Reporter cell lines to screen for inhibitors or regulators of the KRAS-RAF-MEK1/2-ERK1/2 pathway.

The RAS-regulated RAF-MEK1/2-ERK1/2 signalling pathway is activated in cancer due to mutations in RAS proteins (especially KRAS), BRAF, CRAF, MEK1 and MEK2. Whilst inhibitors of KRASG12C (lung adenocarcinoma) and BRAF and MEK1/2 (melanoma and colorectal cancer) are clinically approved, acquired resistance remains a problem. Consequently, the search for new inhibitors (especially of RAS proteins), new inhibitor modalities and regulators of this pathway, which may be new drug targets, continues and increasingly involves cell-based screens with small molecules or genetic screens such as RNAi, CRISPR or protein interference. Here we describe cell lines that exhibit doxycycline-dependent expression KRASG12V or BRAFV600E and harbour a stably integrated EGR1:EmGFP reporter gene that can be detected by flow cytometry, high-content microscopy or immunoblotting. KRASG12V or BRAFV600E-driven EmGFP expression is inhibited by MEK1/2 or ERK1/2 inhibitors (MEKi and ERKi). BRAFi inhibit BRAFV600E-driven EmGFP expression but enhance the response to KRASG12V, recapitulating paradoxical activation of wild type RAF proteins. In addition to small molecules, expression of iDab6, encoding a RAS-specific antibody fragment inhibited KRASG12V- but not BRAFV600E-driven EmGFP expression. Finally, substitution of EmGFP for a bacterial nitroreductase gene allowed KRASG12V or BRAFV600E to drive cell death in the presence of a pro-drug, which may allow selection of pathway inhibitors that promote survival. These cell lines should prove useful for cell-based screens to identify new regulators of KRAS- or BRAF-dependent ERK1/2 signalling (drug target discovery) as well as screening or triaging 'hits' from drug discovery screens.

Double Strain-Promoted Macrocyclization for the Rapid Selection of Cell-Active Stapled Peptides.

Peptide stapling is a method for designing macrocyclic alpha-helical inhibitors of protein-protein interactions. However, obtaining a cell-active inhibitor can require significant optimization. We report a novel stapling technique based on a double strain-promoted azide-alkyne reaction, and exploit its biocompatibility to accelerate the discovery of cell-active stapled peptides. As a proof of concept, MDM2-binding peptides were stapled in parallel, directly in cell culture medium in 96-well plates, and simultaneously evaluated in a p53 reporter assay. This in situ stapling/screening process gave an optimal candidate that showed improved proteolytic stability and nanomolar binding to MDM2 in subsequent biophysical assays. α-Helicity was confirmed by a crystal structure of the MDM2-peptide complex. This work introduces in situ stapling as a versatile biocompatible technique with many other potential high-throughput biological applications.

Linear aliphatic dialkynes as alternative linkers for double-click stapling of p53-derived peptides.

We investigated linear aliphatic dialkynes as a new structural class of i,i+7 linkers for the double-click stapling of p53-based peptides. The optimal combination of azido amino acids and dialkynyl linker length for MDM2 binding was determined. In a direct comparison between aliphatic and aromatic staple scaffolds, the aliphatic staples resulted in superior binding to MDM2 in vitro and superior p53-activating capability in cells when using a diazidopeptide derived from phage display. This work demonstrates that the nature of the staple scaffold is an important factor that can affect peptide bioactivity in cells.

Investigating peptide sequence variations for 'double-click' stapled p53 peptides.

Stapling peptides for inhibiting the p53/MDM2 interaction is a promising strategy for developing anti-cancer therapeutic leads. We evaluate double-click stapled peptides formed from p53-based diazidopeptides with different staple positions and azido amino acid side-chain lengths, determining the impact of these variations on MDM2 binding and cellular activity. We also demonstrate a K24R mutation, necessary for cellular activity in hydrocarbon-stapled p53 peptides, is not required for analogous 'double-click' peptides.

Interrogation of the protein-protein interactions between human BRCA2 BRC repeats and RAD51 reveals atomistic determinants of affinity.

