GLUT1-INHIBITORS: SYNTHESIS AND ANALYSIS OF LIGAND-RECEPTOR INTERACTIONS

The family of glucose transporters (GLUTs) embraces a group of isoforms of trans-membrane channel-proteins which allows the entry of D-glucose into the cells. These isoforms differ for localization and for substrate affinity. Isoform GLUT-1 was the object of this thesis, since it was found to be overexpressed in carcinoma cells, and it is the main responsible for the entrance of D-glucose into the central nervous system. The over-expression of this protein on the tumor cells membranes allows this type of cells to accumulate and use a large amount of D-glucose and, consequently, to produce enough energy for their survival. In fact, tumor cells predominantly produce energy by a high rate of glycolysis, followed by lactic acid fermentation in the cytosol, rather than by a comparatively low rate of glycolysis followed by oxidation of pyruvate in mitochondria, such as that occurring in most normal cells. This metabolic switch is known as the Warburg Effect. Thus, selective inhibitors of GLUT1, may represent valid anticancer agents that selectively counteract tumor glycolysis. So far, several molecules have been reported to be able to block glucose uptake. Most of them are natural molecules such as flavonoids, flavones, chalcones, but there are also examples of synthetic molecules, such as tamoxifen, fulvestrant, 17-β-estradiol. The interesting thing is that the majority of these molecules are also active on estrogen receptor beta (ERβ). Based on this concept, some salycilaldoxime and salicylketoxime derivatives, that had been previously designed and synthetized as potential ERβ-ligands in the research group where I have carried out my thesis project, were also tested in cellular assays on GLUT-1, and some of them displayed good inhibitory activities on glucose uptake. The recent crystallization (resolution 2.9Å) of the xylose transporter of E. Coli, XyLE, coordinated with D-glucose provided a valid starting point for a more accurate analysis of human GLUT1 and to study the putative interaction modes of the most active salycilketoxime molecules and flavonoids.This transporter is formed by 492 amino acids and its folder was carried out with the homology model made with online server HHpred. This model showed that protein GLUT1 is formed by 12 transmembranes and 4 intracellular helices and both the N-terminus and C-terminus are located in the intracellular part of the protein. The aqueous channel seems to be divided into two parts by two residues that form a gate in the middle of the channel: Trp388 and His154. The homology model so obtained was inserted in a lipid bilayer of POPC 1-palmitoyl,2-oleoyl-sn-glycero-3-phosphocholine built with software VMD to simulate the biological environment. The resulting complex underwent a molecular dynamic simulation using AMBER 11, to remove all the constraints given by the crystallization process and to evaluate conformational changes that happened when the complex was heated. As the complex was refined, the analysis of the putative binding sites for D-glucose, for the most active ketoximes PGL12 and PGL14, and for flavonoid Myrcetin was carried out with docking programs GOLD and GLIDE. This analysis gave three putative binding sites: one situated in the extracellular portion of the channel, one situated in the middle of the channel and the last one located between the intracellular helices of the protein. The best binding pose for each ligand in each putative binding site was submitted to a molecular dynamic simulation to evaluate the interactions energy (using the AMBER 11 functionality MMPB(GB)SA) and the hydrogen bonds (using AMBER 11 functionality PTRAJ). The trajectories resulting from the dynamic process were also analysed with USCF CHIMERA to evaluate hydrophobic interaction. From the crossing of all these results, the best putative binding site for all the studied inhibitors was the intracellular site.A steered molecular dynamic simulation was also run to evaluate the putative pathway of D-glucose through the channel. Considering the pharmacophoric features required to have a good inhibitory activity on GLUT1, some substitutions were made on the starting hits PGL12 and PGL14 to evaluate how these changes might influence the inhibitory activity. My project was dedicated to study the introduction of various substituents in the phenolic ring. The synthesis of these analogues started from commercial product 3-bromophenol, which was carbamoylated on the hydroxyl group. Then, a remarkably regioselective ortho-lithiation process, followed by a quenching with hexachloroethane, produced the insertion of a chlorine atom in position 2. The corresponding 2-chloro-3-bromo-carbamoylphenol was hydrolysed under basic conditions, to obtain 3-bromo-2-chloro-phenol. This molecule was acetylated and than the acetyl group was transposed by a Fries rearrangement reaction to afford 3-bromo-2-chloro-6-acetylphenol. This molecule underwent a cross-coupling reaction under Suzuki conditions using different boronic-acids to obtain variously substituted ketone intermediates. The metoxy group, if presents, was deprotected with BBr3 in CH2Cl2 and the ketone group was finally transformed into the desired ketoxime

