In vivo screening identifies GATAD2B as a metastasis driver in KRAS-driven lung cancer.

Genetic aberrations driving pro-oncogenic and pro-metastatic activity remain an elusive target in the quest of precision oncology. To identify such drivers, we use an animal model of KRAS-mutant lung adenocarcinoma to perform an in vivo functional screen of 217 genetic aberrations selected from lung cancer genomics datasets. We identify 28 genes whose expression promoted tumor metastasis to the lung in mice. We employ two tools for examining the KRAS-dependence of genes identified from our screen: 1) a human lung cell model containing a regulatable mutant KRAS allele and 2) a lentiviral system permitting co-expression of DNA-barcoded cDNAs with Cre recombinase to activate a mutant KRAS allele in the lungs of mice. Mechanistic evaluation of one gene, GATAD2B, illuminates its role as a dual activity gene, promoting both pro-tumorigenic and pro-metastatic activities in KRAS-mutant lung cancer through interaction with c-MYC and hyperactivation of the c-MYC pathway.

The OncoPPi Portal: an integrative resource to explore and prioritize protein-protein interactions for cancer target discovery.

Motivation: As cancer genomics initiatives move toward comprehensive identification of genetic alterations in cancer, attention is now turning to understanding how interactions among these genes lead to the acquisition of tumor hallmarks. Emerging pharmacological and clinical data suggest a highly promising role of cancer-specific protein-protein interactions (PPIs) as druggable cancer targets. However, large-scale experimental identification of cancer-related PPIs remains challenging, and currently available resources to explore oncogenic PPI networks are limited. Results: Recently, we have developed a PPI high-throughput screening platform to detect PPIs between cancer-associated proteins in the context of cancer cells. Here, we present the OncoPPi Portal, an interactive web resource that allows investigators to access, manipulate and interpret a high-quality cancer-focused network of PPIs experimentally detected in cancer cell lines. To facilitate prioritization of PPIs for further biological studies, this resource combines network connectivity analysis, mutual exclusivity analysis of genomic alterations, cellular co-localization of interacting proteins and domain-domain interactions. Estimates of PPI essentiality allow users to evaluate the functional impact of PPI disruption on cancer cell proliferation. Furthermore, connecting the OncoPPi network with the approved drugs and compounds in clinical trials enables discovery of new tumor dependencies to inform strategies to interrogate undruggable targets like tumor suppressors. The OncoPPi Portal serves as a resource for the cancer research community to facilitate discovery of cancer targets and therapeutic development. Availability and implementation: The OncoPPi Portal is available at http://oncoppi.emory.edu. Contact: andrey.ivanov@emory.edu or hfu@emory.edu.

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.

MEDICI: Mining Essentiality Data to Identify Critical Interactions for Cancer Drug Target Discovery and Development.

Protein-protein interactions (PPIs) mediate the transmission and regulation of oncogenic signals that are essential to cellular proliferation and survival, and thus represent potential targets for anti-cancer therapeutic discovery. Despite their significance, there is no method to experimentally disrupt and interrogate the essentiality of individual endogenous PPIs. The ability to computationally predict or infer PPI essentiality would help prioritize PPIs for drug discovery and help advance understanding of cancer biology. Here we introduce a computational method (MEDICI) to predict PPI essentiality by combining gene knockdown studies with network models of protein interaction pathways in an analytic framework. Our method uses network topology to model how gene silencing can disrupt PPIs, relating the unknown essentialities of individual PPIs to experimentally observed protein essentialities. This model is then deconvolved to recover the unknown essentialities of individual PPIs. We demonstrate the validity of our approach via prediction of sensitivities to compounds based on PPI essentiality and differences in essentiality based on genetic mutations. We further show that lung cancer patients have improved overall survival when specific PPIs are no longer present, suggesting that these PPIs may be potentially new targets for therapeutic development. Software is freely available at https://github.com/cooperlab/MEDICI. Datasets are available at https://ctd2.nci.nih.gov/dataPortal.

AKT1, LKB1, and YAP1 Revealed as MYC Interactors with NanoLuc-Based Protein-Fragment Complementation Assay.