The breast cancer suppressor BRCA2 controls the recombinase RAD51 in the reactions that mediate homologous DNA recombination, an essential cellular process required for the error-free repair of DNA double-stranded breaks. The primary mode of interaction between BRCA2 and RAD51 is through the BRC repeats, which are ∼35 residue peptide motifs that interact directly with RAD51 in vitro. Human BRCA2, like its mammalian orthologues, contains 8 BRC repeats whose sequence and spacing are evolutionarily conserved. Despite their sequence conservation, there is evidence that the different human BRC repeats have distinct capacities to bind RAD51. A previously published crystal structure reports the structural basis of the interaction between human BRC4 and the catalytic core domain of RAD51. However, no structural information is available regarding the binding of the remaining seven BRC repeats to RAD51, nor is it known why the BRC repeats show marked variation in binding affinity to RAD51 despite only subtle sequence variation. To address these issues, we have performed fluorescence polarisation assays to indirectly measure relative binding affinity, and applied computational simulations to interrogate the behaviour of the eight human BRC-RAD51 complexes, as well as a suite of BRC cancer-associated mutations. Our computational approaches encompass a range of techniques designed to link sequence variation with binding free energy. They include MM-PBSA and thermodynamic integration, which are based on classical force fields, and a recently developed approach to computing binding free energies from large-scale quantum mechanical first principles calculations with the linear-scaling density functional code onetep. Our findings not only reveal how sequence variation in the BRC repeats directly affects affinity with RAD51 and provide significant new insights into the control of RAD51 by human BRCA2, but also exemplify a palette of computational and experimental tools for the analysis of protein-protein interactions for chemical biology and molecular therapeutics.

Synaptic NMDA receptor activity boosts intrinsic antioxidant defenses.

Intrinsic antioxidant defenses are important for neuronal longevity. We found that in rat neurons, synaptic activity, acting via NMDA receptor (NMDAR) signaling, boosted antioxidant defenses by making changes to the thioredoxin-peroxiredoxin (Prx) system. Synaptic activity enhanced thioredoxin activity, facilitated the reduction of overoxidized Prxs and promoted resistance to oxidative stress. Resistance was mediated by coordinated transcriptional changes; synaptic NMDAR activity inactivated a previously unknown Forkhead box O target gene, the thioredoxin inhibitor Txnip. Conversely, NMDAR blockade upregulated Txnip in vivo and in vitro, where it bound thioredoxin and promoted vulnerability to oxidative damage. Synaptic activity also upregulated the Prx reactivating genes Sesn2 (sestrin 2) and Srxn1 (sulfiredoxin), via C/EBPbeta and AP-1, respectively. Mimicking these expression changes was sufficient to strengthen antioxidant defenses. Trans-synaptic stimulation of synaptic NMDARs was crucial for boosting antioxidant defenses; chronic bath activation of all (synaptic and extrasynaptic) NMDARs induced no antioxidative effects. Thus, synaptic NMDAR activity may influence the progression of pathological processes associated with oxidative damage.

G-quadruplex-binding benzo[a]phenoxazines down-regulate c-KIT expression in human gastric carcinoma cells.

There is considerable interest in the structure and function of G-quadruplex nucleic acid secondary structures, their cellular functions, and their potential as therapeutic targets. G-Quadruplex sequence motifs are prevalent in gene promoter regions and it has been hypothesized that G-quadruplex structure formation is associated with the transcriptional status of the downstream gene. Using a functional cell-based assay, we have identified two novel G-quadruplex ligands that reduce the transcription of a luciferase reporter driven from the G-quadruplex-containing c-KIT promoter. We have further shown that endogenous c-KIT expression in a human gastric carcinoma cell line is also reduced on treatment with these molecules. Biophysical analysis using surface plasmon resonance has shown that these molecules preferentially bind with high affinity to one of the two G-quadruplex sequences in the c-KIT promoter over double-stranded DNA. This work highlights the utility of cell-based reporter assays to identify new G-quadruplex binding molecules that modulate transcription and identifies benzo[a]phenoxazine derivatives as potential antitumor agents.

Computational analysis of phosphopeptide binding to the polo-box domain of the mitotic kinase PLK1 using molecular dynamics simulation.

