Field of the invention.

The present invention relates to pharmaceutical compositions comprising novel compounds exhibiting anti-cancer activity; particularly, compounds that specifically inhibit the mutated GTPase KRAS4b, and that are useful for the effective treatment of pancreatic cancer; more particularly, the compounds of the invention act as stabilizers of the KRAS4b-PDe5 complex, decreasing the proliferation of cancer cells in pancreatic cancer and inducing cell death through apoptosis.

Background of the invention.

Pancreatic ductal adenocarcinoma (PD AC) is one of the most lethal malignant tumors, with a 5- year survival rate less than 6% [1] To increase the survival rate of pancreatic cancer patients, it is necessary to search for improved tumor markers for earlier diagnosis and for new molecular targets for drug development. In most cases, PDAC is initiated by mutation (codon G12) of the KRas4B GTPase, which has been shown to drive pancreatic neoplasia. KRas4B plays a critical role in human cancer cell biology, with mutationally activated KRas4B shown in 95% of PDAC [2] cases. Mouse models in which the KRas4B oncogene can be switched on and off have impressively demonstrated that continuous oncogenic KRas4B signaling is essential for both the progression and maintenance of PDAC [3] It is also became evident that sustained oncogenic KRas4B signaling is necessary for the growth and maintenance of metastatic lesions [4] KRas4B transport to the PDE-modulated plasma membrane provides the opportunity to interfere with the Ras pathway. PDE65 sustains the correct intracellular organization of KRas4B, and siRNA-mediated knockdown of the PDE65 gene leads to reduced Ras signaling and ERK phosphorylation [5] The PDE65 structure has a large hydrophobic cavity that accommodates the farnesyl group of Ras family GTPases. Some inhibitors have been developed to interfere with KRas4B localization. For example, Deltarasin at nanomolar concentrations disrupts the KRas4B-PDE65 interaction and induces relocalization of Ras family proteins to endomembranes. This inhibitor perturbs the KRas4B-PDE65 interaction, reduces proliferation and ERK1 phosphorylation in KRas4B- transformed pancreatic cancer cell lines, as well as tumor growth in xenografts of human pancreatic carcinoma cells [6] However, since PDE65 shuttles at least 37 other proteins [7], the function of other farnesylated proteins may be affected (http://www.innatedb.com) by Deltarasin. On the other hand, the farnesyl binding site of PDE5 is druggable, and Deltazinone 1, an analog of Deltarasin, has a higher affinity for the hydrophobic cavity of PDE5 [8] However, this drug fails to improve the ability of Deltarasin to induce apoptosis and inhibit ERK phosphorylation in KRas4B-dependent cell lines. This effect is attributed to a more efficient displacement of Deltazinone 1 of PDE65 by the activity of Arl2. Mice rapidly metabolize this compound, so it is not suitable for in vivo experiments [8] Hence, it is necessary to search for new mechanisms and compounds that can affect the molecular mechanisms of KRas4B regulation by PDE65.

In an attempt to provide effective treatments for pancreatic cancer, the following documents are mentioned:

Patent EP3055290B1 describes compounds capable of modulating the G12C mutation of KRAS, HRAS and NRAS proteins, which function as electrophiles capable of forming covalent bonds with the cysteine residue in position 12 of these mutated proteins, with the purpose to treat cancers associated with such mutations, such as colorectal or pancreatic cancer; the compounds described inhibit cell proliferation of cells with natural mutations in KRAS, however the effects are not observed in animal models affected with such cancers.

Patent RET2014112198 describes compositions with synergistic effect comprising a mixture of a phosphatidyllinositol-3 -kinase inhibitor (PI3K) and a mitogen-activated kinase protein (MEK) for the treatment of colorectal cancer, pancreatic cancer, lung non-small cells cancer, as well as melamoma, that have a mutation in KRAS.

Patent application WO2019071875A1 describes Rubiaceae-type cyclic peptides that function as autophagy inhibitors to inhibit KRAS mutations by promoting cell apoptosis, which are useful for the treatment of colorectal, lung and pancreatic cancer.

Patent CN109550044 describes a universal polypeptide vaccine comprising peptides with 6 mutations in KRAS (KRAS-G12R, KRAS-G12V, CDKN2A-H83Y, KRAS-Q61H, TP53-R248W y CDKN2A-P94L), which is useful for the prevention of pancreatic cancer since exhibiting good immunotherapeutic effects on pancreatic cancer.

Finally, patent application MX2018001095 describes the use of the monoclonal antibody Nimotuzumab for the treatment of patients with ductal pancreatic adenocarcinoma (PDAC) expressing KRAS, HRAS or wild NRAS, where such pancreatic cancer does not contain KRAS mutations at positions 12, 16, 61 and 154.

However, these solutions are not effective for the treatment of pancreatic cancer. Therefore, it is important to have more and better solutions and/or active compounds to provide effective treatments for pancreatic cancer. Brief description of the invention.

The GTPase KRas4B has been utilized as a principal target in the development of anticancer drugs. PDE65 transports this protein toward the plasma membrane, where it is released to activate various signaling pathways required for the initiation and maintenance of cancer. Therefore, identifying new small molecules that prevent activation of GTPase by stabilizing the KRas4B-PDE65 molecular complex is a practical strategy to fight against cancer.

The crystal structure of the KRas4B-PDE65 heterodimer was employed to locate possible specific binding sites at the protein-protein interface region. Virtual screening of Enamine-database compounds was performed on the located potential binding sites to identify ligands able to simultaneously bind to both KRas4B-PDE65 heterodimer. A molecular dynamics approach was used to estimate the binding free-energy of complex. Cell viability and cell apoptosis were measured by flow cytometry. G-LISA was used to measure Ras inactivation. Western blot was used to measure of ART and ERK activation. MIA PaCa-2 cells implanted subcutaneously into nude mice, were treated with compounds D14 or C22 and tumor volume were recorded.

According to the binding affinity estimation, compounds D14 and C22 stabilized the protein- protein interaction in the KRas4B-PDE65 complex based on in vitro evaluation of the 38 compounds showing antineoplastic activity against pancreatic MIA PaCa-2 cancer cells. In this invention, we further investigated the antineoplastic cellular properties of two of them, called as D14 and C22, which reduced the viability in the human pancreatic cancer MIA PaCa-2, PanC-l and BxPC-3 cells lines but not in the normal pancreatic hTERT-HPNE cell line. Compounds D14 and C22 induced cellular death via apoptosis. The evaluation of the biochemical and antineoplastic properties of D14 and C22 showed a significantly decreased Ras-GTP activity by 33% in MIA PaCa-2 cells and that D 14 decreased ART phosphorylation by 70% and ERK phosphorylation by 51%, while compound C22 modified AKT phosphorylation by 60% and ERK phosphorylation by 36%. In addition, compounds C22 and D14 significantly reduced tumor growth by 88.6% and 65.9%, respectively in a mouse xenograft model.

