Discovery Of Potent Inhibitors Research Articles

Discovery of [1,2,3]Triazolo[4,5-c]quinoline Derivatives as a New Class of Ataxia-Telangiectasia Mutated Kinase Inhibitors.

Ataxia-telangiectasia mutated (ATM) is an atypical serine/threonine protein kinase which is implicated in the repair of DNA double-strand breaks. Numerous reports have shown that ATM inhibition is an attractive target for radiotherapy and chemotherapy sensitization. Herein we report a new series of ATM kinase inhibitors containing the 1H-[1,2,3]triazolo[4,5-c]quinoline scaffold, which was obtained by virtual screening, structural optimization, and structure-activity relationship studies. Among the inhibitors, A011 was one of the most potent, with an IC50 value of 1.0 nM against ATM. In colorectal cancer cells (SW620 and HCT116), A011 was able to inhibit activation of ATM signaling induced by irinotecan (CPT-11) and ionizing radiation and then increased the sensitivity of colorectal cancer cells to irinotecan and ionizing radiation through increasing G2/M arrest and inducing apoptosis. In the SW620 human colorectal adenocarcinoma tumor xenograft model, A011 sensitized SW620 to CPT-11 by inhibiting ATM activity. Collectively, this work has identified a promising lead in the discovery of potent inhibitors against ATM.

Structure-based assessment and druggability classification of protein–protein interaction sites

The featureless interface formed by protein–protein interactions (PPIs) is notorious for being considered a difficult and poorly druggable target. However, recent advances have shown PPIs to be druggable, with the discovery of potent inhibitors and stabilizers, some of which are currently being clinically tested and approved for medical use. In this study, we assess the druggability of 12 commonly targeted PPIs using the computational tool, SiteMap. After evaluating 320 crystal structures, we find that the PPI binding sites have a wide range of druggability scores. This can be attributed to the unique structural and physiochemical features that influence their ligand binding and concomitantly, their druggability predictions. We then use these features to propose a specific classification system suitable for assessing PPI targets based on their druggability scores and measured binding-affinity. Interestingly, this system was able to distinguish between different PPIs and correctly categorize them into four classes (i.e. very druggable, druggable, moderately druggable, and difficult). We also studied the effects of protein flexibility on the computed druggability scores and found that protein conformational changes accompanying ligand binding in ligand-bound structures result in higher protein druggability scores due to more favorable structural features. Finally, the drug-likeness of many published PPI inhibitors was studied where it was found that the vast majority of the 221 ligands considered here, including orally tested/marketed drugs, violate the currently acceptable limits of compound size and hydrophobicity parameters. This outcome, combined with the lack of correlation observed between druggability and drug-likeness, reinforces the need to redefine drug-likeness for PPI drugs. This work proposes a PPI-specific classification scheme that will assist researchers in assessing the druggability and identifying inhibitors of the PPI interface.

Assays Used for Discovering Small Molecule Inhibitors of YAP Activity in Cancers.

Simple SummaryCancer is a disease in which cells grow in an uncontrolled manner. This can be due to excessive cell proliferation or reduced cell death or a combination of the two. The Hippo signaling pathway, when misregulated, promotes excessive growth and cancer development by inducing uncontrolled cell proliferation and inhibiting cell death. This is achieved due to unregulated activity of the oncogenic effector of this pathway, YAP/TAZ. Therefore, it is critical to develop inhibitors to disrupt YAP activity in cancers. This article reviews the different types of assays that are used in development of small molecule inhibitors for YAP activity in cancers.YAP/TAZ are transcriptional coactivators that function as the key downstream effectors of Hippo signaling. They are commonly misregulated in most human cancers, which exhibit a higher level of expression and nuclear localization of YAP/TAZ, and display addiction to YAP-dependent transcription. In the nucleus, these coactivators associate with TEA domain transcription factors (TEAD1-4) to regulate the expression of genes that promote cell proliferation and inhibit cell death. Together, this results in an excessive growth of the cancerous tissue. Further, YAP/TAZ play a critical role in tumor metastasis and chemotherapy resistance by promoting cancer stem cell fate. Furthermore, they affect tumor immunity by promoting the expression of PD-L1. Thus, YAP plays an important role in multiple aspects of cancer biology and thus, provides a critical target for cancer therapy. Here we discuss various assays that are used for conducting high-throughput screens of small molecule libraries for hit identification, and subsequent hit validation for successful discovery of potent inhibitors of YAP-transcriptional activity. Furthermore, we describe the advantages and limitations of these assays.