The Polo-Like Kinase 1 (PLK1) acts as a central regulator of mitosis and is over-expressed in a wide range of human tumours where high levels of expression correlate with a poor prognosis. PLK1 comprises two structural elements, a kinase domain and a polo-box domain (PBD). The PBD binds phosphorylated substrates to control substrate phosphorylation by the kinase domain. Although the PBD preferentially binds to phosphopeptides, it has a relatively broad sequence specificity in comparison with other phosphopeptide binding domains. We analysed the molecular determinants of recognition by performing molecular dynamics simulations of the PBD with one of its natural substrates, CDC25c. Predicted binding free energies were calculated using a molecular mechanics, Poisson-Boltzmann surface area approach. We calculated the per-residue contributions to the binding free energy change, showing that the phosphothreonine residue and the mainchain account for the vast majority of the interaction energy. This explains the very broad sequence specificity with respect to other sidechain residues. Finally, we considered the key role of bridging water molecules at the binding interface. We employed inhomogeneous fluid solvation theory to consider the free energy of water molecules on the protein surface with respect to bulk water molecules. Such an analysis highlights binding hotspots created by elimination of water molecules from hydrophobic surfaces. It also predicts that a number of water molecules are stabilized by the presence of the charged phosphate group, and that this will have a significant effect on the binding affinity. Our findings suggest a molecular rationale for the promiscuous binding of the PBD and highlight a role for bridging water molecules at the interface. We expect that this method of analysis will be very useful for probing other protein surfaces to identify binding hotspots for natural binding partners and small molecule inhibitors.

Target identification for small-molecule discovery in the FOXO3a tumor-suppressor pathway using a biodiverse peptide library.

Genetic screening technologies to identify and validate macromolecular interactions (MMIs) essential for complex pathways remain an important unmet need for systems biology and therapeutics development. Here, we use a library of peptides from diverse prokaryal genomes to screen MMIs promoting the nuclear relocalization of Forkhead Box O3 (FOXO3a), a tumor suppressor more frequently inactivated by post-translational modification than mutation. A hit peptide engages the 14-3-3 family of signal regulators through a phosphorylation-dependent interaction, modulates FOXO3a-mediated transcription, and suppresses cancer cell growth. In a crystal structure, the hit peptide occupies the phosphopeptide-binding groove of 14-3-3ε in a conformation distinct from its natural peptide substrates. A biophysical screen identifies drug-like small molecules that displace the hit peptide from 14-3-3ε, providing starting points for structure-guided development. Our findings exemplify "protein interference," an approach using evolutionarily diverse, natural peptides to rapidly identify, validate, and develop chemical probes against MMIs essential for complex cellular phenotypes.

A small-molecule inhibitor of the BRCA2-RAD51 interaction modulates RAD51 assembly and potentiates DNA damage-induced cell death.

BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted α-methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionizing radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.

The pharmaceutical solvent N-methyl-2-pyrollidone (NMP) attenuates inflammation through Krüppel-like factor 2 activation to reduce atherogenesis.

N-methyl-2-pyrrolidone (NMP) is a versatile water-miscible polar aprotic solvent. It is used as a drug solubilizer and penetration enhancer in human and animal, yet its bioactivity properties remain elusive. Here, we report that NMP is a bioactive anti-inflammatory compound well tolerated in vivo, that shows efficacy in reducing disease in a mouse model of atherosclerosis. Mechanistically, NMP increases the expression of the transcription factor Kruppel-like factor 2 (KLF2). Monocytes and endothelial cells treated with NMP express increased levels of KLF2, produce less pro-inflammatory cytokines and adhesion molecules. We found that NMP attenuates monocyte adhesion to endothelial cells inflamed with tumor necrosis factor alpha (TNF-α) by reducing expression of adhesion molecules. We further show using KLF2 shRNA that the inhibitory effect of NMP on endothelial inflammation and subsequent monocyte adhesion is KLF2 dependent. Enhancing KLF2 expression and activity improves endothelial function, controls multiple genes critical for inflammation, and prevents atherosclerosis. Our findings demonstrate a consistent effect of NMP upon KLF2 activation and inflammation, biological processes central to atherogenesis. Our data suggest that inclusion of bioactive solvent NMP in pharmaceutical compositions to treat inflammatory disorders might be beneficial and safe, in particular to treat diseases of the vascular system, such as atherosclerosis.

