Approaches Toward Improving the Prognosis of Pediatric Patients With Glioma: Pursuing Mutant Drug Targets With Emerging Small Molecules

Gliomas represent approximately 70% of all pediatric brain tumors and most of these are of astrocytic lineage, furthermore, malignant or high-grade astrocytoma account for approximately 20% of pediatric astrocytoma. Treatment options for pediatric glioma patients are limited. Although low-grade astrocytoma are relatively slow-growing tumors which can often be cured through surgical resection, a significant proportion of cases will recur, as such new treatments are desperately needed. This review covers the various approaches that are currently being made towards improving the prognosis of pediatric glioma patients by pursuing pediatric-selective mutant drug targets with emerging small molecules

Role of EPHA2 Serine 897 phosphorylation in thyroid cancer: molecular mechanisms and biological properties of a novel player in thyroid tumorigenesis

EPH (Erythropoietin-Producing Hepatocellular carcinoma cell line) receptor tyrosine kinases (RTK) belong to the largest subfamily of RTKs counting 14 genes in humans. Among them, EPHA2 is often overexpressed in a variety of human cancers, including thyroid carcinoma. Thyroid carcinomas are commonly driven by genetic lesions targeting the MAPK signaling cascade including rearrangements of several RTKs, such as RET and NTRK, or point mutations in RAS or BRAF. We have previously demonstrated, through a siRNA-based genetic screen of the human kinome, that EPHA2 expression is essential for viability of thyroid cancer cells in culture. To gain insight into the EPHA2 function in thyroid tumorigenesis, we studied the role of the intracellular domain of EPHA2 and, in particular, of its phosphorylation on Serine 897 (pSer897). Ser897 phosphorylation has been previously reported to mediate EPHA2 oncogenic activity. Ser897 is embedded in the consensus phosphorylation sequence for AGC (PKA, PKG, PKC) family kinases, including p90RSK, a direct MAPK target. Here we show that in thyroid cancer cells bearing oncogenic lesions in the MAPK signaling cascade, EPHA2 is robustly phosphorylated on Ser897. Treatment with chemical inhibitors targeting p90RSK or other MAPK pathway components blunts Ser897 phosphorylation of EPHA2. Recombinant p90RSK phosphorylates in vitro EPHA2 Ser897. Finally, RNA interference-mediated knock-down combined with rescue experiments demonstrate that Ser897 phosphorylation of EPHA2 mediates thyroid cancer cell proliferation and motility. Collectively, these findings point to EPHA2 pSer897 as a novel crucial mediator of the oncogenic MAPK signaling cascade, and in particular of p90RSK, in thyroid cancer

Methods to Improve Virtual Screening of Potential Drug Leads for Specific Pharmacodynamic and Toxicological Properties

Ph.DDOCTOR OF PHILOSOPH

Identification of Potential Kinase Inhibitors within the PI3K/AKT Pathway of Leishmania Species

Leishmaniasis is a public health disease that requires the development of more effective treatments and the identification of novel molecular targets. Since blocking the PI3K/AKT pathway has been successfully studied as an effective anticancer strategy for decades, we examined whether the same approach would also be feasible in Leishmania due to their high amount and diverse set of annotated proteins. Here, we used a best reciprocal hits protocol to identify potential protein kinase homologues in an annotated human PI3K/AKT pathway. We calculated their ligandibility based on available bioactivity data of the reported homologues and modelled their 3D structures to estimate the druggability of their binding pockets. The models were used to run a virtual screening method with molecular docking. We found and studied five protein kinases in five different Leishmania species, which are AKT, CDK, AMPK, mTOR and GSK3 homologues from the studied pathways. The compounds found for different enzymes and species were analysed and suggested as starting point scaffolds for the design of inhibitors. We studied the kinases’ participation in protein–protein interaction networks, and the potential deleterious effects, if inhibited, were supported with the literature. In the case of Leishmania GSK3, an inhibitor of its human counterpart, prioritized by our method, was validated in vitro to test its anti-Leishmania activity and indirectly infer the presence of the enzyme in the parasite. The analysis contributes to improving the knowledge about the presence of similar signalling pathways in Leishmania, as well as the discovery of compounds acting against any of these kinases as potential molecular targets in the parasite.Fil: Ochoa, Rodrigo. Universidad de Antioquia; ColombiaFil: Ortega Pajares, Amaya. University of Melbourne; AustraliaFil: Castello, Florencia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Cálculo; ArgentinaFil: Serral, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Cálculo; ArgentinaFil: Fernández Do Porto, Darío Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Cálculo; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Villa Pulgarin, Janny A.. Coorporación Universitaria Remington; ColombiaFil: Varela M., Rubén E.. Universidad Santiago de Cali; ColombiaFil: Muskus, Carlos. Universidad de Antioquia; Colombi