The pharmaceutical compositions comprising compounds D14 and C22 of the invention, are useful as therapeutic drugs for pancreatic ductal adenocarcinoma treatment.

Brief description of the figures.

Figure 1. Interaction of ligands that stabilize the KRas4B-PDE66 molecular complex in silico. The interprotein surface of interaction marked with gray color was used for the accomplishment of docking, being this the region with greater contact between KRas4B GTPase (pink and yellow) and PDE65 (blue). The compounds D14 (black) and C22 (aqua) were identified in the same inter-protein interactions sites.

Figure 2. Identification and evaluation on the cellular viability of the compounds with greater interaction energy on the interprotein region compared with the effect of Deltarasin.

Evaluation of 38 compounds at 200 mM, Deltarasin at 5 pM and DMSO as vehicle, on the MIA PaCa-2 and hTERT-HPNE cell lines.

Figure 3. In silico interaction of compounds D14 and C22 at the interface of the protein complex KRas4B-PDE66. (a) Binding poses of lead molecule D14. (b) Compound C22 interacting with the complex. Both compounds establish different contacts at the inter-protein region of the molecular complex.

Figure 4. Compounds D14 and C22 decrease the cellular viability of KRas4B-dependent pancreatic cancer cells (a and b) Effect of compounds D14 and C22 at various concentrations (0, 6.25, 12.5, 25, 50, 100 and 200 pM) for 72 h on MIA PaCa-2, BxPC-3 and PanC-l pancreatic cancer cells and on hTERT-HPNE normal pancreatic cells. The results show the viability inhibition of pancreatic cancer cell induced by the treatments (c) Morphological visualization of the MIA PaCa-2 and hTERT-HPNE cell lines treated at 200 pM of compounds D14 and C22. Figure 5. Compounds D14 and C22 induce apoptosis in the independent cell line of KRas4B. (a, b and c) Cell death of MIA PaCa-2 and hTERT-HPNE cells was determined by apoxin V / 7- AAD / CytoCalcein Violet and analyzed by flow cytometry (d) Compounds D14 and C22 induced apoptosis by being reflected in the presence of the phosphorylation of P53, Cytochrome-c, procaspase 3, and Smac/Diablo on the MIA PaCa-2 cell line of KRas4B-deoendent. The phosphorylation of these proteins was significantly compared with the control. The total protein extract (300 pg) was used for the apoptosis kit. The dots of the matrix were visualized according to the manufacturer's instructions. The intensity of each point was measured as described in "Material and methods". The upper panel provides matrix analysis of MIA PaCa-2 without compounds (control); the central panel shows the matrix analysis of the MIA PaCa-2 cell line treated with compound D14; the lower panel shows the matrix analysis of the MIA PaCa-2 cell line treated with the C22 compulsion (e) Normalized quantification graph shows the relative change of the phosphorylation differences with respect to the control.

Figure 6. Compounds D14 and C22 decrease the activation of ras in independent cells of KRas4B and the phosphorylation of AKT and ERK promoting the increased phosphorylation of p53. (a) Ras activation (Ras-GTP) decreases in the MIA PaCa-2 cell line treated with the compounds D14 (99.33 mM), C22 (137.5 pM) and Deltarasin at 5 pM for 3 h. (b) Representative immunoblot of whole protein extracts from MIA PaCa-2 treated with D14, C22 and deltarasin for 3h, detected phosphorylation inhibition of ART and ERK using GAPDH with load control (c and d) The quantitative results of the immunoblot of 3 independent studies are shown in graphs e. D14 and C22 compounds induced inhibition of the phosphorylation of ERK1 (T202/Y204), ERK2 (T185/Y187) in human MIA PaCa-2 cancer cell and promoted an increased in the P53 phosphorylation (e and f). The phosphorylation of those proteins were significantly inhibited compared with the control. Protein extract (300 pg) were used for human phospho- MAPK array kit. Array spots were visualized in accordance with the manufactures’s instructions. The intensity of each spot was measured as described in“Material and Methods”. The upper panel gives the array analysis from MIA PaCa-2 without compounds (control); the middle panel shows the array analysis of MIA PaCa-2 cell line treated with D14 compound; the lower panel shows the array analysis of MIA PaCa-2 cell line treated with the C22 compound (e) Normalized quantitation graph that shows the relative fold change of phosphorylation differences upon for control.

Figure 7. Compounds D14 and C22 decrease tumor growth in vivo (a) subcutaneous xenograft of cell MIA PaCa-2, the tumor volume was evaluated during the 15 days of treatment (b) The Weight of the mice was measured throughout the treatment. The mice were treated with the vehicle and compounds D14 and C22 at the two indicated in the scatter plot. NuNu mice were treated with vehicle (10% DMSO, 0.05% carboxy methyl cellulose and 0.02% Tween 80), D14 at 20 mg kg-l or C22 at 10 mg kg-l or 20 mg kg- 1 administered by intraperitoneal injection every two days (n = 10 for DMSO, n = 7 for D14 at 10 mg kg-l, n = 5 for C22 at 10 mg kg-l and n = 10 for C22 at 20 mg kg-l). Changes in tumor volume are given in relation to the initial volume before treatment (the dotted line indicates the initial size of the tumor) (c) hematoxylin-eosin and immunohistochemistry of the tumor sections. The 2 pm sections of the tumors were stained with hematoxylin-eosin (H & E) and analyzed by immunohistochemistry with antibodies directed against cytokeratin 19 (CK19) and Ki-67.

Detailed description of the invention.

Whit the intention to find better compounds that can affect the molecular mechanisms of KRas4B regulation by PDE65 to provide an efficient treatment to PDAC, according with the present invention we propose as a novel strategy for the stabilization of the KRas4B-PDE5 complex to prevent the molecular complex from dissociating to target in PDAC. As a consequence, KRas4B could not be released into the plasma membrane, thus inhibiting Ras signaling. The advantage of this strategy is that a small compound stabilizing the complex would exclusively recognize the KRas4B-PDE65 heterodimer without affecting other molecules that are regulated by PDE65. Our goal is to find small molecules directed to the interface residues between KRas4B and PDE65 through virtual screening in order to promote a more stable union between the targets of these molecules and evaluate their impact on KRas4B signaling in vitro and on a tumor model in vivo. We describe here the structure-based discovery of small molecules with high affinity for the KRas4B-PDE65 complex, their impact in the KRas4B signaling pathway, and the tumor growth inhibition in xenografted mice.