Development of a Novel Cell-Permeable Protein-Protein Interaction Inhibitor for the Polo-box Domain of Polo-like Kinase 1.

Polo-like kinase 1 (PLK1) is a key regulator of mitosis and a recognized drug target for cancer therapy. Inhibiting the polo-box domain of PLK1 offers potential advantages of increased selectivity and subsequently reduced toxicity compared with targeting the kinase domain. However, many if not all existing polo-box domain inhibitors have been shown to be unsuitable for further development. In this paper, we describe a novel compound series, which inhibits the protein-protein interactions of PLK1 via the polo-box domain. We combine high throughput screening with molecular modeling and computer-aided design, synthetic chemistry, and cell biology to address some of the common problems with protein-protein interaction inhibitors, such as solubility and potency. We use molecular modeling to improve the solubility of a hit series with initially poor physicochemical properties, enabling biophysical and biochemical characterization. We isolate and characterize enantiomers to improve potency and demonstrate on-target activity in both cell-free and cell-based assays, entirely consistent with the proposed binding model. The resulting compound series represents a promising starting point for further progression along the drug discovery pipeline and a new tool compound to study kinase-independent PLK functions.

A cryptic hydrophobic pocket in the polo-box domain of the polo-like kinase PLK1 regulates substrate recognition and mitotic chromosome segregation.

The human polo-like kinase PLK1 coordinates mitotic chromosome segregation by phosphorylating multiple chromatin- and kinetochore-binding proteins. How PLK1 activity is directed to specific substrates via phosphopeptide recognition by its carboxyl-terminal polo-box domain (PBD) is poorly understood. Here, we combine molecular, structural and chemical biology to identify a determinant for PLK1 substrate recognition that is essential for proper chromosome segregation. We show that mutations ablating an evolutionarily conserved, Tyr-lined pocket in human PLK1 PBD trigger cellular anomalies in mitotic progression and timing. Tyr pocket mutations selectively impair PLK1 binding to the kinetochore phosphoprotein substrate PBIP1, but not to the centrosomal substrate NEDD1. Through a structure-guided approach, we develop a small-molecule inhibitor, Polotyrin, which occupies the Tyr pocket. Polotyrin recapitulates the mitotic defects caused by mutations in the Tyr pocket, further evidencing its essential function, and exemplifying a new approach for selective PLK1 inhibition. Thus, our findings support a model wherein substrate discrimination via the Tyr pocket in the human PLK1 PBD regulates mitotic chromosome segregation to preserve genome integrity.

Notch ligation by Delta1 inhibits peripheral immune responses to transplantation antigens by a CD8+ cell-dependent mechanism.

Notch signaling plays a fundamental role in determining the outcome of differentiation processes in many tissues. Notch signaling has been implicated in T versus B cell lineage commitment, thymic differentiation, and bone marrow hematopoietic precursor renewal and differentiation. Notch receptors and their ligands are also expressed on the surface of mature lymphocytes and APCs, but the effects of Notch signaling in the peripheral immune system remain poorly defined. The aim of the studies reported here was to investigate the effects of signaling through the Notch receptor using a ligand of the Delta-like family. We show that Notch ligation in the mature immune system markedly decreases responses to transplantation antigens. Constitutive expression of Delta-like 1 on alloantigen-bearing cells renders them nonimmunogenic and able to induce specific unresponsiveness to a challenge with the same alloantigen, even in the form of a cardiac allograft. These effects could be reversed by depletion of CD8+ cells at the time of transplantation. Ligation of Notch on splenic CD8+ cells results in a dramatic decrease in IFN-gamma with a concomitant enhancement of IL-10 production, suggesting that Notch signaling can alter the differentiation potential of CD8+ cells. These data implicate Notch signaling in regulation of peripheral immunity and suggest a novel approach for manipulating deleterious immune responses.

Computationally-guided optimization of small-molecule inhibitors of the Aurora A kinase-TPX2 protein-protein interaction.

Free energy perturbation theory, in combination with enhanced sampling of protein-ligand binding modes, is evaluated in the context of fragment-based drug design, and used to design two new small-molecule inhibitors of the Aurora A kinase-TPX2 protein-protein interaction.

Modulating Protein-Protein Interactions of the Mitotic Polo-like Kinases to Target Mutant KRAS.

