Andrei A. Ivanov

Evaluation of Homology Modeling of G Protein-Coupled Receptors in Light of the A2A Adenosine Receptor Crystallographic Structure

Homology modeling of the human A(2A) adenosine receptor (AR) based on bovine rhodopsin predicted a protein structure that was very similar to the recently determined crystallographic structure. The discrepancy between the experimentally observed orientation of the antagonist and those obtained by previous antagonist docking is related to the loop structure of rhodopsin being carried over to the model of the A(2A) AR and was rectified when the beta(2)-adrenergic receptor was used as a template for homology modeling. Docking of the triazolotriazine antagonist ligand ZM241385 1 was greatly improved by including water molecules of the X-ray structure or by using a constraint from mutagenesis. Automatic agonists docking to both a new homology modeled receptor and the A(2A) AR crystallographic structure produced similar results. Heterocyclic nitrogen atoms closely corresponded when the docked adenine moiety of agonists and 1 were overlaid. The cumulative mutagenesis data, which support the proposed mode of agonist docking, can be reexamined in light of the crystallographic structure. Thus, homology modeling of GPCRs remains a useful technique in probing the structure of the protein and predicting modes of ligand docking.

Development of selective agonists and antagonists of P2Y receptors.

Although elucidation of the medicinal chemistry of agonists and antagonists of the P2Y receptors has lagged behind that of many other members of group A G protein-coupled receptors, detailed qualitative and quantitative structure–activity relationships (SARs) were recently constructed for several of the subtypes. Agonists selective for P2Y1, P2Y2, and P2Y6 receptors and nucleotide antagonists selective for P2Y1 and P2Y12 receptors are now known. Selective nonnucleotide antagonists were reported for P2Y1, P2Y2, P2Y6, P2Y11, P2Y12, and P2Y13 receptors. At the P2Y1 and P2Y12 receptors, nucleotide agonists (5′-diphosphate derivatives) were converted into antagonists of nanomolar affinity by altering the phosphate moieties, with a focus particularly on the ribose conformation and substitution pattern. Nucleotide analogues with conformationally constrained ribose-like rings were introduced as selective receptor probes for P2Y1 and P2Y6 receptors. Screening chemically diverse compound libraries has begun to yield new lead compounds for the development of P2Y receptor antagonists, such as competitive P2Y12 receptor antagonists with antithrombotic activity. Selective agonists for the P2Y4, P2Y11, and P2Y13 receptors and selective antagonists for P2Y4 and P2Y14 receptors have not yet been identified. The P2Y14 receptor appears to be the most restrictive of the class with respect to modification of the nucleobase, ribose, and phosphate moieties. The continuing process of ligand design for the P2Y receptors will aid in the identification of new clinical targets.

Molecular modeling of a PAMAM-CGS21680 dendrimer bound to an A2A adenosine receptor homodimer

The theoretical possibility of bivalent binding of a dendrimer, covalently appended with multiple copies of a small ligand, to a homodimer of a G protein-coupled receptor was investigated with a molecular modeling approach. A molecular model was constructed of a third generation (G3) poly(amidoamine) (PAMAM) dendrimer condensed with multiple copies of the potent A(2A) adenosine receptor agonist CGS21680. The dendrimer was bound to an A(2A) adenosine receptor homodimer. Two units of the nucleoside CGS21680 could occupy the A(2A) receptor homodimer simultaneously. The binding mode of CGS21680 moieties linked to the PAMAM dendrimer and docked to the A(2A) receptor was found to be similar to the binding mode of a monomeric CGS21680 ligand.

Design, synthesis, and bioactivity of putative tubulin ligands with adamantane core

Several adamantane-based taxol mimetics were synthesized and found to be cytotoxic at micromolar concentrations and to cause tubulin aggregation. The extent of the aggregation is maximal for N-benzoyl-(2R,3S)-phenylisoseryloxyadamantane (5) and is very sensitive to the structural modifications. A hybrid compound (15), combining adamantane-based taxol mimetic with colchicine was synthesized and found to possess both microtubule depolymerizing and microtubule bundling activities in A549 human lung carcinoma cells.

The OncoPPi network of cancer-focused protein–protein interactions to inform biological insights and therapeutic strategies

As genomics advances reveal the cancer gene landscape, a daunting task is to understand how these genes contribute to dysregulated oncogenic pathways. Integration of cancer genes into networks offers opportunities to reveal protein–protein interactions (PPIs) with functional and therapeutic significance. Here, we report the generation of a cancer-focused PPI network, termed OncoPPi, and identification of >260 cancer-associated PPIs not in other large-scale interactomes. PPI hubs reveal new regulatory mechanisms for cancer genes like MYC, STK11, RASSF1 and CDK4. As example, the NSD3 (WHSC1L1)–MYC interaction suggests a new mechanism for NSD3/BRD4 chromatin complex regulation of MYC-driven tumours. Association of undruggable tumour suppressors with drug targets informs therapeutic options. Based on OncoPPi-derived STK11-CDK4 connectivity, we observe enhanced sensitivity of STK11-silenced lung cancer cells to the FDA-approved CDK4 inhibitor palbociclib. OncoPPi is a focused PPI resource that links cancer genes into a signalling network for discovery of PPI targets and network-implicated tumour vulnerabilities for therapeutic interrogation.

Minor groove dimeric bisbenzimidazoles inhibit in vitro DNA binding to eukaryotic DNA topoisomerase I.