Pancreatic cancer is one of the most lethal cancers in the world, and it has been observed that the expression of mutated KRas4B is sufficient for the development and tumor growth in pancreatic cancer as well as for one-third of other types of cancers. Mutations at the l2th, l3th and 61 st residues in the small GTPase KRas4B limit the function of its molecular negative regulator RasGAP, so the activity of the GTPase KRas4B, as well as the signaling pathways dependent on this GTPase, remains constitutively active. The possibility of identifying compounds that bind directly to KRas4B and block its function has been studied for more than three decades; however, the efforts made to find compounds that inhibit the activity of mutated KRas4B activity have not been successful, which is mainly due to the lack of small-molecule binding sites on its KRas4B molecular target. Thus, in the present invention, the most important goal was to detect in silico compounds stabilizing the molecular complex KRas4B-PDE65, as well as to evaluate the antineoplastic properties of these compounds on the pancreatic cancer MIA PaCa-2 cell line, for the first time. For this purpose, we detected 38 compounds. In the present invention, we only reported the in vitro and in vivo evaluation of compounds D14 and C22.

The chemical formula of compound D14 is shown below:

while the chemical formula of compound C22 is shown below:

However, it is important to point out that we have deepened the study on other compounds identified in the present invention at the preclinical stage, and the results have been consistent and promising regarding specific antineoplastic properties, since these compounds not only affected the pancreatic cancer cells but also showed KRas4B dependent activity on colon cancer cells. These compounds and their pharmacological effects will be patented soon. Regarding the in silico analysis of compounds D14 and C22, it was possible to determine the binding free energy (AGbind) for the protein-protein and receptor-ligand complexes, and the binding of all the systems were found to be thermodynamically favorable. Table 3 illustrates that the primary energetic contribution to AGbmd for protein-protein and protein-ligand systems was guided by nonpolar contributions (DEhoh-poiar). In contrast, the polar contributions (AE _po iar) showed unfavorable energy influences on all the protein-protein complexes but not the protein-ligand contacts. Comparison among the different protein-protein systems demonstrates that in our computer simulations, C22 is somewhat more efficient in promoting the affinity between KRas4B and PDE65 than D14. Mutated KRas4B increased its affinity to PDE66 with respect to wild-type, the association of D14 to mutated KRas4B-PDE5 complex contributed to decrease the affinity of the mutated KRas4B- PDE5 complex, whereas an increased in the affinity is observed when C22 is bound to mutated heterodimer. In overall, comparison of results for wild-type and mutated systems point out that the two compounds are efficient increasing the protein-protein association of wild-type KRas4B- PDE65, complex, whereas that only C22 is able to increase the protein-protein association in wild- type and mutated KRas4B-PDE65 complex. Although the in silico results indicate that these compounds may bind to the KRas4B-PDE65 complex, increasing its interaction and thus affecting its function, we also demonstrate these effects experimentally. The results show that compounds D14 and C22 decrease the total Ras activity of (Ras-GTP) and directly impact the ART and ERK. Our experimental data suggest that compounds D14 and C22 impact cellular processes related to cell survival, cell cycle, protein synthesis and cell growth. The inhibition of the ART signaling pathway could explain the induction of apoptosis by compounds D14 and C22 in the MIA PaCa- 2 cell line, whereas the inhibition of ERK by compound D14 and C22 would influence transcriptional regulation signaling pathways and cell cycle regulation. Despite the differential effects of compounds D14 and C22, these compounds directly impact the phosphorylation status of at least two signaling pathways that have been reported as vital in pancreatic cancer cells and whose its constitutive activation is associated with a bad prognosis in patients with pancreatic cancer [25] On the other hand the D14 and C22 compounds promoted a differential activation of elements related with the apoptosis signaling pathways in the pancreatic cancer cells such as p53, procaspase 3, Smac/Diablo and cytochrome-c, in this sense was detected in the present invention that D 14 compound promoted an increase in the phosphorylation of XIAP protein, which has been reported that is an inhibitor of apoptosis mechanisms [24], with this information, we consider that C22 compound could be better than D14. The antineoplasic activity of D14 and C22 compounds detected in vitro also revealed its inhibitory activity against tumor growth in a xenograft murine model. In these trials, it is important to note that the mice did not exhibit weight loss during the treatment, suggesting that the compound does not have a toxic effect in animals. It will also be important to explore the combined effect of compounds D 14 and C22 and determine whether there is a synergistic antineoplastic effect between the two compounds on pancreatic cancer cells. It is also important to note that we have selected and deepened knowledge of the antineoplasic properties of other organic compounds presented in this invention, which have shown specific and improved antineoplasic potency against pancreatic cancer cells, as well as analogs of these compounds. These results will also be patented soon. All these data show the great potential antineoplasic properties of the compounds evaluated in the present invention.

For the first time, we describe two small molecules that stabilize the KRas4B-PDE65 molecular complex explored under in silico procedure. The antineoplastic evaluation of these compounds showed that they affected Ras activation pathways and tumor growth in xenografted mice. The antineoplastic activity was specifically against pancreatic cancer cells, as normal pancreatic cells were not affected. Compounds D14 and C22 present a new pharmacological alternative for suppressing the Ras signaling of pancreatic cancer cells and developing novel drugs against KRas4B -dependent pancreas cancer cells.

The results of the present invention show that the compositions described here are useful as a new therapeutic alternative for patients with pancreatic cancer; so it is part of the embodiments of the invention the use of compounds D14 and C22 as such or its pharmaceutically acceptable salts to obtain compositions for pharmaceutical use that include it.

Therefore, it is the object of the invention to provide pharmaceutical compositions comprising the compound D14 and/or C22, either together or separately, with pharmaceutically acceptable excipients suitable to be administered to the patient who requires it. It’s an embodiment of the invention the adaptation of the active principles to be used in pharmaceutical compositions for enteral, parenteral, and topical use, including inhalation. The patient's effective doses of the active ingredient shall also be adjusted in accordance with appropriate pre-clinical and clinical studies, but on the basis of the findings of the present invention.

Examples of pharmaceutically acceptable excipients accompanying the active ingredient of the invention for oral administration as tablets or capsules are namely: agents comprising thinners, binders, stabilizers, and loading agents; thickening agents such as povidone, microcrystalline cellulose, lactose, etc.; disintegrating agents such as cross-linked carboxymethylcellulose; surfactants such as sodium lauryl sulphate; lubricating or sliding agents such as magnesium stearate, colloidal silicon dioxide, etc., where such excipients may be formulated for preferably slow or prolonged release for a systemic effect.