The c-Myc (MYC) transcription factor is a major cancer driver and a well-validated therapeutic target. However, directly targeting MYC has been challenging. Thus, identifying proteins that interact with and regulate MYC may provide alternative strategies to inhibit its oncogenic activity. In this study, we report the development of a NanoLuc-based protein-fragment complementation assay (NanoPCA) and mapping of the MYC protein interaction hub in live mammalian cells. The NanoPCA system was configured to enable detection of protein-protein interactions (PPI) at the endogenous level, as shown with PRAS40 dimerization, and detection of weak interactions, such as PINCH1-NCK2. Importantly, NanoPCA allows the study of PPI dynamics with reversible interactions. To demonstrate its utility for large-scale PPI detection in mammalian intracellular environment, we have used NanoPCA to examine MYC interaction with 83 cancer-associated proteins in live cancer cell lines. Our new MYC PPI data confirmed known MYC-interacting proteins, such as MAX, GSK3A, and SMARCA4, and revealed a panel of novel MYC interaction partners, such as RAC-α serine/threonine-protein kinase (AKT)1, liver kinase B (LKB)1, and Yes-associated protein (YAP)1. The MYC interactions with AKT1, LKB1, and YAP1 were confirmed by coimmunoprecipitation of endogenous proteins. Importantly, AKT1, LKB1, and YAP1 were able to activate MYC in a transcriptional reporter assay. Thus, these vital growth control proteins may represent promising MYC regulators, suggesting new mechanisms that couple energetic and metabolic pathways and developmental signaling to MYC-regulated cellular programs.

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.

Targeting protein-protein interactions as an anticancer strategy.

The emergence and convergence of cancer genomics, targeted therapies, and network oncology have significantly expanded the landscape of protein-protein interaction (PPI) networks in cancer for therapeutic discovery. Extensive biological and clinical investigations have led to the identification of protein interaction hubs and nodes that are critical for the acquisition and maintenance of characteristics of cancer essential for cell transformation. Such cancer-enabling PPIs have become promising therapeutic targets. With technological advances in PPI modulator discovery and validation of PPI-targeting agents in clinical settings, targeting of PPI interfaces as an anticancer strategy has become a reality. Future research directed at genomics-based PPI target discovery, PPI interface characterization, PPI-focused chemical library design, and patient-genomic subpopulation-driven clinical studies is expected to accelerate the development of the next generation of PPI-based anticancer agents for personalized precision medicine. Here we briefly review prominent PPIs that mediate cancer-acquired properties, highlight recognized challenges and promising clinical results in targeting PPIs, and outline emerging opportunities.

Saccharomyces cerevisiae Apn1 mutation affecting stable protein expression mimics catalytic activity impairment: implications for assessing DNA repair capacity in humans.

Apurinic/apyrimidinic (AP) endonucleases play a major role in the repair of AP sites, oxidative damage and alkylation damage in DNA. We employed Saccharomyces cerevisiae in an unbiased forward genetic screen to identify amino acid substitutions in the major yeast AP endonuclease, Apn1, that impair cellular DNA repair capacity by conferring sensitivity to the DNA alkylating agent methyl methanesulfonate. We report here the identification and characterization of the Apn1 V156E amino acid substitution mutant through biochemical and functional analysis. We found that steady state levels of Apn1 V156E were substantially decreased compared to wild type protein, and that this decrease was due to more rapid degradation of mutant protein compared to wild type. Based on homology to E. coli endonuclease IV and computational modeling, we predicted that V156E impairs catalytic ability. However, overexpression of mutant protein restored DNA repair activity in vitro and in vivo. Thus, the V156E substitution decreases DNA repair capacity by an unanticipated mechanism via increased degradation of mutant protein, leading to substantially reduced cellular levels. Our study provides evidence that the V156 residue plays a critical role in Apn1 structural integrity, but is not involved in catalytic activity. These results have important implications for elucidating structure-function relationships for the endonuclease IV family of proteins, and for employing simple eukaryotic model systems to understand how structural defects in the major human AP endonuclease APE1 may contribute to disease etiology.

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.