Application of the LAD-LASSO as a dimensional reduction technique in the ANN-based QSAR study: Discovery of potent inhibitors using molecular docking simulation

In this study, the combination of the least absolute deviation-least absolute shrinkage and selection operator (LAD-LASSO) was introduced as a new variable selection method for the artificial neural network (ANN)-based quantitative structure-activity relationship (QSAR) studies. The biological activity of various chemical compounds was predicted using an ANN-based QSAR model combined with the efficient LAD-LASSO variable selection method. In this study, 3224 computed DRAGON descriptors were reduced to a smaller number using preprocessing methods. The descriptors with the most significant relevance to biological activities were chosen using the LAD-LASSO variable selection method. The selected descriptors were defined as ANN inputs and optimized the designed models. The biological activity of the test set compounds was predicted using the optimum ANN models. The coefficients of determination (R2) for the test data in the different datasets were equal to 0.87, 0.84, and 0.87. Also, the MSE value of the test set is equal to 0.13, 0.07, and 0.11, respectively. The high R2 and low MSE values demonstrate the good prediction ability of the constructed QSAR models. The applicability domain (AD) and Y-randomization test also proved the efficiency of the developed models. Finally, The performance of the QSAR model was evaluated by the identification of novel compounds with high potency. As a result, the weak structure of the dataset was identified and modified using the effect of selected descriptors on the biological activity, resulting in the establishment of new compounds with significant potency. The response value of the new suggested compounds was predicted using the optimum ANN models. Receptor-ligand interactions were extracted for all proposed compounds. The presence of different hydrophilic and hydrophobic interactions in the active site of the respective receptor indicates the high potential of suggested chemical compounds.

Molecular Dynamics Simulations Reveal the Interaction Fingerprint of Remdesivir Triphosphate Pivotal in Allosteric Regulation of SARS-CoV-2 RdRp.

The COVID-19 pandemic has now strengthened its hold on human health and coronavirus’ lethal existence does not seem to be going away soon. In this regard, the optimization of reported information for understanding the mechanistic insights that facilitate the discovery towards new therapeutics is an unmet need. Remdesivir (RDV) is established to inhibit RNA-dependent RNA polymerase (RdRp) in distinct viral families including Ebola and SARS-CoV-2. Therefore, its derivatives have the potential to become a broad-spectrum antiviral agent effective against many other RNA viruses. In this study, we performed comparative analysis of RDV, RMP (RDV monophosphate), and RTP (RDV triphosphate) to undermine the inhibition mechanism caused by RTP as it is a metabolically active form of RDV. The MD results indicated that RTP rearranges itself from its initial RMP-pose at the catalytic site towards NTP entry site, however, RMP stays at the catalytic site. The thermodynamic profiling and free-energy analysis revealed that a stable pose of RTP at NTP entrance site seems critical to modulate the inhibition as its binding strength improved more than its initial RMP-pose obtained from docking at the catalytic site. We found that RTP not only occupies the residues K545, R553, and R555, essential to escorting NTP towards the catalytic site, but also interacts with other residues D618, P620, K621, R624, K798, and R836 that contribute significantly to its stability. From the interaction fingerprinting it is revealed that the RTP interact with basic and conserved residues that are detrimental for the RdRp activity, therefore it possibly perturbed the catalytic site and blocked the NTP entrance site considerably. Overall, we are highlighting the RTP binding pose and key residues that render the SARS-CoV-2 RdRp inactive, paving crucial insights towards the discovery of potent inhibitors.

Constitutional Dynamic Inhibition/Activation of Carbonic Anhydrases.