Solutions can be prepared for intravenous or intraperitoneal administration of the active ingredient dissolved first in an organic solvent such as DMSO, ethanol, or dimethylformamide and subsequently in aqueous buffers such as PBS.

Special preference is given to pharmaceutical forms designed for local administration to the site of colorectal cancer, where liquid or solid compositions may be formulated, suitable for local administration, for example, from components compatible with that pharmaceutical form namely from a lipid or peptide nature, or peptidomimetics known in the state of the art as non- immunogenic, and which may preferably remain attached to the active principles of the invention to improve its bioavailability; propelling agents such as propane, butane, or permissible chlorofluorocarbons; pH regulators such as sulphuric acid; chelating agents such as EDTA. Active principles can also be formed into micronized particles contained in gelatine capsules or other forms known in the technical field, which help to release the active principle to its target site of action; e.g., through solid pharmaceutical forms such as tablets or pellets, including those formulated for prolonged release.

According to the present invention, the compositions described here can be obtained by combining the compounds D14 and/or C22 with pharmaceutically compatible vehicles known in the art, in corresponding quantities and/or concentrations as described here; and compounds known in the art can be included for obtaining such compositions. Also, the administration of such compositions can be made depending on the patient's conditions, which will determine the dose and frequency of administration necessary to achieve an effective treatment of the ailment in each particular case. The following examples are shown reflecting the methods by which it is feasible to reproduce the effects of the invention. These examples only have an illustrative character and do not limit the scope of the invention.

Example 1. Methodology.

Structure of the KRas4B-PDE5 complex. At the earliest stages of the present invention, the 3D structure of the KRas4B-PDE5 complex was unknown. Homology modeling of this complex was carried out using the Molecular Operating Environment package [9] employing as a template the previously reported RHEB-PDE65 crystallographic structure (PDB ID 3T5G). A large set of structures was modeled resulting from different side-chain rotamers of newly incorporated residues. The structure with the best packing index was subjected to a global energy -minimization analysis with the CHARMM27 force field to yield the final model. Later on, two different crystallographic structures of the KRas4B-PDE65 complex were reported and deposited at PDB ID: 5TAR and 5TB5. They differ in the KRas4B C terminus close to the farnesylation site, the former shows an ordered structure, while the latter has a partially disordered segment. We used both, reported 3D models and our model built, the 5TAR and 5TB5 structures to represent KRas4B-PDE5 intermolecular contacts and to guide the search for small organic compounds capable to simultaneously form interactions to both proteins, thus acting on the complex as molecular staples. Since in 3T5G structure RHEB is in contact to PDE65 we directly used solvent- exposed cavities composed of atoms from both proteins as targets for potential-ligand search. In the case of 5TAR, we used as targets the pockets close to the KRas4B-PDE65 interface as found in the crystallographic lattice. KRas4BGl2C mutant corresponding with the predominant mutation present in MIA PaCa cell line was modeled with PyMOL v0.99 (http://www.pymol.org).

Virtual Screenning. ENAMINE ' s Discovery Diversity Set database (DDS) containing 50,240 low molecular weight compounds was selected for virtual screening. The 2D structures was translated into 3D structures using MOE -Import Search. Hydrogens and partial charges were assigned according to MMFF94 force field. Strong acids and bases are deprotonated and protonated, respectively. In order to simulate de molecular flexibility shown in real systems, structural conform ers were constructed for each compound in DDS with MOE-Conformer Search and using a conformational energy cut-off of 3 kcal/mol respect to minimum energy conformer of each compound, according calculated to MMFF94 force field. The new database was then used for virtual screening. Potential binding sites, i.e. concave pockets at the protein-protein interface region in the KRas4B-PDE65 model and crystallographic structures, were identified with MOE- SiteFinder and CASTp server [10] Previous, all crystallographic water and other organic molecules were removed. Hydrogen atoms and partial charges were added to the KRas4B-PDE65 complex using the CHARMM27 force field. Virtual screening was carried out using MOE Dock function and setting the Alpha-Site -Triangle and the London dG as the methods to bias the orientation search on potential binding sites and docking scoring function, respectively. At least 10 000 different orientations or poses on potential binding sites were proved and evaluated for each conformer, and the ten best coupling score for each conformer were saved for further analysis. Finally, the KRas4B-PDE5-ligand complexes with the best binding energies and frequency were selected and evaluated with respect to the specific contacts that the compounds and the binding strengths, with preference given to the more polar compounds. Molecular Dynamics (MD) Simulations and Binding Free Energy Calculations. MD simulations of protein-protein and protein-ligand complexes were performed using AMBER 16 package [1 1] and the ffl4SB forcefield [12] Ligand charges for ligands and for no parameterized residues in proteins were determined using the AM1-BCC level and the general Amber force field (GAFF) [13] For protein-protein and protein-ligand complexes a 15 A and 12 A, respectively, a rectangular-shaped box of TIP3P water model [14] was applied to solvate the complex and Cl and Na ⁺ ions for protein-protein and protein-ligand systems were placed to neutralize the positive or negative charges around the complex models at pH 7. Before MD simulations, each molecular system was minimized through 3000 steps of steepest descent minimization followed by 3000 steps of conjugate gradient minimization. Then, systems were heated from 0 to 310 K during 500 picoseconds (ps) of MD with restrained positions under an NVT ensemble. Next, MD simulations for 500 ps, in an isothermal-isobaric ensemble (NPT), were carried out to adjust the solvent density, followed by 600 ps of constant pressure equilibration at 310K, using the SHAKE algorithm [15] on hydrogen atoms, and Langevin dynamics for temperature control. Equilibration runs were tailed by 100 ns-long MD simulations without position restraints, under periodic boundary conditions using an NPT ensemble at 310 K. The particle mesh Ewald method was utilized to describe the electrostatic term [16], and a 10 A cut-off was used for the van der Waals interactions. Temperature and pressure were preserved using the weak-coupling algorithm [17] with coupling constants tT and rP of 1.0 and 0.2 ps, respectively. The time step of the MD simulations was set to 2.0 femtoseconds, and the SHAKE algorithm [15] was used to constrain bond lengths at their equilibrium values. Coordinates were saved for analyses every 50 ps. AmberToolsl4 was used to examine the time-dependence of the root mean squared deviation (RMSD), and the radius of gyration (RG), as well as for clustering analysis to identify the most populated conformation during the equilibrated simulation time.

