Application of Binding Free Energy Calculations to Prediction of Binding Modes and Affinities of MDM2 and MDMX Inhibitors

Molecular docking is widely used to obtain binding modes and binding affinities of a molecule to a given target protein. Despite considerable efforts, however, prediction of both properties by docking remains challenging mainly due to protein’s structural flexibility and inaccuracy of scoring functions. Here, an integrated approach has been developed to improve the accuracy of binding mode and affinity prediction, and tested for small molecule MDM2 and MDMX antagonists. In this approach, initial candidate models selected from docking are subjected to equilibration MD simulations to further filter the models. Free energy perturbation molecular dynamics (FEP/MD) simulations are then applied to the filtered ligand models to enhance the ability in predicting the near-native ligand conformation. The calculated binding free energies for MDM2 complexes are overestimated compared to experimental measurements mainly due to the difficulties in sampling highly flexible apo-MDM2. Nonetheless, the FEP/MD binding free energy calculations are more promising for discriminating binders from nonbinders than docking scores. In particular, the comparison between the MDM2 and MDMX results suggests that apo-MDMX has lower flexibility than apo-MDM2. In addition, the FEP/MD calculations provide detailed information on the different energetic contributions to ligand binding, leading to a better understanding of the sensitivity and specificity of protein-ligand interactions

Rigorous Computational and Experimental Investigations on MDM2/MDMX-Targeted Linear and Macrocyclic Peptides

There is interest in peptide drug design, especially for targeting intracellular protein–protein interactions. Therefore, the experimental validation of a computational platform for enabling peptide drug design is of interest. Here, we describe our peptide drug design platform (CMDInventus) and demonstrate its use in modeling and predicting the structural and binding aspects of diverse peptides that interact with oncology targets MDM2/MDMX in comparison to both retrospective (pre-prediction) and prospective (post-prediction) data. In the retrospective study, CMDInventus modules (CMDpeptide, CMDboltzmann, CMDescore and CMDyscore) were used to accurately reproduce structural and binding data across multiple MDM2/MDMX data sets. In the prospective study, CMDescore, CMDyscore and CMDboltzmann were used to accurately predict binding affinities for an Ala-scan of the stapled α-helical peptide ATSP-7041. Remarkably, CMDboltzmann was used to accurately predict the results of a novel D-amino acid scan of ATSP-7041. Our investigations rigorously validate CMDInventus and support its utility for enabling peptide drug design

Computational Studies of Difference in Binding Modes of Peptide and Non-Peptide Inhibitors to MDM2/MDMX Based on Molecular Dynamics Simulations

Inhibition of p53-MDM2/MDMX interaction is considered to be a promising strategy for anticancer drug design to activate wild-type p53 in tumors. We carry out molecular dynamics (MD) simulations to study the binding mechanisms of peptide and non-peptide inhibitors to MDM2/MDMX. The rank of binding free energies calculated by molecular mechanics generalized Born surface area (MM-GBSA) method agrees with one of the experimental values. The results suggest that van der Waals energy drives two kinds of inhibitors to MDM2/MDMX. We also find that the peptide inhibitors can produce more interaction contacts with MDM2/MDMX than the non-peptide inhibitors. Binding mode predictions based on the inhibitor-residue interactions show that the π–π, CH–π and CH–CH interactions dominated by shape complimentarity, govern the binding of the inhibitors in the hydrophobic cleft of MDM2/MDMX. Our studies confirm the residue Tyr99 in MDMX can generate a steric clash with the inhibitors due to energy and structure. This finding may theoretically provide help to develop potent dual-specific or MDMX inhibitors

Applications of nuclear magnetic resonance spectroscopy: from drug discovery to protein structure and dynamics.