Approaches Towards the Inhibition of Anti-Apoptotic Proteins

Anti-apoptotic proteins play a fundamental role in cell survival. Under physiological conditions, such proteins trigger apoptosis in defective or damaged cells only; under pathological conditions, however, they can be dysregulated allowing the cells to survive despite being harmful. Considering the importance of anti-apoptotic proteins in many physio-pathological roles, their specific inhibition is an attractive strategy to develop safe therapeutics. This thesis describes the inhibition of two classes of anti-apoptotic proteins: 1) Inhibition of the anti-apoptotic protein CK2 to develop novel anti-cancer molecules targeting pockets outside the well-conserved ATP-binding site: -Using a Fragment-Based-Drug-Discovery (FBDD) approach twelve small molecule inhibitors of CK2 were developed. The lead molecule, 3l, inhibited the catalytic activity of CK2α by binding in the cryptic αD pocket with a Kd of 4 μM. 3l stopped proliferation of colorectal cancer cells with a GI50 of 10 μM and presented improved drug-like properties and selectivity compared to previously reported inhibitors. Remarkably, 3l has the potential to be developed into a potent and selective anticancer drug. -Using a combination of rational-based approach and peptide stapling, twenty-two conformationally-constrained peptides were generated to target the protein-protein interaction (PPI) of CK2 and affect its function. The lead peptide, P7-F1C5, presented a novel, highly-functionalised constraint that allowed the molecule to become cell-permeable, exert its anti-proliferative activity in cancerous cells, and to become resistant to serum proteases. P7-F1C5 is the first macromolecule reported in the literature that binds to CK2α with sub-micromolar affinity (Kd 150 nM), and that can act as a chemical probe for targeting the PPI of CK2. 2) Inhibition of the anti-apoptotic Bcl-2 proteins to dissect their role in platelet activation and apoptosis. Bcl-2 proteins regulate cell lifespan; however, their role in non-nucleated platelets is not fully understood. The elucidation of these pathways in platelets is crucial to the development of selective anti-platelet therapeutics. To this end, this thesis describes the development and the first application of twenty-seven BH3-only peptides in human platelets highlighting how peptides can provide an alternative to conventional methodologies to study PPIs in platelets. The most promising peptide, P9-F5C5, engaged the anti-apoptotic protein Bcl-xL with 26 nM affinity and reviled a new role for the protein Bim in platelet activation.Trinity Colleg

Antiproliferative oxime derivatives that inhibit glucose transporter 1 (GLUT1) in cancer cells

The Warburg effect, consisting in alterations of the glucose metabolism in cancer cells, where glucose mostly undergoes glycolysis with production of lactate, is currently being considered as one of the most intriguing hallmarks of cancer [1]. Therefore, the discovery of new agents able to block the glycolytic processes in tumor cells holds promise for developing relatively nontoxic anticancer treatments [2]. In terms of energy (ATP) production, glycolysis is dramatically less efficient than oxidative phosphorylation (OXPHOS). In fact, most normal cells rely on OXPHOS for glucose degradation, since they are generally well-oxygenated. On the contrary, invasive tumor tissues are often exposed to more-or-less transient hypoxia, which cannot guarantee the proper functioning of OXPHOS. Under these hypoxic conditions glycolysis leading to lactate production is mainly preferred, since it does not depend on oxygen availability. However, due to the lower efficiency of the glycolytic process, cancer cells commonly show a remarkably high glucose uptake, which is supported by the overexpression of the glucose transporters (GLUTs). GLUT1 is one of the most commonly transporters that are overexpressed by cancer cells and, therefore, represent a potential target for selectively hitting them [3], although only a very limited number of GLUT1-inhibitors have been reported so far [4]. On the basis of an analysis of the pharmacophoric features displayed by some previously reported GLUT1-inhibitors, we have identified a series of oxime derivatives [5] as potentially active on this transporter. A preliminary screening of these compounds in H1299 lung cancer cells demonstrated that some of them are able to effectively counteract glucose uptake and cell growth, displaying IC50 values in the low micromolar range. We have then developed a new computational model of GLUT1, which provided us with valuable clues about the possible binding site and the most important interactions occurring with some representative oxime derivatives and GLUT1. These indications may prove to be very valuable for the future development of novel potent and selective GLUT1-inhibitors. References: [1] Hanahan, D.; Weinberg, R. A. Cell 2011, 144, 646-674. [2] Granchi, C.; Minutolo, F. ChemMedChem 2012, 7, 1318-1350. [3] Rastogi, S.; Banerjee, S.; Chellappan, S.; Simon, G. R. Cancer Lett. 2007, 257, 244-251. [4] Liu, Y.; Cao, Y.; Zhang, W.; Bergmeier, S.; Qian, Y.; Akbar, H.; Colvin, R.; Ding, J.; Tong, L.; Wu, S.; Hines, J.; Chen, X. Mol. Cancer Ther. 2012, 11, 1672-1682, and references therein. [5] Minutolo, F.; Bertini, S.; Granchi, C.; Marchitiello, T.; Prota, G.; Rapposelli, S.; Tuccinardi, T.; Martinelli, A.; Gunther, J. R.; Carlson, K. E.; Katzenellenbogen, J. A.; Macchia, M. J. Med. Chem. 2009, 52, 858-867

Targeted covalent inhibitors of MDM2 using electrophile-bearing stapled peptides.