Calculation of binding free energies. Calculation of binding free energies was carried out using the MMGBSA approach [18, 19,20] provided in the Amberl6 suite [11] 500 snapshots were chosen at time intervals of 100 ps from the last 50 ns of MD simulations, using a salt concentration of 0.1 M and the Generalized Born (GB) implicit solvent model [21] The binding free energy of protein-protein and protein-ligand systems was determined as follows:

AGbind ⁼ G ^COmplex - G ^recept0r - G ^ligand

AGbind ⁼ AEMM + AGsolvation- TAS

wherein AEMM represents the total energy of the molecular mechanical force field that includes the electrostatic (AEeie) and van der Waals (AEvdw) interaction energies. AGsolvation signifies the desolvation free energy price upon complex formation, estimated from GB implicit model and solvent-accessible surface area (SASA) calculation that yield AGcic/soi and AGnpoi/soi. Whilst, -TAS is the solute entropy rising from structural changes that occur in the degrees of freedom of the free solutes and when are forming the protein-protein or protein-ligand complex.

Reagents. Small organic compounds identified by virtual screening were purchased from ENAMINE (www.enamine.net) (Kyiv, ETkraine), the compounds were dissolved in DMSO 1.5% (SIGMA-ALDRIHC, catalog No. 276855-1L). Deltarasin (hydrochloride) (Cayman Chemical, catalog No. 1440898-82-7).

Cell Culture. Human pancreatic cancer cell line, MIA PaCa-2, PanC-l, BxPC-3 and human pancreatic cell line, hTERT-HPNE, were obtained from the American Type Culture Collection (ATCC; Manassas, VA). Cell lines were grown as monolayers in the specific medium suggest by the ATCC.

Cell viability Assay. Cell lines were seeded at a density of 30,000 cells per well in a 96-well microtiter plate in growth medium and allowed to adhere 24 h. Then, they were treated with 200 mM of each of the 38 compounds. Cell proliferation was assessed every 24 h during 3 days. Cell viability was determined by MTT (MTT Cell Proliferation Assay ATCC ^{0 I0 I0K} ), by adding 10 pL of MTT per well, in dark conditions and incubated for 4 h. To solubilize the formazan crystals, 100 pL of acid isopropanol (50 mL of Triton X-100, 4 mL of HC1, 446 mL of isopropanol) was added, stirred continuously at room temperature and darkness for 3-4 h. The absorbance was measured in a spectrophotometer (Infinite F500 TEC AN) at a wavelength of 570 nm. Each concentration was evaluated in triplicate, the solvent of the fractions and the untreated cells were taken as negative controls. The data are presented as the average percentage of proliferation and the standard deviation of the mean.

IC50 determination. Cell lines were seeded at a density of 20,000 cells per well in a 96-well microtiter plate in growth medium and allowed to adhere 24 h. Following the treatment with 200, 100, 50, 25, 12.5 and 6.25 pL of D14 and C22, cell viability was assessed for 5 days each 24 h. At the end of treatment, cell viability was determined by the CellTiter-Glo Luminescent Cell Viability Assay (Promega, catalog No. G7573). The dose-response curve was used to calculate the concentration of drug resulting in 50% inhibition of cell viability (IC50). The assays were repeated 5 times. Apoptosis Assay. Approximately 5 X 10 ⁵ cells were seeded in 6-well plates for 24 h. Then, cells were treated with an ICso concentration of D14 and C22 compounds and vehicle for 24 h. Cells were harvested with 0.25% trypsin, washed with phosphate buffered saline (PBS), and collected together by centrifugation. Apoptosis was determined using the Apoptosis/Necrosis Detection kit (Abeam, catalog No. ab 176749, Cambridge, England) according to the manufacturer’s instructions and analyzed by flow cytometer on a FACSCalibur instrument (BD Biosciences) followed by data analysis using FlowJo software (Tree Star Inc). All experiments were performed in triplicate. Proteome Profiler Apoptosis Array (R&D Systems: ARY009) were used to evaluated the activity of D14 and C22 compounds on the MIA-PaCa-2 cancer cells to determine the signaling pathways associated with cell death via Kras4B inhibition, which were done following the manufacturer’s instructions.

Ras Activation assay. The inactivation of Ras by D14 and C22 was determined using a G-LISA Ras activation assay kit (Cytoskeleton, catalog No. # BK131). The cells were cultured in serum- starved for 16 h and pre-treated with the D 14 and C22 to 99.3 mM and 137.5 pM respectively for 1 h or Deltarasin to 5 pM for 3 h. Subsequently, the cells were stimulated with the epidermal growth factor (EGF) (100 ng/mL) for 10 min. Lysates (1 mg/ml) were added to 96-well plates coated with Ras GTP -binding protein (Raf-RBD), following the manufacturer’s instructions. Experiments for each cell type were repeated three times.

Western blot. The cells were serum-starved for 16 h and pre-treated with D14: 99.3 pM or C22: 137.5 pM for 1 h or Deltarasin: 5 pM for 3 h. After pre-treatment, cells were stimulated with EGF at 100 ng/mL for 10 min. Whole-cell extracts were obtained by lysis of the Mia PaCa-2 cells in lysis buffer [20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1 mM NaVCh, 1 mM NaF, 10 mM b-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, and 1.2 mg/ml complete™ Lysis-M (Roche, Mannheim Germany) protease inhibitor cocktail]. The protein extracts were forced through a 22-gauge needle 10 times and centrifuged for 10 min at 14,000 rpm at 4°C, and the protein concentration was determined by the Pierce™ BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, EISA). Approximately 25 pg of protein was separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Then it was incubated with the following primary antibodies: Total ERK (Cell Signaling-9102; 1 : 1000), pERK (Cell Signaling- 9101; 1 : 1000), Total ART (Cell Signaling-9272 1 : 1000), pAKT(Cell Signaling-4060 1 : 1000), and anti-GAPDH (Gene Tex-GTXl00l l8 1 : 100,000). Immunodetection was performed using a ChemiDoc™ Imaging Systems (BIO-RAD). Densitometry analysis was performed using the software ImageJ version 1.45 (National Institute of Health, USA).

