasing tumor burden and poor prognosis. Current antibody-based immunochemical tests for cancer biomarkers of ovarian (CA125), breast (CA27.29 or CA15-3), pancreatic, gastric, colonic, and carcinoma (CA19-9) target highly glycosylated mucin proteins. However, these tests lack the specificity and sensitivity for use in early detection. This glycomics approach to find glycan biomarkers of breast cancer involves chemically cleaving oligosaccharides (glycans) from glycosylated proteins that are shed or secreted by breast cancer tumor cell lines. The resulting free glycan species are analyzed by MALDI-FT-ICR MS. Further structural analysis of the glycans can be performed in FTMS through the use of tandem mass spectrometry with infrared multiphoton dissociation. Glycan profiles were generated for each cell line and compared. These methods were then used to analyze sera obtained from a mouse model of breast cancer and a small number of serum samples obtained from human patients diagnosed with breast cancer or patients with no known history of breast cancer. In addition to the glycosylation changes detected in mice as mouse mammary tumors developed, glycosylation profiles were found to be sufficiently different to distinguish patients with cancer from those without. Although the small number of patient samples analyzed so far is inadequate to make any legitimate claims at this time, these promising but very preliminary results suggest that glycan profiles may contain distinct glycan biomarkers that may correspond to glycan "signatures of cancer."

Adult↗
PubMed2006

Combinatorial chemistry identifies high-affinity peptidomimetics against alpha4beta1 integrin for in vivo tumor imaging.

Small peptide-based agents have attracted wide interest as cancer-targeting agents for diagnostic imaging and targeted therapy. There is a need to develop new high-affinity and high-specificity peptidomimetic or small-molecule ligands against cancer cell surface receptors. Here we report on the identification of a high-affinity peptidomimetic ligand (LLP2A; IC50 = 2 pM) against alpha4beta1 integrin using both diverse and highly focused one-bead-one-compound combinatorial peptidomimetic libraries in conjunction with high-stringency screening. We further demonstrate that LLP2A can be used to image alpha4beta1-expressing lymphomas with high sensitivity and specificity when conjugated to a near infrared fluorescent dye in a mouse xenograft model. Thus, LLP2A provides an important tool for noninvasive monitoring of alpha4beta1 expression and activity during tumor progression, and it shows great potential as an imaging and therapeutic agent for alpha4beta1-positive tumors.

Animals↗
PubMed2006

Slow-binding human serine racemase inhibitors from high-throughput screening of combinatorial libraries.

One-bead one-compound combinatorial chemistry together with a high-throughput screen based on fluorescently labeled enzyme allowed the identification of slow binding inhibitors of human serine racemase (hSR). A peptide library of topographically segregated encoded resin beads was synthesized, and several hSR-binding compounds were isolated, identified, and resynthesized for further kinetic study. Of these, several showed inhibitory effects with moderate potency (high micromolar K(I)s) toward hSR. A clear structural motif was identified consisting of 3-phenylpropionic acid and histidine moieties. Importantly, the inhibitors identified showed no structural similarities to the natural substrate, L-serine. Detailed kinetic analyses of the properties of selected inhibitors show that the screening protocol used here selectively identifies slow binding inhibitors. They provide a pharmacophore for the future isolation of more potent ligands that may prove useful in probing and understanding the biological role of hSR.

Combinatorial Chemistry Techniques↗
PubMed2006

Structure-activity relationship studies of a series of peptidomimetic ligands for alpha(4) beta(1) integrin on Jurkat T-leukemia cells.

alpha(4)beta(1) integrin is a therapeutic target for inflammation, autoimmune diseases, and lymphoid cancers. A series of peptidomimetic ligands based on the Nle-D-I motif have been synthesized and their binding affinities (IC(50)) to activated alpha(4)beta(1) integrin on Jurkat T-leukemia cells have been determined using a cell adhesion assay. One of the 51 ligands, 18, has been determined to have an IC(50) of 0.6 nM and has a more than twofold increase of binding affinity than the initial lead compound 1. Extensive SAR studies provide important information for further ligand optimization, which has served as a foundation for studies that ultimately led to identification of a potent ligand with an IC(50) of 2 pM.

Antineoplastic Agents↗
PubMed2006

Profiling of glycans in serum for the discovery of potential biomarkers for ovarian cancer.

A glycomic approach is developed to identify oligosaccharide markers for ovarian cancer by rapidly profiling globally released oligosaccharides. Glycoproteins shed by cancer cells are found in the supernatant (or conditioned media) of cultured cells. In this approach, shed glycoproteins are profiled for their oligosaccharide content using beta-elimination conditions. Changes in glycosylation are monitored by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS). Because shed glycoproteins would also be found in serum, similar glycan profiling was performed to observe potential oligosaccharide markers. Oligosaccharide profiling data on a limited set of patient and normal serum samples were studied to determine potential glycan markers in ovarian cancer. We were able to demonstrate the presence of at least 15 unique serum glycan markers in all patients but absent in normal individuals. To determine the structure of the glycan biomarkers, a number of the ions were isolated and further analyzed using infrared multiphoton dissociation (IRMPD). One major advantage of this approach is that glycans are examined directly from patient sera without the need to obtain cancer biopsy specimens or to purify glycosylated proteins from these specimens.

Biomarkers, Tumor↗
PubMed2005

Discovery of selective aldo-keto reductase ligands--an on-bead assay strategy.