The potential of six dimeric bisbenzimidazoles bound to scDNA to inhibit eukaryotic DNA topoisomerase (topo-I) was studied chemically; the tested compounds differed in linker structure and length. All the compounds inhibited topo-I, DB(7) being the most efficient; its inhibitory activity in vitro was 50-fold higher than that of camptothecin. It is noteworthy that inhibitory properties of nearly all the tested compounds increased many times if they were preincubated with scDNA for three days.

Application of the bridgehead fragments for the design of conformationally restricted melatonin analogues.

Conformationally constrained analogues of the hormone melatonin with a side chain incorporated into the bicyclic bridgehead core were synthesized based on the homology modeling and molecular docking studies performed for the MT(2) melatonin receptor. The methoxy-indole derivative fused with exo-N-acetamino-substituted bicyclo[2.2.2]octane was found to possess nanomolar MT(2) receptor affinity.

Enabling systematic interrogation of protein–protein interactions in live cells with a versatile ultra-high-throughput biosensor platform

Large-scale genomics studies have generated vast resources for in-depth understanding of vital biological and pathological processes. A rising challenge is to leverage such enormous information to rapidly decipher the intricate protein-protein interactions (PPIs) for functional characterization and therapeutic interventions. While a number of powerful technologies have been employed to detect PPIs, a singular PPI biosensor platform with both high sensitivity and robustness in a mammalian cell environment remains to be established. Here we describe the development and integration of a highly sensitive NanoLuc luciferase-based bioluminescence resonance energy transfer technology, termed BRET(n), which enables ultra-high-throughput (uHTS) PPI detection in live cells with streamlined co-expression of biosensors in a miniaturized format. We further demonstrate the application of BRET(n) in uHTS format in chemical biology research, including the discovery of chemical probes that disrupt PRAS40 dimerization and pathway connectivity profiling among core members of the Hippo signaling pathway. Such hippo pathway profiling not only confirmed previously reported PPIs, but also revealed two novel interactions, suggesting new mechanisms for regulation of Hippo signaling. Our BRET(n) biosensor platform with uHTS capability is expected to accelerate systematic PPI network mapping and PPI modulator-based drug discovery.

Molecular Modeling the Human A1 Adenosine Receptor and Study of the Mechanisms of Its Selective Ligand Binding

Adenosine receptors (classified as G-protein-coupled receptors) are present in the majority of human and mammalian cells and tissues and are involved in many key biological processes. Depending on their biochemical and pharmacologic properties, four subtypes of adenosine receptors (A1, A2a, A2b, and A3) are distinguished. All of them contain a typical transmembrane domain formed by seven α helices linked pairwise with three extracellular and three intracellular hydrophilic loops. The ligand-binding site of the receptor is located inside the transmembrane domain [1]. It is known that activation of the A1 and A3 receptors decreases the cAMP level, whereas activation of the A2a and A2b subtypes increases it. In addition, stimulation of A1 receptors causes activation of potassium channels and inhibition of calcium channels [2]. The receptors ligands of adenosine are widely used in pharmacology and medicine for treatment of some psychoneurological and cardiovascular diseases [3]. Although numerous agonists and antagonists of adenosine receptors are currently known [4], the majority of them do not exhibit sufficient selectivity and efficiency. In addition, no model of adenosine receptors containing not only the transmembrane α helices, but also the hydrophilic loops, has been designed thus far. In view of this, a thorough study of the structure of adenosine receptors and the mechanisms of ligand binding to the receptors, as well as the search for new highly selective and efficient ligands of these receptors is now a topical problem. The purpose of this work was to design a molecular model of the human A1 adenosine receptor and to study the mechanisms of selective binding of ligands to this receptor. Adenosine receptors are membrane proteins; they are difficult to crystallize and study by X-ray analysis. For this reason, the structure of these receptors is studied using molecular modeling based on homology with a template protein (usually, rhodopsin) that is also G-protein-coupled and that was studied by X-ray analysis [5]. To identify the amino acids forming each of the seven transmembrane α helices of the A1 receptor, we performed multiple alignment of the amino acid sequences of the four known subtypes of adenosine receptors and rhodopsin: TM1

Molecular modeling study of the mechanism of ligand binding to human melatonin receptors.

The epiphysis hormone melatonin plays a key role in the regulation of circadian rhythms of mammals, as well as in the functioning of the cardiovascular, immune, and digestive systems and retina [1]. The involvement of melatonin in the regulation of a great number of physiological processes has determined its use as a drug. However, when used as a drug, melatonin has certain disadvantages—a short degradation period and low solubility (which causes difficulties with preparation of the pharmaceutical dosage form), as well as a too broad spectrum of action. In view of this, the search for melatonin analogs that would be deprived of the above-listed disadvantages is very important. For this purpose, it is necessary to study the characteristic features of the structure of melatonin receptors and the main mechanisms of ligand‐receptor interactions. Melatonin receptors belong to the rhodopsin family of G protein-coupled receptors. Because the representatives of this family are located in the lipid bilayer of cell membrane, crystallization of these receptors and experimental study of their structure are hampered. Xray data were obtained for only one representative of this family, rhodopsin [2]. Since all receptors of the rhodopsin family have a similar structure, the most effective way of constructing molecular models of melatonin receptors is modeling based on their homology with rhodopsin. Two subtypes (Mel1a and Mel1b) of human melatonin receptors are known. Similar to other representatives of the rhodopsin family, the melatonin receptor consists of seven transmembrane α -helices, three intracellular and three extracellular hydrophilic loops, as well as an intracellular and an extracellular terminal domains.