In this review we consider one important member of the metalloenzymes family, the carbonic anhydrase (CA), involved in the treatment of several common diseases. Different approaches have emerged to regulate the activity of CA, mostly acting on the inner catalytic active site or outer microenvironment of the enzyme, leading to inhibition or activation of CA. In recent years, gradually increased attention has focused on the adoption of constitutional dynamic chemistry (CDC) strategies for the screening and discovery of potent inhibitors or activators. The participation of reversible covalent bonds enabled the enzyme itself to select the optimal ligands obtained from diverse building blocks with comparatively higher degree of variety, resulting in the fittest recognition of enzyme ligands from complex dynamic systems. With the increasing implementation of CDC for enzyme targets, it shows great potential for drug discovery or CO2 capture applications.

Discovery of potential inhibitors against New Delhi metallo-\u03b2-lactamase-1 from natural compounds: in silico-based methods

New Delhi metallo-β-lactamase variants and different types of metallo-β-lactamases have attracted enormous consideration for hydrolyzing almost all β-lactam antibiotics, which leads to multi drug resistance bacteria. Metallo-β-lactamases genes have disseminated in hospitals and all parts of the world and became a public health concern. There is no inhibitor for New Delhi metallo-β-lactamase-1 and other metallo-β-lactamases classes, so metallo-β-lactamases inhibitor drugs became an urgent need. In this study, multi-steps virtual screening was done over the NPASS database with 35,032 natural compounds. At first Captopril was extracted from 4EXS PDB code and use as a template for the first structural screening and 500 compounds obtained as hit compounds by molecular docking. Then the best ligand, i.e. NPC120633 was used as templet and 800 similar compounds were obtained. As a final point, ten compounds i.e. NPC171932, NPC100251, NPC18185, NPC98583, NPC112380, NPC471403, NPC471404, NPC472454, NPC473010 and NPC300657 had proper docking scores, and a 50 ns molecular dynamics simulation was performed for calculation binding free energy of each compound with New Delhi metallo-β-lactamase. Protein sequence alignment, 3D conformational alignment, pharmacophore modeling on all New Delhi metallo-β-lactamase variants and all types of metallo-β-lactamases were done. Quantum chemical perspective based on the fragment molecular orbital (FMO) method was performed to discover conserved and crucial residues in the catalytic activity of metallo-β-lactamases. These residues had similar 3D coordinates of spatial location in the 3D conformational alignment. So it is posibble that all types of metallo-β-lactamases can inhibit by these ten compounds. Therefore, these compounds were proper to mostly inhibit all metallo-β-lactamases in experimental studies.

Three new acrylic acid derivatives from Achillea mellifolium as potential thymidine phosphorylase inhibitor: molecular docking and MD simulation studies

Discovery of potent inhibitors of thymidine phosphorylase (TP) can offer appropriate approach in cancer treatment owing to it’s over expression in various human tumors compared to normal healthy tissues. Thymidine phosphorylase alongside 2-deoxy-D-ribose are reported as promoters of unwanted angiogenesis in cancerous cells. In this study, three new acrylic acid derivatives (1–3) have been isolated from ethyl acetate fraction of Achillea mellifolium. The characterization of these compounds (1–3) was done using UV, IR, 1 D and 2 D-NMR spectroscopy (1H-NMR, 13C-NMR, HMBC, NOESY) and mass spectrometry. The structure of these acrylic acid derivatives were ethyl (E)-3-((1S,5R)-5-methoxy-2,6,6-trimethyl-4-oxocyclohex-2-en-1-yl)acrylate (1), methyl (E)-3-((1S,5R)-5-methoxy-2,6,6-trimethyl-4-oxocyclohex-2-en-1-yl)acrylate (2) and (4S,6R)-6-methoxy-3,5,5-trimethyl-4-((E)-3-oxobut-1-en-1-yl)cyclohex-2-en-1-one (3). Thymidine phosphorylase (TP) inhibition studies showed compound 3 as most active inhibitor of TP with IC50 value 57.81 ± 3.41 while compound 1 and 2 showed IC50 value as 158.9 ± 0.97 and 89.92 ± 0.37, respectively. In addition, molecular docking studies of compound (1–3) were performed to shed light on their binding interaction patterns for binding into active pocket of TP. Similarly, all compounds (1–3) were evaluated for their anti-oxidant potential showing anti-oxidant activities with IC50 value ranging from 49.73 ± 0.41 to 79.81 ± 0.39. Later, these compound-protein (1–3) complexes were further subjected to MD simulations studies (50 ns) involving root mean square deviation, root mean square fluctuation, and secondary structure analysis to explore their binding mode stability inside active pocket. Communicated by Ramaswamy H. Sarma