IDENTIFICATION OF NOVEL POTENTIAL CANCER THERAPIES BY SYNTHETIC LETHAL SCREENING

There is an urgent need for novel effective drug regimens for the treatment of cancer. Current chemotherapy suffers from a slim therapeutic index, with significant toxicity from effective drug doses or tumor recurrence at low drug doses. Identifying synergistic interactions between drugs is a difficult process. To accelerate the discovery of potential drug combinations, I have developed a druggable genome siRNA synthetic lethal screen capable of rapidly identifying novel drug targets that would sensitize cancer cells to sublethal concentrations of microtubule destabilizing agents. I employed a high-throughput cell-based 16,560-siRNA screen to isolate a high-confidence list of genes that, when silenced, enhanced glioblastoma multiforme cancer cell chemosensitivity. Two gene products that were the major focus of my work were midline2 and the neurokinin receptor NK1R. Silencing of midline2, a PP2A-microtubule tether, sensitized cells to two microtubule destabilizing agents, vinblastine and disorazole C1, suggesting a mechanistic dependency of the phosphatidylinositol 3-kinase pathway on microtubule functionality. Combinations of phosphatidylinositol 3-kinase inhibitors with disorazole C1 and several vinca alkaloids confirmed this hypothesis. To verify microtubule destabilizing agent sensitization by NK1R silencing, I demonstrated a significant collaboration of neurokinin receptor NK1R antagonists with low concentrations of vinca alkaloids. These assay results and subsequent novel combination strategies demonstrate the tremendous ability of this synthetic lethal screen to predict potent collaborations between different classes of drugs, as well as identifying molecular constituents mediating those interactions

Iterative in Situ Click Chemistry Assembles a Branched Capture Agent and Allosteric Inhibitor for Akt1

We describe the use of iterative in situ click chemistry to design an Akt-specific branched peptide triligand that is a drop-in replacement for monoclonal antibodies in multiple biochemical assays. Each peptide module in the branched structure makes unique contributions to affinity and/or specificity resulting in a 200 nM affinity ligand that efficiently immunoprecipitates Akt from cancer cell lysates and labels Akt in fixed cells. Our use of a small molecule to preinhibit Akt prior to screening resulted in low micromolar inhibitory potency and an allosteric mode of inhibition, which is evidenced through a series of competitive enzyme kinetic assays. To demonstrate the efficiency and selectivity of the protein-templated in situ click reaction, we developed a novel QPCR-based methodology that enabled a quantitative assessment of its yield. These results point to the potential for iterative in situ click chemistry to generate potent, synthetically accessible antibody replacements with novel inhibitory properties

Doctor of Philosophy

dissertationAcute lymphoblastic leukemia (ALL) is the most common cancer of childhood, with approximately 2000 cases diagnosed annually in the US. Although cure rates for childhood ALL are currently ~80%, T-cell ALL (T-ALL) is still more difficult to treat than B-cell ALL, requiring harsher treatments with concomitant harsher side effects. The goal of this study was to identify more targeted therapies for treating T-ALL with the intent of reducing harsh treatment side effects, thus preserving both lives and long-term quality of life. To meet this goal, 26,400 compounds from the ChemBridge library were screened utilizing zebrafish larvae since they have the combined attributes of vertebrate physiology and small size. The transgenic lck:eGFP zebrafish line with T-cell specific GFP was chosen since compounds which eliminate immature T-cells in the thymus might also eliminate developmentally arrested leukemic blasts. The screen identified five "hit" compounds that cause reduction in GFP without sickening the larvae or causing general cell cycle effects. Of these five compounds, one compound, "Lenaldekar" (LDK), was effective in killing human Jurkat T-ALL without harming healthy lymphocytes. In vivo, LDK shows efficacy in treating leukemia in both zebrafish and mouse xenograft models of T-ALL without observable toxicity or endorgan damage. Furthermore, expanded leukemia testing showed that T-ALL, B-ALL, and CML are all largely LDK-sensitive, including most treatment-refractory relapsed Ph+ leukemias and primary patient samples. Moreover, some AML and multiple myeloma cell lines also show LDK sensitivity. Molecular characterization shows that LDK down-regulates the PI3K/AKT/mTOR (P/A/mT) pathway, which pathway is up-regulated in ~50% of T-ALL cases. Recent results suggest that LDK may achieve this effect via inactivation of the insulin-like growth factor 1 receptor (IGF1-R), which activates the P/A/mT pathway. In addition, LDK treatment elicits a second activity of G2/M arrest in most sensitive cell lines, which arrest appears to be independent of P/A/mT pathway inhibition. Future directions include identifying and modeling LDK's direct biochemical target(s) with the intent of utilizing structure-activity relationships to optimize LDK's chemical structure and efficacy. This study's ultimate goal is to bring LDK into clinical trials for the treatment of T-ALL in both monotherapy and combination therapy applications

