Discovery of cell active macrocyclic peptides with on-target inhibition of KRAS signaling.

Macrocyclic peptides have the potential to address intracellular protein-protein interactions (PPIs) of high value therapeutic targets that have proven largely intractable to small molecules. Here, we report broadly applicable lessons for applying this modality to intracellular targets and specifically for advancing chemical matter to address KRAS, a protein that represents the most common oncogene in human lung, colorectal and pancreatic cancers yet is one of the most challenging targets in human disease. Specifically, we focused on KRpep-2d, an arginine-rich KRAS-binding peptide with a disulfide-mediated macrocyclic linkage and a protease-sensitive backbone. These latter redox and proteolytic labilities obviated cellular activity. Extensive structure-activity relationship studies involving macrocyclic linker replacement, stereochemical inversion, and backbone α-methylation, gave a peptide with on-target cellular activity. However, we uncovered an important generic insight - the arginine-dependent cell entry mechanism limited its therapeutic potential. In particular, we observed a strong correlation between net positive charge and histamine release in an ex vivo assay, thus making this series unsuitable for advancement due to the potentially fatal consequences of mast cell degranulation. This observation should signal to researchers that cationic-mediated cell entry - an approach that has yet to succeed in the clinic despite a long history of attempts - carries significant therapy-limiting safety liabilities. Nonetheless, the cell-active molecules identified here validate a unique inhibitory epitope on KRAS and thus provide valuable molecular templates for the development of therapeutics that are desperately needed to address KRAS-driven cancers - some of the most treatment-resistant human malignancies.

Resistance to anti-microtubule drug-induced cell death is determined by regulation of BimEL expression.

Anti-microtubule agents are frequently used as anticancer therapeutics. Cell death induced by these agents is considered to be due to sustained mitotic arrest caused by the activation of spindle assembly checkpoint (SAC). However, some cell types are resistant to mitotic cell death. Cells' ability to escape mitotic arrest (mitotic slippage) is thought to be a major mechanism contributing to this resistance. Here, we show that resistance to cell death induced by anti-mitotic agents is not linked to cells' capacity to undergo mitotic slippage as generally believed but is dependent on the state of BimEL regulation during mitosis. While transcriptional repression of BimEL in the mitotic death-resistant cells involves polycomb repressive complex 2 (PRC2)-mediated histone trimethylation, the BimEL protein is destabilized by cullin 1/4A-ßTrCP-dependent degradation involving activation of cullin 1/4A by neddylation. These results imply that pharmacological augmentation of BimEL activity in anti-microtubule drug-resistant tumors may have important therapeutic implications.

Conformational landscape of the epidermal growth factor receptor kinase reveals a mutant specific allosteric pocket.

Activating mutations within the epidermal growth factor receptor (EGFR) kinase domain give rise to several cancers including Non-Small Cell Lung Cancer (NSCLC). Small molecule inhibitors targeted at these mutants have proven to be clinically successful drugs. These molecules are ATP competitive and rapidly result in the emergence of resistance. Recently Jia et al. [Nature, 2016, 534, 129-132] reported a small molecule inhibitor (called EAI045) that binds at an allosteric pocket, does not compete with ATP and displays high potency and selectivity towards certain activating mutants (L858R, T790M, L858R/T790M) of EGFR, with IC50 values ranging from 3 nM to 49 nM. We present here a study combining extensive molecular dynamics simulations with binding assays to provide a structural basis underlying the mechanism of binding of this molecule. It appears that in mutants, conformational destabilization of the short helix (that carries Leu858 in the wildtype), is key to the exposure of the allosteric pocket which otherwise is occluded by a set of sidechains including L858. We extend this hypothesis to show that a similar mechanism would enable the molecule to inhibit EGFRL861Q which is another oncogenic mutant and validate this with binding experiments. The screening of the human structural kinome revealed at least 12 other oncogenic kinases which carry at least one activating mutant in this disorder-prone region and hence would be amenable to allosteric inhibition by molecules such as EAI045. Our study characterizes a druggable allosteric pocket which appears to be specific to certain oncogenic mutants of the EGFR and holds therapeutic potential.

Replication stress-induced endogenous DNA damage drives cellular senescence induced by a sub-lethal oxidative stress.

Although oxidative stress has been shown to induce senescence and replication stress independently, no study has implicated unresolved replication stress as the driver for cellular senescence in response to oxidative stress. Using cells exposed to increasing concentrations of hydrogen peroxide, we show that sub-lethal amount of exogenous hydrogen peroxide induces two waves of DNA damage. The first wave is rapid and transient while the second wave coincides with the cells transition from the S to the G2/M phases of cell cycle. Subsequently, cells enter growth arrest accompanied by the acquisition of senescence-associated characteristics. Furthermore, a p53-dependent decrease in Rad51, which is associated with the formation of DNA segments with chromatin alterations reinforcing senescence, and Lamin B1 that is involved in chromatin remodeling, is observed during the establishment of the senescent phenotype. On the other hand, increase in senescence associated-ß-Gal activity, a classical marker of senescence and HMGA2, a marker of the senescence-associated heterochromatin foci, is shown to be independent of p53. Together, our findings implicate replication stress-induced endogenous DNA damage as the driver for the establishment of cellular senescence upon sub-lethal oxidative stress, and implicate the role of p53 in some but not all hallmarks of the senescent phenotype.