Medicinal chemistry of the A3 adenosine receptor: agonists, antagonists, and receptor engineering.

A(3) adenosine receptor (A(3)AR) ligands have been modified to optimize their interaction with the A(3)AR. Most of these modifications have been made to the N(6) and C2 positions of adenine as well as the ribose moiety, and using a combination of these substitutions leads to the most efficacious, selective, and potent ligands. A(3)AR agonists such as IB-MECA and Cl-IB-MECA are now advancing into Phase II clinical trials for treatments targeting diseases such as cancer, arthritis, and psoriasis. Also, a wide number of compounds exerting high potency and selectivity in antagonizing the human (h)A(3)AR have been discovered. These molecules are generally characterized by a notable structural diversity, taking into account that aromatic nitrogen-containing monocyclic (thiazoles and thiadiazoles), bicyclic (isoquinoline, quinozalines, (aza)adenines), tricyclic systems (pyrazoloquinolines, triazoloquinoxalines, pyrazolotriazolopyrimidines, triazolopurines, tricyclic xanthines) and nucleoside derivatives have been identified as potent and selective A(3)AR antagonists. Probably due to the "enigmatic" physiological role of A(3)AR, whose activation may produce opposite effects (for example, concerning tissue protection in inflammatory and cancer cells) and may produce effects that are species dependent, only a few molecules have reached preclinical investigation. Indeed, the most advanced A(3)AR antagonists remain in preclinical testing. Among the antagonists described above, compound OT-7999 is expected to enter clinical trials for the treatment of glaucoma, while several thiazole derivatives are in development as antiallergic, antiasthmatic and/or antiinflammatory drugs.

Enhanced potency of nucleotide-dendrimer conjugates as agonists of the P2Y14 receptor: multivalent effect in G protein-coupled receptor recognition.

The P2Y(14) receptor is a G protein-coupled receptor activated by uridine-5'-diphosphoglucose and other nucleotide sugars that modulates immune function. Covalent conjugation of P2Y(14) receptor agonists to PAMAM (polyamidoamine) dendrimers enhanced pharmacological activity. Uridine-5'-diphosphoglucuronic acid (UDPGA) and its ethylenediamine adduct were suitable functionalized congeners for coupling to several generations (G2.5-6) of dendrimers (both terminal carboxy and amino). Prosthetic groups, including biotin for avidin complexation, a chelating group for metal complexation (and eventual magnetic resonance imaging), and a fluorescent moiety, also were attached with the eventual goals of molecular detection and characterization of the P2Y(14) receptor. The activities of conjugates were assayed in HEK293 cells stably expressing the human P2Y(14) receptor. A G3 PAMAM conjugate containing 20 bound nucleotide moieties (UDPGA) was 100-fold more potent (EC(50) 2.4 nM) than the native agonist uridine-5'-diphosphoglucose. A molecular model of this conjugate docked in the human P2Y(14) receptor showed that the nucleotide-substituted branches could extend far beyond the dimensions of the receptor and be available for multivalent docking to receptor aggregates. Larger dendrimer carriers and greater loading favored higher potency. A similar conjugate of G6 with 147 out of 256 amino groups substituted with UDPGA displayed an EC(50) value of 0.8 nM. Thus, biological activity was either retained or dramatically enhanced in the multivalent dendrimer conjugates in comparison with monomeric P2Y(14) receptor agonists, depending on size, degree of substitution, terminal functionality, and attached prosthetic groups.

Functionalized congeners of A3 adenosine receptor-selective nucleosides containing a bicyclo[3.1.0]hexane ring system.