Structure-based Virtual Screening Approach for the Discovery of Potent Inhibitors of Aminoglycoside 6'-N-Acetyltransferase Type Ib [AAC(6')-Ib] against K. pneumoniae Infections

Background: Aminoglycoside 6'-N-acetyltransferase type Ib (AAC(6')-Ib) from Klebsiella pneumoniae is an established drug target and has conferred insensitivity to aminoglycosides. Aminoglycosides are often inactivated by aminoglycoside modifying enzymes encoded by genes present in the chromosome, plasmids, and other genetic elements. The AAC(6′)- Ib is an enzyme of clinical importance found in a wide variety of gram-negative pathogens. The AAC(6′)-Ib enzyme is of interest not only because of its ubiquity but also because of other characteristics e.g., it presents significant microheterogeneity at the N-termini and the aac(6′)-Ib gene is often present in integrons, transposons, plasmids, genomic islands, and other genetic structures. The majority of the reported potent inhibitors against the target are substrate analogs. Therefore, there is a need to develop or discover new scaffolds other than substrate analogs as AAC(6')-Ib inhibitor. Objective: The objective of this study is to set optimum parameters for the structure-based virtual screening by multiple docking and scoring methods. The multiple scoring of each ligand also incorporates the ‘Induced Fit’ docking effect that helps to build further confidence in the shortlisted compounds. The method eventually is able to predict the potential inhibitors that bind to the active site and can potentially inhibit the activity of the Aminoglycoside 6′-N-acetyltransferase type Ib [AAC(6’)-Ib] from Klebsiella pneumoniae. Methods: Using the available three-dimensional structure of enzyme AAC(6')-Ib inhibitor complex, a structure-based virtual screening was performed with the hope of prioritizing the promising leads. In order to set up the protocol, 30,000 drug-like molecules were selected from the ChemBridge library. Multiple docking programs, i.e. UCSF DOCK6 and AutoDock Vina have been applied in the current study so that a consensus is developed to the predicted binding modes and thus the docking accuracy. The Amber scores of the Dock6 – a secondary scoring function was also used to perform the ‘Induced Fit’ effect and correspondingly re-rank the compounds. Results: The top 30 ranked compounds of the most frequent scored were selected from the histogram. The 2D interactions of those 30 compounds were drawn from the Ligplot+ tool. Six of the compounds were prioritized as potential inhibitors as they are representing the maximum number of interactions from the rest of the compounds and also possess the drug-likeness as predicted by the estimated ADMET properties. Conclusion: This study provided useful insight that the proposed compounds have the potential to bind to the aminoglycoside binding site of AAC(6′)-Ib that may eventually inhibit the Klebsiella pneumoniae. This study has the potential to propose putative new and novel inhibitors against a resistant drug target of Klebsiella pneumoniae.

Discovery of Potent Inhibitors against P-Glycoprotein-Mediated Multidrug Resistance Aided by Late-Stage Functionalization of a 2-(4-(Pyridin-2-yl)phenoxy)pyridine Analogue.

SIS3 is a specific inhibitor of Smad3 that inhibits the TGFβ1-induced phosphorylation of Smad3. In this article, a variety of SIS3 derivatives were designed and synthesized to discover potential inhibitors against P-glycoprotein-mediated multidrug resistance aided by late-stage functionalization of a 2-(4-(pyridin-2-yl)phenoxy)pyridine analogue. A novel class of potent P-gp reversal agents were investigated, and a lead compound 37 was identified as a potent P-gp reversal agent with strong bioactivity and outstanding affinity for P-gp.