MAPK activation profiling. Cells were rinsed with cold PBS and immediately lysed in buffer supplemented with 4xcOmplete ^™ EDTA-free Ultra Protease Inhibitor Cocktail (Sigma-Aldrich) and lxPhosSTOP ^™ (Sigma-Aldrich) at 4°C for 30 min. Following centrifugation at l4,000xg for 5 min, supernatants were transferred into a clean tube and protein concentrations were determined using the Precision Red Advanced Protein Assay (Cytoskeleton, Inc. ADV02-A). Lysates were diluted and analyzed using the Human Phospho-MAPK Arrays (Proteome Profiler; R&D Systems; Minneapolis, MN, USA) according to the manufacturer ^' s instructions. Nitrocellulose membranes were scanned using a ChemiDoc™ Imaging Systems (BIO-RAD Laboratories, Inc.).

Treatment of Subcutaneous Pancreatic Carcinoma Xenografts. Male immune-deficient Nu/Nu nude mice at 6 weeks of age (CINVESTAV, Mexico) were maintained in pathogen-free conditions with irradiated chow. The animals were subcutaneously injected in the torso with 5 x 10 ⁶ MIA PaCa-2 cells per tumor in 0.1 ml of sterile phosphate-buffered saline. When MIA PaCa-2 cells reached palpable tumors (>l00mm ³ ), mice were divided randomly into three groups receiving vehicle (10% DMSO, 0,05% Carboxy Methyl Cellulose and 0,02% Tween 80 in PBS) (h=10) or D14 at 20 mg kg ¹ (n=7) and C22 at 10 mg kg ¹ (n=5) and 20 mg kg ¹ (h=10) administered by intra- peritoneal injection three times for week. Body weight was measured once a week, whereas tumors were measured twice weekly. Tumor sizes were calculated by the following formula: [(length x width ² )/2 in mm.

Immunohistochemical staining of xenograft tumors. One day after the last treatment, the mice were sacrificed by C02 chamber and the xenograft tumors were resected, fixed in 4% buffered formalin and embedded in paraffin. The tumors were cut with a microtome obtaining 2 pm slices. For hematoxylin and eosin (H & E) staining, the tissues were deparaffmized in xylene, hydrated in dehydrated alcohol starting from absolute ethanol to distilled water, stained for 2 minutes with Harris Hematoxylin, decolorized with 0.5% acid alcohol and fixing the color in lithium carbonate for 1 minute, washed in distilled water, in 96% ethanol and stained with Sigma Eosin, washed and dehydrated in gradual alcohol changes until absolute alcohol was reached, allowed to dry at room temperature, mounted and observed, to identify the site of the injury. For immunohistochemical staining, the tissues were deparaffmized in xylene, hydrated in depleted alcohols starting from absolute ethanol to distilled water, the epitopes were unmasked with 10 mM Buffer Citrate of pH 6.03 in the Tender Cocker, washed with PBS of pH 7.4, endogenous peroxidase was blocked with 0.9% H2O2 for 15 min, the block with 3% BSA for lh, the antibodies Ki-67 (BIOCARE MEDICAL API 3156 AA) and CK 19 (GENETEX GTX110414) was diluted with 1% PBS and 1% BSA, the primary antibody was incubated at room temperature for 40min, washed with PBS for 3min, incubated with the biotinylated secondary antibody for 20min at room temperature, washed with PBS for 3min, incubated with streptavidin for l5min, washes with PBS for 3 min, reactions were revealed with 4% diaminobenzidine (DAB) monitored each reaction under a microscope, were counterstained with Harry's Hematoxylin 30 seconds, they were washed with distilled water, dehydrated in gradual changes of ethanol from distilled water to absolute Ethanol, allowed to dry at room temperature, mounted and observed.

Statistical Analysis. The statistical significances of the differences among the data were determined by Tukey’s multiple comparisons test, using GraphPad Prism ^® 6 software (San Diego, CA, USA) when appropriate. P<0.05 was considered statistically significant. Values are presented as the means ± s.e.m. (standard error of the mean).

Example 2. Interface of KRas4B-PDE66 complex. The primary and natural interaction between KRas4B and PDE65 is through the famesyl group attached to the C terminus of the former [22] Our strategy does not consist in compete with the farnesyl binding site in PDE65 to destabilize the complex, but on the contrary, to strengthen their binding through the design of a small drug-like ligand able to simultaneously bind to both molecules, in the interface of the complex, acting as a molecular staple. Binding of this type of ligand would reduce the dissociation rate of KRas4B and PDE65, and increase the affinity constant, thus affecting their functional role. To identify this type of compounds, we used virtual screening on the KRas4B-PDE65 complex obtained from crystallographic data (PDB ID 5TAR25). The protein-protein interface area consists of 1900 Ά2, with 12 interchain hydrogen bonds and 115 non-bonded contacts. We directed the molecular docking efforts to the groove formed around the interchain contact of the complex (Fig. 1). The surface of this groove was explored by MOE SiteFinder where we identified several binding pockets composed of atoms of both proteins with a potential capacity of binding drug-like compounds.

Example 3. Virtual screening and docking interaction. The database of conformers prepared from ENAMINE diversity dataset (50,240 compounds) was used for docking studies against the interfacial groove of KRas4B-PDE65 complex. The docking results were sorted and analyzed by binding score, frequency of obtaining similar poses, type of interactions, and presence of ligand contacts with both KRas4B and PDE65 proteins. We gathered a group of 38 compounds fulfilling all requirements, 35 of them with optimal docking scores between -13.4 and -16.6 (Table 1).

Table 1. Potential candidates to stabilize the KRas4B-PDE66 complex. Results obtained from the virtual screening analysis. Frequency stands for the number of times a similar pose was obtained from different starting conditions during our docking procedure.

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All 38 compounds were acquired from ENAMINE for in vitro assays (Fig. 2). Also, we selected two of the compounds for further computational and binding-energy characterization by molecular dynamics. The choice of these compounds was based on a compromise of the different requirements using in the original selection, followed by visual inspection of the docked ligands on their interfacial binding-sites of the KRas4B-PDE65 complex to form specific contacts to both proteins. We selected D14 as the compound with the highest molecular weight in the set, and thus with more atoms to be in potential contact to the proteic complex. Also, we chose C22 as a representative compound of many of the poses obtained from docking with the whole set of compounds (Figs. 1 and 3). D14 showed a coupling of -15.2. This molecule can have eleven hydrogen bonds with KRas4B: Glyl5, Serl7, Glu3 l, Glu37, Asp38, Asp57, Gly60, Metl69, Lysl77 and Lysl79, while PDE65 has two hydrogen bonds: Leul05 and Glul07; presents an ionic bond with Asp57 of KRas4B; and six pi bonds with KRas4B: Serl7, Val29, Tyr32, Ile36, Gly60 and Serl8l and a pi bond with Glul07 of PDE65. Similarly, C22 has a score of -13.9. This molecule has six hydrogen bonds with KRas4B: Serl7, Asp30, Metl69, Aspl72, Lysl77 and Lysl78, while PDE65 has a hydrogen bond with Glul07; three pi bonds with KRas4B: Val29, Glyl73 and Lysl78 and a pi bond with Glul07 of PDE65 (Table 2, Fig. 3). These two compounds share a hydrogen bond with PDE6d in Glul07, three hydrogen bonds with KRas4B in Serl7, Metl69 and lysl77 and a pi bond with KRas4B in Val29 (Table 2 and Fig. 3). Piperazine and acetamide groups of D14 are essential to interact with the molecular complex. In the case of the C22 compound, benzamide, the amino group, and are important in the interaction with the complex. These regions generated strong bonds, according to the results obtained in silico.