An enzyme labeling and screening strategy for the discovery of ligands selective in binding two structurally similar members of the aldo-keto reductase family of enzymes is reported. The resulting fluorescence microscope data obtained by screening a 74,088 member library led to the identification of selective ligands for aldose reductase (ALR2) and aldehyde reductase (ALR1). Resynthesis results validate the selectivity of these ligands.

Alcohol Oxidoreductases↗
PubMed2005

Solid-phase synthesis and antibacterial evaluations of N-demethylvancomycin derivatives.

Twenty-five N-demethylvancomycin derivatives were synthesized on solid-support and their structures were determined by LC-MS/MS. Biological evaluation of these compounds indicated that bulky hydrophobic substituent on vancosamine of N-demethylvancomycin can increase antibacterial activity against vancomycin-resistant Enterococcus faecalis.

Anti-Bacterial Agents↗
PubMed2005

Chemical xenobiotics and mitochondrial autoantigens in primary biliary cirrhosis: identification of antibodies against a common environmental, cosmetic, and food additive, 2-octynoic acid.

Emerging evidence has suggested environmental factors as causative agents in the pathogenesis of primary biliary cirrhosis (PBC). We have hypothesized that in PBC the lipoyl domain of the immunodominant E2 component of pyruvate dehydrogenase (PDC-E2) is replaced by a chemical xenobiotic mimic, which is sufficient to break self-tolerance. To address this hypothesis, based upon our quantitative structure-activity relationship data, a total of 107 potential xenobiotic mimics were coupled to the lysine residue of the immunodominant 15 amino acid peptide of the PDC-E2 inner lipoyl domain and spotted on microarray slides. Sera from patients with PBC (n = 47), primary sclerosing cholangitis (n = 15), and healthy volunteers (n = 20) were assayed for Ig reactivity. PBC sera were subsequently absorbed with native lipoylated PDC-E2 peptide or a xenobiotically modified PDC-E2 peptide, and the remaining reactivity analyzed. Of the 107 xenobiotics, 33 had a significantly higher IgG reactivity against PBC sera compared with control sera. In addition, 9 of those 33 compounds were more reactive than the native lipoylated peptide. Following absorption, 8 of the 9 compounds demonstrated cross-reactivity with lipoic acid. One compound, 2-octynoic acid, was unique in both its quantitative structure-activity relationship analysis and reactivity. PBC patient sera demonstrated high Ig reactivity against 2-octynoic acid-PDC-E2 peptide. Not only does 2-octynoic acid have the potential to modify PDC-E2 in vivo but importantly it was/is widely used in the environment including perfumes, lipstick, and many common food flavorings.

Amino Acid Sequence↗
PubMed2005

Peptide and small-molecule microarrays.

Two methods that use chemoselective ligation chemistry to prepare peptide and small-molecule microarrays are described here. The first method involves the functionalization of a glass slide with a glyoxylyl group, followed by chemoselective ligation of small molecules or peptides to the functionalized surface via a covalent bond. In the second method, peptides or small molecules are first conjugated to a macromolecular scaffold. The final ligand-scaffold conjugates are then spotted and adsorbed onto the solid surface. Three different assay methods to screen such chemical microarrays are described.

Animals↗
PubMed2005

Identification of novel targeting peptides for human ovarian cancer cells using "one-bead one-compound" combinatorial libraries.

Using "one-bead one-compound" combinatorial chemistry technology, we generated random peptide libraries containing millions of 90 mum TentaGel beads, each with its own unique amino acid sequence. A cyclic random 8-mer library was screened with CAOV-3 (a human ovarian adenocarcinoma cell line) and beads with a unique ligand that bind to the cell surface receptors were coated by one or more layers of cells. These positive beads were isolated, stripped, and microsequenced. Several peptide motifs were identified from these screenings, some of which were novel and unique, e.g., cDGX(4)GX(6)X(7)c. Structure-activity relationship studies of this peptide revealed that the l-aspartate residue at position 2, the two glycines at positions 3 and 5, and the two d-cysteines at the amino and COOH terminus are critical for activity. In addition, a hydrophobic residue was preferred at position X(4), whereas amino acids at positions X(6) and X(7) were more variable. Binding of this peptide to a number of different cancer cell lines and normal cells was also determined and we observed that peptides with this motif bound preferentially to three other human ovarian cancer cell lines (ES-2, SKOV-3, and OVCAR-3) as well as a human glioblastoma cancer cell line (A172). Structural analysis of the peptides using high-resolution nuclear magnetic resonance spectroscopy revealed strong conformational similarity among all peptides with cX(1)GX(4)GX(6)X(7)c motif. Blocking study with a panel of anti-integrin antibodies strongly suggests alpha3 integrin present on these ovarian adenocarcinoma cells is the target receptor for this peptide.

Adenocarcinoma↗
PubMed2004

OBOC small-molecule combinatorial library encoded by halogenated mass-tags.

[reaction: see text] A bromine-/chlorine-containing mass-tag encoding strategy for a small-molecule OBOC combinatorial library is reported. The resulting MALDI FTMS isotope pattern of each tag clearly defines the component building blocks of each "hit" bead in an 1890-member demonstration library screened on-bead for binding against streptavidin via both enzyme-linked colorimetric and Quantum Dot/COPAS assays.

Journal Article↗