Discovery of potential inhibitors for phosphodiesterase 5A, sodium-potassium pump and beta-adrenergic receptor from Terminalia arjuna: in silico approach

The aim of this work was to perform in silico analysis of selected biomolecules from Terminalia arjuna (T. arjuna) by using virtual screening, molecular docking and pharmacophore modeling. Reported 30 biomolecules of T. arjuna were used as ligands. Grip-based docking was carried out to produce the target-specific complex model using vLife MDS 4.4 software. Docked conformations of the selected T. arjuna biomolecules resulted in eight potential biomolecules namely Casuarinin, Luteolin, Pelargonidin, Arjunin, Castalagin, Punicalagin, Kaempferol and Quercetin with major interactions and exhibited good affinity to the residues of protein targets. Developed pharmacophore models have suggested minimum pharmacophoric features required in the biomolecule so as to show standard like activity. Interestingly, Casuarinin showed multiple inhibitions on phosphodiesterase 5A and sodium-potassium pump whereas Pelargonidin on phosphodiesterase 5A and beta-adrenergic protein targets. Conclusively, this study provides a suitable platform for discovery of novel inhibitors from natural source for heart disorders.

Analysis of Inhibitor Binding Combined with Reactivity Studies to Discover the Potentially Inhibiting Phytochemicals Targeting Chikungunya Viral Replication.

Chikungunya fever is a challenging threat to human health in various parts of the world nowadays. Many attempts have been made for developing an effective drug against this viral disease and no effective antiviral treatment has been developed to control the spread of the Chikungunya virus (CHIKV) in humans. This research is aimed at the discovery of potential inhibitors against this virus by employing computational techniques to study the interactions between non-structural proteins of Chikungunya virus and phytochemicals from plants. Four non-structural proteins were docked with 2035 phytochemicals from various plants. The ligands having binding energies ≥ -8.0 kcal/mol were considered as potential inhibitors for these proteins. ADMET studies were also performed to analyze different pharmacological properties of these docked compounds and to further analyze the reactivity of these phytochemicals against CHIKV, DFT analysis was carried out based on HOMO and LUMO energies. By analyzing the binding energies, Ki, ADMET properties and band energy gaps, it was observed that 13 phytochemicals passed all the criteria to be a potent inhibitor against CHIKV in humans. A total of 13 phytochemicals were identified as potent inhibiting candidates, which can be used against the Chikungunya virus.

Discovery of novel acylhydrazone neuraminidase inhibitors

Neuraminidase (NA) plays a crucial role in the replication and transmission of influenza virus. NA inhibitors have been developed as effective treatments for influenza A and B infections. In this paper, a new lead neuraminidase inhibitor 6a (IC50 = 7.10 ± 0.2 μM) was discovered by ligand-based virtual screening, receptor-based virtual screening, molecular dynamics simulation (MD), and bioassay validation. MD simulation indicates that the morpholinyl group of 6a could be embedded in 430-loop of NA. To exploit the 430-loop in the active site, a series of novel acylhydrazone NA inhibitors 6b-6g were designed and synthesized based on the lead compound 6a. Compound 6e exerts the most potency, with IC50 value of 2.37 ± 0.5 μM against NA, which is lower than that of oseltamivir carboxylate (OC) (IC50 = 3.84 μM). Overall, this work provided unique insights in the discovery of potent inhibitors against NA.

Discovery of C-1 modified oseltamivir derivatives as potent influenza neuraminidase inhibitors

Inspired by our initial discovery about a series of neuraminidase (NA) inhibitors targeting the 150-cavity, in present study, we designed, synthesized, and biologically tested a panel of novel oseltamivir derivatives with C-1 modification, targeting the 430-cavity, an additional binding site which widely and stably existed in both group-1 and group-2 NAs. Some of the synthesized compounds displayed robust anti-influenza potencies against H5N1 and H5N6 viruses. Among them, compound 8b exerted the greatest inhibition, with IC50 values of 0.088 and 0.097 μM and EC50 values of 4.26 and 1.31 μM against H5N1 and H5N6 strains, respectively, which are similar to those of oseltamivir carboxylate (OSC). And its potency against mutant H5N1-H274Y NA was just 7-fold weaker than OSC. Molecular modeling revealed the elongated group at C-1 position being projected toward the 430-cavity. Notably, although compound 8b was not sensitive toward H5N1 strain relative to OSC in the embryonated egg model, it displayed greater anti-influenza virus effect against H5N6 strain than OSC at the concentration of 10 mmol/L. Overall, this work provided unique insights in the discovery of potent inhibitors against both group-1 and group-2 NAs.