Table 2. Useful compounds to stabilize the KRas4B-PDE66 complex. Results obtained from the virtual selection analysis in heterodimeric crystallographic complex.

Table 2 (continue)

Example 4. Molecular dynamics and free energy calculations. Since all atoms of the receptor complex remained fixed during the docking procedure, we performed all-atom molecular dynamics (MD) simulations to introduce the effect of ligands on the heterodimeric complex and estimate the stability of the binding of both C22 and D14 on the native KRas4B-PDE65 complex and containing G12C mutation in KRas4B. The results from these simulations allowed us to calculate the binding free-energy of each ligand to the complex, as well as the effect on KRas4B- PDE65 binding in the presence and absence of the ligands (Table 3).

Table 3. Binding free energy components of protein-protein and protein-ligand complexes

(in kcal/mol units).

Binding free energies and individual energy terms of complexes starting from docked conformations (kcal/mol).

The polar (AEpolar=AEele + AGele,sol) and non-polar (AEnon-polar=AEvwd + AGnpol,sol) contributions are shown. All the energies are averaged over 500 snapshots at time intervals of 100 ps from the last 50 ns-long MD simulations and are in kcal/mol (± standard error of the mean).

Deviations from the starting structure (measured as RMSD) and increases in the molecular size (measured by the radius of gyration, RG) were calculated on snapshots from the MD to determine the equilibrium conditions in the simulations. This step is necessary to perform confident structural and energetic analysis of the protein-protein complex and the ligand-complex systems. RMSD analysis demonstrated that the native and mutant KRas4B-PDE65, KRas4B-PDE65-Dl4 and KRas4B-PDE65-C22 systems reached equilibrated RMSD fluctuations during the first 10 to 50 ns with RMSD values that oscillated between 2.6 to 4.2 A (Table 4). RG analysis showed that the systems mentioned above reached equilibrated radius of gyration fluctuations after 20 to 50 ns with RG values that fluctuated between 22.2 to 22.8 A (Table 4). Correspondingly, RMSD analysis of the PDE65-ligand systems showed that systems reached an equilibrium fluctuation at 10 and 50 ns for PDE65-C22 and PDE65-D14, with average RMSD values of 1.9 ± 0.3 and 1.3 ± 0.2, respectively. RG analysis of the PDE65-C22 and PDE65-D14 systems demonstrated that they reached equilibrated RG oscillations at 50 and 20 ns, respectively, with average RG values of 16.4 ± 0.1 and 16.2 ± 0.1, respectively (Table 4). RMSD and RG analysis of protein-protein and protein- ligand systems shows that despite the differences in simulation time required to reach stable RMSD and RG values, all the systems finally reached equilibrium. Therefore, based on this structural performance, subsequent clustering analysis and binding free energy calculations were performed while excluding the first 50 ns from the lOO-ns-long MD simulations (Table 3).

Table 4. Average geometrical values (A) over the last 50 ns of 100-ns-long MD simulations

(± standard deviation).

Example 5. D14 and C22 compounds decrease the cellular viability of pancreatic cancer cells.

A cell viability assay was performed to determine whether compounds D14 and C22 have effects on cell viability or show cytotoxic effects. These compounds were tested on the hTERT-HPNE and MIA PaCa-2 cell lines at 200 mM, using Deltarasin (a PDE65 inhibitor) as a positive control at 5 pM. After 72 h of incubation, we observed by microscopic analysis of the treated cell lines showed again that these compounds have relatively high activity on the MIA PaCa-2 cell line without affecting the cellular hTERT-HPNE in terms of their growth and activity, such as cell morphology, proliferation, and the amount of living cells up to 72 h (Fig. 4c). D14 caused MIA PaCa-2 cells to peel off the dish surface, and their morphology did not change into a spherical shape. In the hTERT-HPNE cell line, cell detachment did not occur, and no drastic change in cell morphology was observed with respect to DMSO. C22 caused similar damage to D14 in MIA PaCa-2 cells (Fig. 4c). However, the hTERT-HPNE cell line showed cellular detachment and changes in morphology (Fig. 4c). Deltarasin affected the MIA PaCa-2 cell line, presenting round cells and less cellular detachment than compounds D14 and C22. In the hTERT-HPNE cell line treated with Deltarasin, there was more cellular detachment and spherical morphology than in the cells receiving other treatments, possible due to the no specificity of Deltarasin, therefore is possible that other proteins are being affected through a molecular mechanism up to now unknown by the Deltarasin. This effect was also verified by a cell viability assay in MIA PaCa-2 and hTERT- HPNE cells in the presence of different concentrations of D14 and C22 (6.25-200 mM) and DMSO treatments were followed for 5 days and read every 24 h. A dose-response effect was observed (Fig. 4a) with each treatment, and both compounds affected significantly the cell visibility of pancreatic cancer cell line MIA PaCa-2 more than the noncancerous pancreatic cell line hTERT- HPNE. (Figs. 4a and 4b). Also were evaluated both D14 and C22 compounds on cellular viability of BxPC-3 and PanC-l pancreatic cancer cells lines, detecting that both compounds affected the cell viability these pancreatic cancer cells. The ICso values of compounds D14 and C22 on hTERT- HPNE cell line were 431.1 mM and 649.9 mM respectively, while ICsoto MIA PaCa-2, were 99.33 mM and 137.5 pM; to BxPC-3 were 252.85 pM and 97.88 pM; and PanC-l cell lines were 105 pM and 108.5 pM. These results suggest that these compounds may affect the cell viability of other pancreatic cancer cell lines specifically.

Example 6. D14 and C22 compounds induce apoptosis on cancer pancreatic cells lines. One of the objectives proposed in the present invention was to determine the type cell death produced by D14 and C22 compounds in the hTERT-HPNE and MIA PaCa-2 cells lines, in this sense we used two different experimental strategies, one of them was using the Apoptosis/Necrosis Detection kit analyzed by flow cytometry which allows to detect the double labeling of apoxin-V and 7-Aminoactinomycin D (7-AAD). The second was the Human Apoptosis Array kit to determine the impact of D14 and C22 compounds on phosphorylation of different elements associated with the signaling pathways of cell death.