The versatility of nuclear magnetic resonance (NMR) spectroscopy is apparent when presented with diverse applications to which it can contribute. Here, NMR is used i) as a screening/ validation tool for a drug discovery program targeting the Phosphatase of Regenerating Liver 3 (PRL3), ii) to characterize the conformational heterogeneity of p53 regulator, Murine Double Minute X (MDMX), and iii) to characterize the solution dynamics of guanosine monophosphate kinase (GMPK). Mounting evidence suggesting roles for PRL3 in oncogenesis and metastasis has catapulted it into prominence as a cancer drug target. Yet, despite significant efforts, there are no PRL3 small molecule inhibitors currently in clinical trials. This work combines screening of an FDA-approved drug panel and the identification of binders by protein-observed NMR. FDA-approved drugs salirasib and candesartan were identified as potent inhibitors in in vitro inhibition and migration assays while a weak inhibitor, olsalazine, was identified by NMR as the first small molecule inhibitor to directly bind PRL3. NMR was also used to validate the binding of additional compounds identified as experimental PRL3 inhibitors. Thienopyridone, a potent experimental inhibitor, did not show direct binding to PRL3 but instead inhibited phosphatase activity via redox mechanism. NMR also revealed that other experimental inhibitors did not engage PRL3. Thus, there remains a need to identify potent PRL3-directed inhibitors. Meanwhile, molecular modeling revealed a putative druggable site that has not been thoroughly explored before. The current study provides some scaffolds such as candesartan and particularly, olsalazine, the only binder identified, that could be the starting point of further drug discovery efforts, as well as a putative site that can be targeted in silico. MDMX, a negative regulator of p53, is another important therapeutic target in cancer, along with the homologous protein, MDM2. Inhibitors that block the MDM2-p53 interaction have been identified and despite similarities in the binding site of these homologous proteins, these inhibitors are ineffective against MDMX. It is hypothesized that the flexibility of MDMX contributes to this significant difference in response to inhibitors, despite comparable affinity to their endogenous target, p53. Examination of available inhibitor-bound structures of MDMX reveal a conserved pharmacophore but the structures adopt distinct conformations away from the binding site. This implies that global motions of the protein might contribute to molecular recognition. The conformational heterogeneity in MDMX was further confirmed by collecting residual dipolar couplings (RDCs). Further investigations on both MDMX and MDM2 are necessary to uncover whether the flexibility of MDMX contributes to the differential binding to inhibitors. Finally, NMR relaxation methods and state-of-the-art high-power Carr-Purcell-Meiboom Gill (CPMG) relaxation dispersion measurements, the first documented application on an enzyme, were used to characterize the solution dynamics of GMPK and the changes in dynamics upon GMP binding. Substrate binding resulted in restricting the amplitudes of motion for backbone amide bonds within the picosecond-nanosecond timescale. Meanwhile, CPMG showed dispersion in both in the absence and presence of GMP, such that substrate binding did not quench dynamics within the microsecond-millisecond timescale. Interestingly, more residues are observed to have dispersion in the bound form, some near the C-terminal of helix 3, which has previously been proposed to be involved in product release. Current studies show that substrate binding affect different timescales of protein motion. Future work shall follow how motions within different timescales are affected as GMPK processes its substrates – such as, for instance, binding of ATP analogs within the ATP binding site or simultaneous occupancy of both substrate binding pockets. This paves the way for a complete picture of the relationship of function and dynamics in the conformational enzymatic cycle of a bi-substrate enzyme using GMPK as a model. The current work illustrates some of the diverse applications of NMR on three unique systems that are also drug targets. Information collected here can be leveraged on future structure and dynamics studies as well as drug discovery efforts targeting any of these proteins

Insight Into the Binding Mechanism of p53/pDIQ-MDMX/MDM2 With the Interaction Entropy Method

The study of the p53-MDMX/MDM2 binding sites is a research hotspot for tumor drug design. The inhibition of p53-targeted MDMX/MDM2 has become an effective approach in anti-tumor drug development. In this paper, a theoretically rigorous and computationally accurate method, namely, the interaction entropy (IE) method, combined with the polarized protein-specific charge (PPC) force field, is used to explore the difference in the binding mechanism between p53-MDMX and p53-MDM2. The interaction of a 12mer peptide inhibitor (pDIQ), which is similar to p53 in structure, with MDMX/MDM2 is also studied. The results demonstrate that p53/pDIQ with MDM2 generates a stronger interaction than with MDMX. Compared to p53, pDIQ has larger binding free energies with MDMX and MDM2. According to the calculated binding free energies, the differences in the binding free energy among the four complexes that are obtained from the combination of PPC and IE are more consistent with the experimental values than with the results from the combination of the non-polarizable AMBER force field and IE. In addition, according to the decomposition of the binding free energy, the van der Waals (vdW) interactions are the main driving force for the binding of the four complexes. They are also the main source of the weaker binding affinity of p53/pDIQ-MDMX relative to p53/pDIQ-MDM2. Compared with p53-MDMX/MDM2, according to the analysis of the residue decomposition, the predicated total residue contributions are higher in pDIQ-MDMX/MDM2 than in p53-MDMX/MDM2, which explains why pDIQ has higher binding affinity than p53 with MDMX/MDM2. The current study provides theoretical guidance for understanding the binding mechanisms and designing a potent dual inhibitor that is targeted to MDMX/MDM2