Sub-lethal oxidative stress induces lysosome biogenesis via a lysosomal membrane permeabilization-cathepsin-caspase 3-transcription factor EB-dependent pathway.

Here we provide evidence to link sub-lethal oxidative stress to lysosome biogenesis. Exposure of cells to sub-lethal concentrations of exogenously added hydrogen peroxide resulted in cytosol to nuclear translocation of the Transcription Factor EB (TFEB), the master controller of lysosome biogenesis and function. Nuclear translocation of TFEB was dependent upon the activation of a cathepsin-caspase 3 signaling pathway, downstream of lysosomal membrane permeabilization and accompanied by a significant increase in lysosome numbers as well as induction of TFEB-dependent lysosome-associated genes expression such as Ctsl, Lamp2 and its spliced variant Lamp2a, Neu1and Ctsb and Sqstm1 and Atg9b. The effects of sub-lethal oxidative stress on lysosomal gene expression and biogenesis were rescued upon gene silencing of caspase 3 and TFEB. Notably, caspase 3 activation was not associated with phenotypic hallmarks of apoptosis, evidenced by the absence of caspase 3 substrate cleavage, such as PARP, Lamin A/C or gelsolin. Taken together, these data demonstrate for the first time an unexpected and non-canonical role of a cathepsin-caspase 3 axis in the nuclear translocation of TFEB leading to lysosome biogenesis under conditions of sub-lethal oxidative stress.

OpenComet: an automated tool for comet assay image analysis.

Reactive species such as free radicals are constantly generated in vivo and DNA is the most important target of oxidative stress. Oxidative DNA damage is used as a predictive biomarker to monitor the risk of development of many diseases. The comet assay is widely used for measuring oxidative DNA damage at a single cell level. The analysis of comet assay output images, however, poses considerable challenges. Commercial software is costly and restrictive, while free software generally requires laborious manual tagging of cells. This paper presents OpenComet, an open-source software tool providing automated analysis of comet assay images. It uses a novel and robust method for finding comets based on geometric shape attributes and segmenting the comet heads through image intensity profile analysis. Due to automation, OpenComet is more accurate, less prone to human bias, and faster than manual analysis. A live analysis functionality also allows users to analyze images captured directly from a microscope. We have validated OpenComet on both alkaline and neutral comet assay images as well as sample images from existing software packages. Our results show that OpenComet achieves high accuracy with significantly reduced analysis time.

PPARγ disease gene network and identification of therapeutic targets for prostate cancer.

Peroxisome proliferator-activated receptor (PPAR) belongs to the nuclear hormone receptor superfamily. Recently published reports demonstrate the importance of a direct repeat 2 (DR2) as a PPARγ-responsive element in addition to the canonical direct repeat 1 (DR1) Peroxisome proliferator response elements (PPREs). However, a comprehensive and systematic approach to constructing de novo disease-specific gene networks for PPARγ is lacking, especially one that includes PPARγ target genes containing either DR1 or DR2 site within their promoter region. Here, we computationally identified 1154 PPARγ direct target genes and constructed the PPARγ disease gene network, which revealed 138 PPARγ target genes that are associated with 65 unique diseases. The network shows that PPARγ target genes are highly associated with cancer and neurological diseases. Thirty-eight PPARγ direct target genes were found to be involved in prostate cancer and two key (hub) PPARγ direct target genes, PRKCZ and PGK1, were experimentally validated to be repressed upon PPARγ activation by its natural ligand, 15d-PGJ(2) in three prostrate cancer cell lines. We proposed that PRKCZ and PGK1 could be novel therapeutic targets for prostate cancer. These investigations would not only aid in understanding the molecular mechanisms by which PPARγ regulates disease targets but would also lead to the identification of novel PPARγ gene targets.

Computational identification and experimental validation of PPRE motifs in NHE1 and MnSOD genes of human.

BACKGROUND: Activation of PPARs has been reported to inhibit the proliferation of malignant cells from different lineages. They are involved in transcription regulation of genes upon activation by a ligand. The binding of PPARs to the promoter sequence either represses or activates the gene. Hence, PPARs represent promising targets for cancer treatment because of their anti-proliferative and pro-apoptotic activities. Here we computationally identified PPAR binding regions in NHE1 and MnSOD. We further validated the predictions in vitro. RESULTS: Our results computationally predicted the presence of 2 PPRE motifs in NHE1 and 3 PPRE motifs in MnSOD. We experimentally confirmed the true motifs and their regulation by PPAR. CONCLUSION: Our results suggest that both NHE1 and MnSOD have PPRE binding motif in their upstream/promoter region and hence are regulated by PPAR upon ligand binding.