Insight into the key features for ligand binding in Y1230 mutated c-Met kinase domain by molecular dynamics simulations

c-Met kinase has been considered as an attractive target for developing antitumor agents. The strong interactions between Tyr1230 and the inhibitors emphasized its importance for ligand binding. The clinically related Tyr1230 mutations have made negative impacts on current c-Met kinase inhibitors, especially the exquisitely selective ones, like PF-04217903, while the multi-targeted inhibitors, like Crizotinib, were not affected so much. In this study, the protein–ligand interactions between c-Met kinase domain (wild, Y1230C and Y1230H) and these inhibitors were compared. The binding site was expanded and the post-mutated regions became solvent accessible. The heavy dependency of PF-04217903 on the interactions with Tyr1230 resulted in the steep decrease of its potency against the Y1230 mutants. It was found that the ligand entrance region contributed consistently to the binding of Crizotinib, but not PF-04217903. Additional groups substituted in the ligand entrance region with stable interactions should be beneficial for improving the inhibitory activity of PF-04217903 against the Y1230 mutants. These findings will facilitate the discovery of potent inhibitors against Y1230 mutated c-Met kinase.

Discovery of potential inhibitor against human acetylcholinesterase: a molecular docking and molecular dynamics investigation

Alzheimer’s disease (AD) is a progressive neurodegenerative disease of central nervous system among elderly people. Human acetylcholinesterase (hAChE), an important enzyme in neuronal signaling, is responsible for the degradation of acetylcholine which in turn prevents the post synaptic signal transmissions. hAChE has been an attractive target of drug discovery for the search of therapeutics against AD. In the recent past hAChE has become hot target for the investigation of new potential therapeutics. We performed virtual screening of entire database against hAChE. Further, the extra precision molecular docking was carried out to refine the docking results and the best complex was passed for molecular dynamics simulations in order of understanding the hAChE dynamics and its behavior in complex with the ligand which corroborate the outcomes of virtual screening. This also provides binding free energy data that establishes the ligands efficiency for inhibiting hAChE. The computational findings discussed in this paper provide initial information of inhibitory effects of ligand, (drugbank entry DB00983), over hAChE.

Discovery of acylguanidine oseltamivir carboxylate derivatives as potent neuraminidase inhibitors

In search of novel anti-influenza agents with higher potency, a series of acylguanidine oseltamivir carboxylate analogues were synthesized and evaluated against influenza viruses (H1N1 and H3N2) in vitro. The representative compounds with strong inhibitory activities (IC50 <40nM) against neuraminidase (NA) were further tested against the NA from oseltamivir-resistant strain (H259Y). Among them, compounds 9 and 17 were potent NA inhibitors that exhibited a 5 and 11-fold increase in activity comparing with oseltamivir carboxylate (2, OC) against the H259Y mutant, respectively. Furthermore, the effect against influenza virus H259Y mutant (H1N1) replication and cytotoxicity assays indicated that compounds 9 and 17 exhibited a 20 and 6-fold increase than the parent compound 2, and had no obvious cytotoxicity in vitro. Moreover, the molecular docking studies revealed that the docking modes of compounds 9 and 17 were different from that of oseltamivir, and the new hydrogen bonds and hydrophobic interaction were formed in this case. This work provided unique insights in the discovery of potent inhibitors against NAs from wild-type and oseltamivir-resistant strains.

Inhibitors of BRD4 Protein from a Marine-Derived Fungus Alternaria sp. NH-F6.