Predicting and Testing Helix-Mimetic Inhibitors of the p53-Mdm2 Interaction

Aberrant protein-protein interactions (PPIs) are found in many disease states. Consequently, there is a need for PPI inhibitors for use as research tools and pharmaceutical lead compounds. Computational methods could greatly assist with the search for new PPIs. Oligobenzamides are novel PPI inhibitors which can theoretically be produced to display any sequence of side chains. Understanding the nature of oligobenzamide binding is important for identification of the most efficient strategy of predicting oligobenzamide inhibitors. The prediction of oligobenzamide affinities using thermodynamic integration and implicit solvent methods is described. Affinities of oligobenzamides for Mdm2 predicted using implicit solvent methods bore a moderate correlation with measured affinities. Examination of MM-PBSA results using analysis of variance revealed that it is not necessary to run simulations with every member of a large combinatorial library in order to predict their relative affinities because within a particular binding site, the degree of interaction between the side chains is small. However, it could be useful to separate molecules based on their predicted binding pose because oligobenzamides can bind to Mdm2 in many different ways, depending on the choice of side chains. This insight will be valuable for future attempts to predict oligobenzamide affinities. The 1H-15N HSQC NMR spectrum peaks of 15N-labelled Mdm2 L33E were assigned to facilitate the future validation of binding poses. An oligoamide was shown using NMR to bind in the correct place. However, NMR testing revealed that oligobenzamides can aggregate in aqueous solution despite being soluble. A novel FRET-based method was also developed which can be used to test potential inhibitors with a low solubility and high absorbance during their development. It was adapted for a microwell plate to facilitate future high throughput screening and an assay involving Cherry-labelled Mdm2 was tested which could be developed into an in vivo assay in the future

The effect of ethanolic leaves extract of soursop (Annona muricata L.) on human colorectal cancer cell line: cell viability and in silico study to cyclin D1 protein