Mutations activating KRAS underlie many forms of cancer, but are refractory to therapeutic targeting. Here, we develop Poloppin, an inhibitor of protein-protein interactions via the Polo-box domain (PBD) of the mitotic Polo-like kinases (PLKs), in monotherapeutic and combination strategies to target mutant KRAS. Poloppin engages its targets in biochemical and cellular assays, triggering mitotic arrest with defective chromosome congression. Poloppin kills cells expressing mutant KRAS, selectively enhancing death in mitosis. PLK1 or PLK4 depletion recapitulates these cellular effects, as does PBD overexpression, corroborating Poloppin's mechanism of action. An optimized analog with favorable pharmacokinetics, Poloppin-II, is effective against KRAS-expressing cancer xenografts. Poloppin resistance develops less readily than to an ATP-competitive PLK1 inhibitor; moreover, cross-sensitivity persists. Poloppin sensitizes mutant KRAS-expressing cells to clinical inhibitors of c-MET, opening opportunities for combination therapy. Our findings exemplify the utility of small molecules modulating the protein-protein interactions of PLKs to therapeutically target mutant KRAS-expressing cancers.

Cellular Notch responsiveness is defined by phosphoinositide 3-kinase-dependent signals.

BACKGROUND: Notch plays a wide-ranging role in controlling cell fate, differentiation and development. The PI3K-Akt pathway is a similarly conserved signalling pathway which regulates processes such as differentiation, proliferation and survival. Mice with disrupted Notch and PI3K signalling show phenotypic similarities during haematopoietic cell development, suggesting functional interaction between these pathways. RESULTS: We show that cellular responsiveness to Notch signals depends on the activity of the PI3K-Akt pathway in cells as diverse as CHO cells, primary T-cells and hippocampal neurons. Induction of the endogenous PI3K-Akt pathway in CHO cells (by the insulin pathway), in T-cells (via TCR activation) or in neurons (via TrKB activation) potentiates Notch-dependent responses. We propose that the PI3K-Akt pathway exerts its influence on Notch primarily via inhibition of GSK3-beta, a kinase known to phosphorylate and regulate Notch signals. CONCLUSION: The PI3K-Akt pathway acts as a "gain control" for Notch signal responses. Since physiological levels of intracellular Notch are often low, coincidence with PI3K-activation may be crucial for induction of Notch-dependent responses.

Allosteric modulation of AURKA kinase activity by a small-molecule inhibitor of its protein-protein interaction with TPX2.

The essential mitotic kinase Aurora A (AURKA) is controlled during cell cycle progression via two distinct mechanisms. Following activation loop autophosphorylation early in mitosis when it localizes to centrosomes, AURKA is allosterically activated on the mitotic spindle via binding to the microtubule-associated protein, TPX2. Here, we report the discovery of AurkinA, a novel chemical inhibitor of the AURKA-TPX2 interaction, which acts via an unexpected structural mechanism to inhibit AURKA activity and mitotic localization. In crystal structures, AurkinA binds to a hydrophobic pocket (the 'Y pocket') that normally accommodates a conserved Tyr-Ser-Tyr motif from TPX2, blocking the AURKA-TPX2 interaction. AurkinA binding to the Y- pocket induces structural changes in AURKA that inhibit catalytic activity in vitro and in cells, without affecting ATP binding to the active site, defining a novel mechanism of allosteric inhibition. Consistent with this mechanism, cells exposed to AurkinA mislocalise AURKA from mitotic spindle microtubules. Thus, our findings provide fresh insight into the catalytic mechanism of AURKA, and identify a key structural feature as the target for a new class of dual-mode AURKA inhibitors, with implications for the chemical biology and selective therapeutic targeting of structurally related kinases.

Modulation of the notch pathway for immunotherapy.

Since its initial description as a neurogenic locus in Drosophila, the Notch pathway has been shown to play a central role in cell fate decisions across species, including vertebrates, guiding the differentiation of multiple cell types. In the immune system, its function was first demonstrated during lymphopoiesis, but in recent years this pathway has been shown to still be active in peripheral T-cells. Therapeutic opportunities that could arise from the manipulation of Notch signaling in immune disorders such as autoimmunity, allergy and in cancer immunotherapy and transplantation are discussed.