Exploration of Alternative Scaffolds for P2Y14 Receptor Antagonists Containing a Biaryl Core.

Various heteroaryl and bicyclo-aliphatic analogues of zwitterionic biaryl P2Y14 receptor (P2Y14R) antagonists were synthesized, and affinity was measured in P2Y14R-expressing Chinese hamster ovary cells by flow cytometry. Given this series' low water solubility, various polyethylene glycol derivatives of the distally binding piperidin-4-yl moiety of moderate affinity were synthesized. Rotation of previously identified 1,2,3-triazole attached to the central m-benzoic acid core (25) provided moderate affinity but not indole and benzimidazole substitution of the aryl-triazole. The corresponding P2Y14R region is predicted by homology modeling as a deep, sterically limited hydrophobic pocket, with the outward pointing piperidine moiety being the most flexible. Bicyclic-substituted piperidine ring derivatives of naphthalene antagonist 1, e.g., quinuclidine 17 (MRS4608, IC50 ≈ 20 nM at hP2Y14R/mP2Y14R), or of triazole 2, preserved affinity. Potent antagonists 1, 7a, 17, and 23 (10 mg/kg) protected in an ovalbumin/Aspergillus mouse asthma model, and PEG conjugate 12 reduced chronic pain. Thus, we expanded P2Y14R antagonist structure-activity relationship, introducing diverse physical-chemical properties.

Structure-Guided Modification of Heterocyclic Antagonists of the P2Y14 Receptor.

The P2Y14 receptor (P2Y14R) mediates inflammatory activity by activating neutrophil motility, but few classes of antagonists are known. We have explored the structure-activity relationship of a 3-(4-phenyl-1 H-1,2,3-triazol-1-yl)-5-(aryl)benzoic acid antagonist scaffold, assisted by docking and molecular dynamics (MD) simulation at a P2Y14R homology model. A computational pipeline using the High Throughput MD Python environment guided the analogue design. Selection of candidates was based upon ligand-protein shape and complementarity and the persistence of ligand-protein interactions over time. Predictions of a favorable substitution of a 5-phenyl group with thiophene and an insertion of a three-methylene spacer between the 5-aromatic and alkyl amino moieties were largely consistent with empirical results. The substitution of a key carboxylate group on the core phenyl ring with tetrazole or truncation of the 5-aryl group reduced affinity. The most potent antagonists, using a fluorescent assay, were a primary 3-aminopropyl congener 20 (MRS4458) and phenyl p-carboxamide 30 (MRS4478).

Usage of cancer associated autoantibodies in the detection of disease.

Efforts toward deciphering the complexity of the tumor specific proteome by profiling immune responses generated against tumor associated antigens (TAAs) holds great promise for predicting the presence of cancer long before the development of clinical symptoms. The immune system is capable of sensing aberrant expression of certain cellular components involved in tumorigenesis and the resultant autoantibody response provides insights to the targets that are responsible for eliciting immunogenicity to these cellular components. Analysis of the cancer-specific humoral immune response has led to panels of biomarkers that are specific and sensitive biomarkers of disease. Using multianalyte-based in vitro analytical discovery platforms which can be easily adapted into clinical diagnostic screening tests, body fluids such as serum, plasma saliva, or urine can be interrogated to detect autoantibodies against natural or recombinant antigens, which may possess etiologic significance to cancer. Non-invasive screening tests exhibiting high specificity and sensitivity to detect early stage cancer in the heterogeneous population of cancer patients potentially have the greatest impact in decreasing mortality rates. Overall, this review summarizes different experimental approaches in the development of diagnostic screening tests for the early detection of cancer and their implementation in the development of clinical multianalyte biomarker assays.

Detecting tumor-specific autoantibodies for cancer diagnosis: a technology overview.

BACKGROUND: Studies on the autoantibody (humoral immune) response to tumor-associated aberrant cellular components have provided critical information about an individual's disease state. Tumor-specific autoantibodies detected in human serum samples are being studied extensively to determine their utility in developing serological diagnostic assays for cancer. The development of accurate panels of diagnostic markers for cancer detection is now being pursued using new technologies and tailored computational methods capable of analyzing data generated from large-scale biomarker discovery projects. OBJECTIVE: In this technical overview, current methodologies being applied to the identification and characterization of tumor-specific autoantibodies in cancer patients' sera are reviewed. METHODS: A variety of research approaches are presented that are now being used or have the potential to evaluate large numbers of patient sera for the presence of tumor-specific autoantibodies. Each approach is discussed regarding its primary attributes (advantages and limitations) that could lead to serological diagnostic assays for the early detection of cancer. CONCLUSION: Preliminary results in the development of serological diagnostic assays have demonstrated that the basic experimental tools to accomplish this goal exist. In the future, autoantibody patterns against tumor-specific proteins may achieve high specificity and sensitivity for diagnosing disease in screening populations. The development of highly accurate reliable assays is a prerequisite for this technology to be integrated into clinically applicable strategies in patient care.

Expression of human intestinal mucin is modulated by the beta-galactoside binding protein galectin-3 in colon cancer.

BACKGROUND & AIMS: Alterations in the production of the beta-galactoside binding protein galectin-3 and of MUC2 intestinal mucin have been independently correlated with the malignant behavior of human colon cancer cells. MUC2 mucin is a major ligand for galectin-3, and colon cancer cells that differ quantitatively in MUC2 expression may also vary in expression of galectin-3. The current study was designed to investigate the relationship between galectin-3 production and MUC2 mucin synthesis by human colon cancer cells. METHODS: The effect of galectin-3 on MUC2 mucin production was assessed by stable transfection of sense and antisense galectin-3 expression constructs under the control of constitutive or tetracycline-inducible promoters into human colon cancer cells. Galectin-3 and MUC2 expression were determined by fluorescence-activated cell sorter (cell surface galectin-3), Western and Northern analysis (galectin-3, MUC2), and gel filtration of secreted high-weight glycoprotein (MUC2). In vitro results were confirmed in vivo by analysis of cecal xenografts in athymic mice. RESULTS: Colon cancer cells with high levels of galectin-3 also had high levels of MUC2 mucin, whereas those with low galectin-3 levels had low MUC2 levels. Alterations in galectin-3 levels by expression of sense or antisense galectin-3 constructs resulted in parallel alterations of MUC2 protein and RNA. Induction of antisense to galectin-3 in vivo was associated with decreases in both galectin-3 and MUC2 protein in cecal xenografts. CONCLUSIONS: The beta-galactoside binding protein galectin-3 modulates the expression of its major ligand MUC2 mucin in human colon cancer cells. This may have important implications for understanding the role of galectin-3 in colon cancer metastasis.