(N)-Methanocarba nucleosides containing bicyclo[3.1.0]hexane replacement of the ribose ring previously demonstrated selectivity as A(3) adenosine receptor (AR) agonists (5'-uronamides) or antagonists (5'-truncated). Here, these two series were modified in parallel at the adenine C2 position. N(6)-3-Chlorobenzyl-5'-N-methyluronamides derivatives with functionalized 2-alkynyl chains of varying length terminating in a reactive carboxylate, ester, or amine group were full, potent human A(3)AR agonists. Flexibility of chain substitution allowed the conjugation with a fluorescent cyanine dye (Cy5) and biotin, resulting in binding K(i) values of 17 and 36 nM, respectively. The distal end of the chain was predicted by homology modeling to bind at the A(3)AR extracellular regions. Corresponding l-nucleosides were nearly inactive in AR binding. In the 5'-truncated nucleoside series, 2-Cl analogues were more potent at A(3)AR than 2-H and 2-F, functional efficacy in adenylate cyclase inhibition varied, and introduction of a 2-alkynyl chain greatly reduced affinity. SAR parallels between the two series lost stringency at distal positions. The most potent and selective novel compounds were amine congener 15 (K(i) = 2.1 nM) and truncated partial agonist 22 (K(i) = 4.9 nM).

Molecular recognition in the P2Y(14) receptor: Probing the structurally permissive terminal sugar moiety of uridine-5'-diphosphoglucose.

The P2Y(14) receptor, a nucleotide signaling protein, is activated by uridine-5'-diphosphoglucose 1 and other uracil nucleotides. We have determined that the glucose moiety of 1 is the most structurally permissive region for designing analogues of this P2Y(14) agonist. For example, the carboxylate group of uridine-5'-diphosphoglucuronic acid proved to be suitable for flexible substitution by chain extension through an amide linkage. Functionalized congeners containing terminal 2-acylaminoethylamides prepared by this strategy retained P2Y(14) activity, and molecular modeling predicted close proximity of this chain to the second extracellular loop of the receptor. In addition, replacement of glucose with other sugars did not diminish P2Y(14) potency. For example, the [5'']ribose derivative had an EC(50) of 0.24muM. Selective monofluorination of the glucose moiety indicated a role for the 2''- and 6''-hydroxyl groups of 1 in receptor recognition. The beta-glucoside was twofold less potent than the native alpha-isomer, but methylene replacement of the 1''-oxygen abolished activity. Replacement of the ribose ring system with cyclopentyl or rigid bicyclo[3.1.0]hexane groups abolished activity. Uridine-5'-diphosphoglucose also activates the P2Y(2) receptor, but the 2-thio analogue and several of the potent modified-glucose analogues were P2Y(14)-selective.

Evaluation of homology modeling of G-protein-coupled receptors in light of the A(2A) 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 P2Y(1), P2Y(2), and P2Y(6) receptors and nucleotide antagonists selective for P2Y(1) and P2Y(12) receptors are now known. Selective nonnucleotide antagonists were reported for P2Y(1), P2Y(2), P2Y(6), P2Y(11), P2Y(12), and P2Y(13) receptors. At the P2Y(1) and P2Y(12) 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 P2Y(1) and P2Y(6) receptors. Screening chemically diverse compound libraries has begun to yield new lead compounds for the development of P2Y receptor antagonists, such as competitive P2Y(12) receptor antagonists with antithrombotic activity. Selective agonists for the P2Y(4), P2Y(11), and P2Y(13) receptors and selective antagonists for P2Y(4) and P2Y(14) receptors have not yet been identified. The P2Y(14) 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.

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.

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.

Probing distal regions of the A2B adenosine receptor by quantitative structure-activity relationship modeling of known and novel agonists.

The binding modes at the A 2B adenosine receptor (AR) of 72 derivatives of adenosine and its 5'- N-methyluronamide with diverse substitutions at the 2 and N (6) positions were studied using a molecular modeling approach. The compounds in their receptor-docked conformations were used to build CoMFA and CoMSIA quantitative structure-activity relationship models. Various parameters, including different types of atomic charges, were examined. The best statistical parameters were obtained with a joint CoMFA and CoMSIA model: R (2) = 0.960, Q (2) = 0.676, SEE = 0.175, F = 158, and R (2) test = 0.782 for an independent test set containing 18 compounds. On the basis of the modeling results, four novel adenosine analogues, having elongated or bulky substitutions at N (6) position and/or 2 position, were synthesized and evaluated biologically. All of the proposed compounds were potent, full agonists at the A 2B AR in adenylate cyclase studies. Thus, in support of the modeling, bulky substitutions at both positions did not prevent A 2B AR activation, which predicts separate regions for docking of these moieties.