Bromodomains (BRD) are readers of the epigenetic code that regulate gene transcription through their recognition of acetyl-lysine modified histone tails. Recently, bromodomain-containing proteins such as BRD4 have been demonstrated to be druggable through the discovery of potent inhibitors. These protein–protein interaction inhibitors have the potential to modulate multiple diseases by their profound anti-inflammatory and antiproliferative effects. In order to explore new BRD4 inhibitors as well as lead compounds for the development of new drugs, the secondary metabolites of Alternaria sp. NH-F6, a fungus isolated from deep-sea sediment samples, were analyzed systematically. Five new compounds including two new perylenequinones (1–2), one new alternaric acid (3), 2-(N-vinylacetamide)-4-hydroxymethyl-3-ene-butyrolactone (4), one new cerebroside (5), together with 19 known compounds (6–24) were isolated from the ethyl acetate extracts of this strain. Their structures were elucidated using nuclear magnetic resonance (NMR) and high resolution electrospray ionization mass spectrometry (HR-ESI-MS) analyses. Finally, all these compounds were evaluated for their inhibitory activity against BRD4 protein, and compound 2 exhibited a potent inhibition rate of 88.1% at a concentration of 10 µM. This research provides a new BRD4 inhibitor which may possess potential antitumoral, antiviral, or anti-inflammatory pharmaceutical values.

Abstract PR04: Small molecule-mediated activation of Ras elicits inhibition of MAPK and PI3K signaling though pathway feedback

Abstract Oncogenic mutation or hyper-activation of Ras results in aberrant cellular signaling and is responsible for approximately 30% of all human tumors. Therefore, Ras is an outstanding candidate for therapeutic inhibition. However, the discovery of potent inhibitors has been challenging. Our laboratory has recently discovered small molecules that perturb Son of Sevenless (SOS) -catalyzed nucleotide exchange on Ras (Burns et al. PNAS 2014). Here we describe experiments conducted to determine the mechanism of compound action. SOS activator compound 4 (C4) induces signaling flux through the MAPK pathway in response to elevated Ras-GTP levels, reminiscent of epidermal growth factor signaling. We used mass spectrometry to show that in response to C4 stimulation, phospho-modifications on SOS1 increased at ERK phosphorylation consensus sequences. We hypothesize that these phospho-modifications can cause delocalization of SOS and membrane bound Ras, and we are currently using proximity-ligation to assess whether the interaction between SOS1 and Ras is affected by C4 treatment. Cross-talk between the MAPK and PI3K signaling pathways may be responsible for the inhibition of phospho-AKT Ser473 levels in response to MAPK signaling flux, or vice versa. Here we show, using MEK and PI3K inhibitor pre-treatments, that events in both pathways are independent after C4 treatment. As both SOS1 and PI3K bind to Ras at a similar site, we are currently testing the hypothesis that C4 disrupts the interaction between Ras and PI3K, whilst allowing signaling flux through the MAPK pathway. Overall, we have discovered small-molecule activators of SOS that modulate Ras-GTP levels, resulting in signaling flux through the MAPK pathway and eventual downstream inhibition of both the MAPK and PI3K pathways in cancer cells. This abstract is also being presented as Poster A15. Citation Format: Jennifer E. Howes, Denis T. Akan, Bethany M. Alicie, Alex G. Waterson, Olivia W. Rossanese, Stephen W. Fesik. Small molecule-mediated activation of Ras elicits inhibition of MAPK and PI3K signaling though pathway feedback. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr PR04.

Insight into drug resistance mechanisms and discovery of potential inhibitors against wild-type and L1196M mutant ALK from FDA-approved drugs.

Anaplastic lymphoma kinase (ALK) plays a crucial role in multiple malignant cancers. It is known as a well-established target for the treatment of ALK-dependent cancers. Even though substantial efforts have been made to develop ALK inhibitors, only crizotinib, ceritinib, and alectinib had been approved by the U.S. Food and Drug Administration for patients with ALK-positive non-small cell lung cancer (NSCLC). The secondary mutations with drug-resistance bring up difficulties to develop effective drugs for ALK-positive cancers. To give a comprehensive understanding of molecular mechanism underlying inhibitor response to ALK tyrosine kinase mutations, we established an accurate assessment for the extensive profile of drug against ALK mutations by means of computational approaches. The molecular mechanics-generalized Born surface area (MM-GBSA) method based on molecular dynamics (MD) simulation was carried out to calculate relative binding free energies for receptor-drug systems. In addition, the structure-based virtual screening was utilized to screen effective inhibitors targeting wild-type ALK and the gatekeeper mutation L1196M from 3180 approved drugs. Finally, the mechanism of drug resistance was discussed, several novel potential wild-type and L1196M mutant ALK inhibitors were successfully identified.