Differentially activating the oncogenic kinase Akt1

The proto-oncogene Akt/protein kinase B plays a pivotal role in cell growth and survival. Phosphorylation of Akt at Thr308 and Ser473 activates the kinase following growth factor stimulation. Delineating specific role of each activation site in Akt1 on kinase activation, inhibition and substrate selection remain elusive. We designed a unique set of tools, relying on genetic code expansion with phosphoserine and in vivo enzymatic phosphorylation, to produce differentially phosphorylated Akt1 variants. We found that having both sites phosphorylated increased the apparent catalytic rate of the enzyme by 1500-fold relative to the unphosphorylated enzyme. This increment was mainly due to the phosphorylation of Thr308 but not Ser473 which was confirmed via live cell imaging. We further found that the traditional use of phosphomimetics was unable to mimic the effect of p-Thr308 in the test tube and in cells. Akt1 activity is also regulated via interactions between the kinase domain and the N-terminal auto-inhibitory pleckstrin homology (PH) domain. Using the same strategy, we produced Akt1 variants containing programmed phosphorylation to probe the interplay between Akt1 phosphorylation status and the auto-inhibitory function of the PH domain. Deletion of the PH domain increased the enzyme activity for all three Akt1 phospho-variants. For the doubly phosphorylated enzyme, deletion of the PH domain relieved auto-inhibition by 295-fold. The robustly active PH domain deleted enzyme variants were used in enzyme inhibition and substrate selectivity studies. We found that phosphorylation at Ser473 provided resistance to chemical inhibition by the Akt inhibitor Akti-1/2. Finally, we used both defined and randomized peptide libraries to map the substrate selectivity of singly and doubly phosphorylated Akt1 variants. The data revealed that each phospho-form of Akt1 has distinct substrate requirements. Surprisingly, phosphorylation of Ser473 in the context of a pAkt1T308 enzyme led to increased activity on some, but not all Akt1 substrates. We also verified a new Akt1 target as the terminal nucleotidyltransferase (germline development 2) Gld2. In conclusion, the site-specifically phosphorylated Akt1 variants that we produced enabled in characterizing phosphorylation-dependent activity, inhibition and substrate selectivity of the oncogenic kinase Akt1. Since phosphorylation status of Akt1 is used as a cancer biomarker, these variants can act as indispensable tools in further characterizing downstream oncogenic pathways and screening potential drug candidates

STRUCTURE BASED DRUG DESIGN OF HIGH AFFINITY KRAS INHIBITORS

RAS, one of the most well characterized membrane-associated small GTPases, is a notorious oncogene with \u3e15% of all tumors harboring RAS mutations. When RAS is mutated it becomes constitutively active sending cell growth, survival and proliferation into overdrive, which subsequently leads to cancer. Although, RAS has been aggressively targeted with drug design efforts for more than 30 years an FDA approved direct inhibitor has not yet been developed. There are three isoforms of RAS in cells; HRAS, NRAS and KRAS. We focused on KRAS since it is the most frequently mutated isoform in cancer. To identify novel non-covalent small molecules that bind to mutant KRAS, we conducted a high-throughput virtual screen of drug-like compounds against a previously characterized allosteric pocket on a molecular dynamics simulation-derived KRASG12D structure. The in silico predicted hits were then validated with a battery of cell-based and biophysical assays. Specifically, the hits effects on KRAS signaling, binding affinities for KRAS and mechanisms of activity were evaluated. We found two key hit compounds (i) a pyrazolopyrimidine based molecule compound 11 and (ii) a indazole based molecule compound M1. Compound 11 exhibited a monotonous dose-dependent inhibition of KRAS signaling, however compound M1 demonstrated a biphasic dose-depended effect. With the potential to elucidate the structure-activity relationships between these molecules and their unique structures we tested both in cell-based and vi biophysical assays. We found that compound 11 binds to KRAS with nanomolar affinity and completely abolishes CRaf binding in vitro, subsequently leading to a significant reduction in RAS dependent CRaf/ERK activation and son of sevenless (SOS) mediated nucleotide exchange. Moreover, treatment at low micromolar concentrations of compound 11 reduced cell proliferation in six cancer cell lines. Further, compound M1, binds to KRAS in NMR-based studies and both reduced and enhanced signaling in cell-based assays as indicated by western blotting. We attributed this to M1 enhancing and disrupting nucleotide exchange through different mechanisms. Additionally, we noted that M1 showed remarkable selectivity towards inhibiting proliferation of MiaPaCa-2 cells. We then screened a second small-molecule library based on M1 and generated additional hits which decreased mutant KRAS signaling. Likewise, we generated two derivatives of compound 11 which were tested and gave insights to critical functional groups. Combining our results with detailed structural analysis, we are able to describe key ligand-receptor interactions that correlate with activity. Thus, showing that our screening techniques were very successful at generating KRAS binders that have effects on signaling in cells. To our knowledge compound 11 is the first known nanomolar binder of KRAS that disrupts interaction with CRaf resulting in decreased p-ERK levels and cell proliferation. Therefore, compound 11 is a promising hit for the development of novel non-covalent KRAS inhibitors