Flow cytometry analysis of stained cells showed that compound D14 promotes cellular death by 53.8% apoptosis and 4.69% necrosis and that compound C22 promotes cellular death by 31.55% apoptosis and 2.81% necrosis, while compounds D14 and C22 caused no cell damage the normal pancreatic hTERT-HPNE cells, with 91.2% and 89.8% cellular viability detected in normal pancreatic cells treated with D14 and C22 compounds, respectively (Figs. 4a, 4b and 4c). The results analysis obtained using the Human Apoptosis Array allowed to detect that D14 and C22 compounds promoted an increase in the phosphorylation of the p53, S15, S46, and S392 residues, procaspase 3, Smac/Diablo and cytochrome-c (Figs. 5d and 5e). These results indicate that D14 and C22 compounds have the property to induce the proapoptotic proteins activation [23] However, D14 compound promoted the phosphorylation XIAP protein, which has been reported that is an inhibitor of apoptosis mechanisms [24] With this information, we consider that the C22 compound is better than D 14.

Example 7. D14 and C22 compounds decrease the Ras activity and inhibit the pAKT and pERK phosphorylation in pancreatic cancer cells. It has been reported that molecules downstream from KRas4B, such as the pAKT and pERK in pancreatic cancer cells, are related to the signaling pathways involved in survival and cell differentiation, and therefore, we decided to analyze and explore the activity of compounds D14 and C22 on these signaling pathways, as well as on KRas4B, and to compare this activity to that of the Deltarasin in the MIA PaCa-2 cell line. At this point, the evaluation of D14 and C22 and Deltarasin compounds showed that total Ras activation, as indicated in methods performed on the MIA PaCa-2 cell line, was reduced by approximately 33% (Fig. 6a). Treatments with the D14 compound also showed a decrease of 70% in ART phosphorylation and 51% in ERK phosphorylation (Figs. 6b and 6c), while compound C22 changed ART phosphorylation by 60% and ERK phosphorylation by 36% (Figs. 6b and 6d). Deltarasin decreased AKT phosphorylation by approximately 75% and ERK phosphorylation by 54% (Figs. 6b, 6c and 6d). Also a phosphor-MAPK array kit was used to explore the possible mechanism of D14 and C22 compounds induced cell viability inhibition we detected that three members of the MAPK pathway were involved in those effects such as pERK, pAKT, which showed a reduction in expression of phosphorylated forms of these enzymes, also was detected an increase in the phosphorylated form of P53 (S46) protein (Figs. 6e and 6f). These data indicate that compounds D14 and C22 negatively impacted the activation of the KRas signaling pathways in the MIA PaCa-2 cancer cell line.

Example 8. Compounds D14 and C22 inhibit tumor growth in a pancreatic cancer xenograft mouse model. To evaluate the antitumor activity of compounds D14 and C22, Nu/Nu mice were inoculated subcutaneously with the pancreatic cancer cell line MIA PaCa-2, and tumor growth was monitored. The different treatments were administered intraperitoneally (i.p.) three times per week with a total of 7 injections in each mouse. Different doses (10 and 20 mg kg ¹ ) were tested (Fig. 7a). The results showed a tumor reduction of 88.6% and 65.9% in mice treated with compounds C22 and D14, respectively. The highest effect on tumor size decrease was observed at a dose of 20 mg kg ¹ of compound C22, and compared to that of DMSO (control), this effect increased on the last day (Fig. 7a). Moreover, compared to the treated mice, no mouse treated only with vehicle showed a reduction in tumor size. The treatment with compounds D14 and C12 did not cause weight loss in mice, and those that were treated lost less than 5% of their total body weight (Fig. 7b). Deltarasin causes a 15% weight decrease in mice during the first two days of treatment (data not shown). The histopathological analysis of in vivo evaluation of D14 and C22 compounds (Table 5), allowed to determine that these compounds reduced the presence of human pancreatic cancer cells in 60% and 70% respectively (Fig. 7c).

Table 5. Summary of histopathological analysis of in vivo evaluation of the effect of compounds D14 and C22 on tumor growth in nude mice.

References.

1. Siegel R, et.al. CA Cancer J Clin. 2014; 64:9-29.

2. Moskaluk C.A, et.al. Cancer Res. l997;57: 2140-2143.

3. Collins M.A, et.al. J Clin Invest. 2012; 122:639-653.

4. Collins M.A, et.al. PLoS One. 20l2;7: e49707.

5. Chandra A, et.al. Nature Cell Biology. 2012; 14:329-329.

6. Zimmermann G, et.al. Nature. 2013;497:638-642.

7. Spiegel J, et.al. Nat Chem Biol. 2014;10:613-622.

8. Papke B, et.al. Nat Commun. 20l6;7:l 1360.

9. Vilar S, et.al. Curr Top Med Chem. 2008;8: 1555-1572.

10. Liang J, et.al. Protein Sci. 1998;7: 1884-1897.

11. Case D. A, et.al. J Comput Chem. 2005;26: 1668-1688.

12. Duan Y, J Comput Chem. 2003;24: 1999-2012.

13. Wang J, et.al. J Comput Chem. 2004; 25: 1157-1174. Erratum in: J Comput Chem. 2005;26: l l4.

14. Jorgensen W.L, et.al. J Chem Phys. 1983;79:926-935.

15. Van Gunsteren W.F, et.al. Mol. Phys. 1977;34: 1311-1327.

16. Darden T, J. Chem. Phys. 1993;98: 10089-10092

17. Berendsen H. J.C, et.al. J. Chem. Phys. 1984;81 :3684-3690.

18. Miller B.R 3rd, et.al. J Chem Theory Comput. 2012;8:3314-3321.

19. Gohlke H, et.al. J Comput Chem. 2004;25:238-250.

20. Kollman P.A, et.al. Acc Chem Res. 2000;33:889-897.

21. Onufriev A, et.al. Proteins. 2004;55:383-394.

22. Dharmaiah S, et.al. Proc Natl Acad Sci U S A. 2016;113:E6766-E6775.

23. Cailleteau C, et.al. J Cell Biochem. 2009;l07(4):785-796.

24. Li S, et.al. Mol Clin Oncol. 20l3;l(2):305-308.

25. Chadha K.S, et.al. Ann Surg Oncol. 2006;13:933-939.