Herein, we describe the development of a novel staple with an electrophilic warhead to enable the generation of stapled peptide covalent inhibitors of the p53-MDM2 protein-protein interaction (PPI). The peptide developed showed complete and selective covalent binding resulting in potent inhibition of p53-MDM2 PPI.Royal Society, Trinity College, A*STAR (IAF-PP H17/01/a0/010

Downfalls of Chemical Probes Acting at the Kinase ATP-Site: CK2 as a Case Study.

Protein kinases are a large class of enzymes with numerous biological roles and many have been implicated in a vast array of diseases, including cancer and the novel coronavirus infection COVID-19. Thus, the development of chemical probes to selectively target each kinase is of great interest. Inhibition of protein kinases with ATP-competitive inhibitors has historically been the most widely used method. However, due to the highly conserved structures of ATP-sites, the identification of truly selective chemical probes is challenging. In this review, we use the Ser/Thr kinase CK2 as an example to highlight the historical challenges in effective and selective chemical probe development, alongside recent advances in the field and alternative strategies aiming to overcome these problems. The methods utilised for CK2 can be applied to an array of protein kinases to aid in the discovery of chemical probes to further understand each kinase's biology, with wide-reaching implications for drug development

Downfalls of Chemical Probes Acting at the Kinase ATP-Site: CK2 as a Case Study

Protein kinases are a large class of enzymes with numerous biological roles and many have been implicated in a vast array of diseases, including cancer and the novel coronavirus infection COVID-19. Thus, the development of chemical probes to selectively target each kinase is of great interest. Inhibition of protein kinases with ATP-competitive inhibitors has historically been the most widely used method. However, due to the highly conserved structures of ATP-sites, the identification of truly selective chemical probes is challenging. In this review, we use the Ser/Thr kinase CK2 as an example to highlight the historical challenges in effective and selective chemical probe development, alongside recent advances in the field and alternative strategies aiming to overcome these problems. The methods utilised for CK2 can be applied to an array of protein kinases to aid in the discovery of chemical probes to further understand each kinase’s biology, with wide-reaching implications for drug development

Downfalls of Chemical Probes Acting at the Kinase ATP-Site: CK2 as a Case Study.

Protein kinases are a large class of enzymes with numerous biological roles and many have been implicated in a vast array of diseases, including cancer and the novel coronavirus infection COVID-19. Thus, the development of chemical probes to selectively target each kinase is of great interest. Inhibition of protein kinases with ATP-competitive inhibitors has historically been the most widely used method. However, due to the highly conserved structures of ATP-sites, the identification of truly selective chemical probes is challenging. In this review, we use the Ser/Thr kinase CK2 as an example to highlight the historical challenges in effective and selective chemical probe development, alongside recent advances in the field and alternative strategies aiming to overcome these problems. The methods utilised for CK2 can be applied to an array of protein kinases to aid in the discovery of chemical probes to further understand each kinase's biology, with wide-reaching implications for drug development

Oxime-based inhibitors of glucose transporter 1 displaying antiproliferative effects in cancer cells

An analysis of the main pharmacophoric features present in the still limited number of inhibitors of glucose transporter GLUT1 led to the identification of new oxime-based inhibitors, which proved to be able to efficiently hinder glucose uptake and cell growth in H1299 lung cancer cells. The most important interactions of a representative inhibitor were indicated by a novel computational model of GLUT1, which was purposely developed to explain these results and to provide useful indications for the design and the development of new and more efficient GLUT1 inhibitors

Binding Mode and Induced Fit Predictions for Prospective Computational Drug Design

Computer-aided drug design plays an important role in medicinal chemistry to obtain insights into molecular mechanisms and to prioritize design strategies. Although significant improvement has been made in structure based design, it still remains a key challenge to accurately model and predict induced fit mechanisms. Most of the current available techniques either do not provide sufficient protein conformational sampling or are too computationally demanding to fit an industrial setting. The current study presents a systematic and exhaustive investigation of predicting binding modes for a range of systems using PELE (Protein Energy Landscape Exploration), an efficient and fast protein–ligand sampling algorithm. The systems analyzed (cytochrome P, kinase, protease, and nuclear hormone receptor) exhibit different complexities of ligand induced fit mechanisms and protein dynamics. The results are compared with results from classical molecular dynamics simulations and (induced fit) docking. This study shows that ligand induced side chain rearrangements and smaller to medium backbone movements are captured well in PELE. Large secondary structure rearrangements, however, remain challenging for all employed techniques. Relevant binding modes (ligand heavy atom RMSD < 1.0 Å) can be obtained by the PELE method within a few hours of simulation, positioning PELE as a tool applicable for rapid drug design cycles