Latar Belakang: Kanker kolorektal merupakan transformasi patologis dari epitel kolon dan rektum normal menjadi massa jaringan abnormal, perubahan ini terjadi karena ekspresi berlebih dari protein cyclin D1 yang menginduksi proliferasi sel kolorektal secara berlebihan. Pengobatan dan pencegahan kanker kolorektal dapat dilakukan secara alami dengan mengonsumsi ekstrak daun Annona muricata L. (sirsak). Sirsak dikenal karena banyak komponen fitokimia yang berfungsi sebagai anti kanker. Metode: Penelitian ini menggunakan sel kanker kolorektal HT-29 yang diberi ekstrak etanol daun sirsak dan 5 Fluorourasil (5-FU). Tujuannya untuk menemukan konsentrasi sitotoksisitas yang dapat menghambat 50% populasi sel HT-29 (CC50) dan konsentrasi yang didapat sebelumnya akan diuji dengan metode uji MTT. Analisis docking molekuler dilakukan antara molekul-molekul dari ekstrak etanol daun sirsak terhadap protein Cyclin D1 menggunakan perangkat lunak molecular operating environment (MOE) 2013.08. Hasil: CC50 ekstrak etanol daun sirsak adalah 278 μg / mL dan 5-FU adalah 88 μg / mL. Persentase terendah sel HT-29 yang layak adalah 2 x CC50 setelah perlakuan ekstrak etanol daun sirsak (40,4 ± 1,3%) dibandingkan dengan 5-FU (52,8 ± 4,3%), kontrol pelarut ( 97,2 ± 1,4%), dan kontrol sel (100%). Analisis docking molekuler untuk protein cyclin D1 diperoleh asam N-hexadecanoic dan molekul phytol sebagai kandidat yang baik untuk menghambat protein cyclin D1. Kesimpulan: Ekstrak etanol daun sirsak dapat menurunkan viabilitas sel kultur kanker kolon HT-29 dan berdasarkan analisis molekular docking dilihat dari energi bebas gibbs (ΔG) dan afinitas tertinggi (pKi) diperoleh N-hexadecanoic dan molekul phytol sebagai penghambat protein cyclin D1. (Health Science Journal of Indonesia 2019;10(2):96-102) Kata Kunci: Kanker kolorektal HT-29, ekstrak etanol daun sirsak, viabilitas sel, molecular docking, cyclin D1   Abstract Introduction: Colorectal cancer is a pathological transformation of normal colon and rectum epithelial that becomes an abnormal tissue mass, due to the overexpression of cyclin D1 protein that inducing excessive proliferation of colorectal cell. The treatment and prevention of colorectal cancer could be done naturally by consuming leaves extract of Annona muricata L. (soursop). Soursop is known for many phytochemical components that serve as an anti-cancer. Methods: This study was used HT-29 colorectal cancer cell that treated with ethanolic leaves extract of soursop and 5-Fluorourasil (5-FU) to find the cytotoxicity concentration that can inhibit 50% of HT-29 cell population (CC50) and the next concentrations of them were treated for next treatment with MTT assay. Molecular docking analysis of the compounds of ethanolic leaves extract of soursop to cyclin D1 protein used molecular operating environment (MOE) 2013.08 software. Results: CC50 of ethanolic leaves extracts of soursop was 278 μg/mL dan 5-FU was 88 μg/mL. The lowest percentage of viable HT-29 cell was 2 x CC50 after ethanolic leaves extract of soursop treatment (40,4±1,3%) was compared to 5-FU (52,8±4,3%), solvent control (97,2±1,4%), and cells control (100%). Analysis of molecular docking to cyclin D1 protein was obtained N-hexadecanoic acid and phytol molecules as good candidates to inhibit cyclin D1 protein. Conclusions: The ethanolic leaves extract of soursop could be a good alternative treatment for colorectal cancer and its compounds had ability to inhibit cyclin D1 protein (the highest gibbs free energy (ΔG) and affinity (pKi)). (Health Science Journal of Indonesia 2019;10(2):96-102) Keywords: Colorectal cancer, ethanolic leaves extract of soursop, cell viability, molecular docking, cyclin D

MDM2 Case Study: Computational Protocol Utilizing Protein Flexibility Improves Ligand Binding Mode Predictions

Recovery of the P53 tumor suppressor pathway via small molecule inhibitors of onco-protein MDM2 highlights the critical role of computational methodologies in targeted cancer therapies. Molecular docking programs in particular, provide a quantitative ranking of predicted binding geometries based on binding free energy allowing for the screening of large chemical libraries in search of lead compounds for cancer therapeutics. This study found improved binding mode predictions of medicinal compounds to MDM2 using the popular docking programs AutoDock and AutoDock Vina, while adopting a rigid-ligand/flexible-receptor protocol. Crystal structures representing small molecule inhibitors bound to MDM2 were selected and a total of 12 rotatable bonds was supplied to each complex and distributed systematically between the ligand and binding site residues. Docking results were evaluated in terms of the top ranked binding free energy and corresponding RMSD values from the experimentally known binding site. Results show lowest RMSD values coincide with a rigid ligand, while the protein retained the majority of flexibility. This study suggests the future implementation of a rigid-ligand/flexible-receptor protocol may improve accuracy of high throughput screenings of potential cancer drugs targeting the MDM2 protein, while maintaining manageable computational costs

Selection of protein conformations for structure-based polypharmacology studies

Several drugs exert their therapeutic effect through the modulation of multiple targets. Structure-based approaches hold great promise for identifying compounds with the desired polypharmacological profiles. These methods use knowledge of the protein binding sites to identify stereoelectronically complementary ligands. The selection of the most suitable protein conformations to be used in the design process is vital, especially for multitarget drug design in which the same ligand has to be accommodated in multiple binding pockets. Herein, we focus on currently available techniques for the selection of the most suitable protein conformations for multitarget drug design, compare the potential advantages and limitations of each method, and comment on how their combination could help in polypharmacology drug design