Structural insight into selectivity of amylin and calcitonin receptor agonists.

Amylin receptors (AMYRs), heterodimers of the calcitonin receptor (CTR) and one of three receptor activity-modifying proteins, are promising obesity targets. A hallmark of AMYR activation by Amy is the formation of a 'bypass' secondary structural motif (residues S19-P25). This study explored potential tuning of peptide selectivity through modification to residues 19-22, resulting in a selective AMYR agonist, San385, as well as nonselective dual amylin and calcitonin receptor agonists (DACRAs), with San45 being an exemplar. We determined the structure and dynamics of San385-bound AMY3R, and San45 bound to AMY3R or CTR. San45, via its conjugated lipid at position 21, was anchored at the edge of the receptor bundle, enabling a stable, alternative binding mode when bound to the CTR, in addition to the bypass mode of binding to AMY3R. Targeted lipid modification may provide a single intervention strategy for design of long-acting, nonselective, Amy-based DACRAs with potential anti-obesity effects.

Acetylene Semi-Hydrogenation at Room Temperature over Pd-Zn Nanocatalyst.

A reaction of fundamental and commercial importance is acetylene semi-hydrogenation. Acetylene impurity in the ethylene feedstock used in the polyethylene industry poisons the Ziegler-Natta catalyst which adversely affects the polymer quality. Pd based catalysts are most often employed for converting acetylene into the main reactant, ethylene, however, it often involves a tradeoff between the conversion and the selectivity and generally requires high temperatures. In this work, bimetallic Pd-Zn nanoparticles capped by hexadecylamine (HDA) have been synthesized by co-digestive ripening of Pd and Zn nanoparticles and studied for semi-hydrogenation of acetylene. The catalyst showed a high selectivity of ~85 % towards ethylene with a high ethylene productivity to the tune of ~4341 µmol g-1 min-1 , at room temperature and atmospheric pressure. It also exhibited excellent stability with ethylene selectivity remaining greater than 85 % even after 70 h on stream. To the best of the authors' knowledge, this is the first report of room temperature acetylene semi-hydrogenation, with the catalyst effecting high amount of acetylene conversion to ethylene retaining excellent selectivity and stability among all the reported catalysts thus far. DFT calculations show that the disordered Pd-Zn nanocatalyst prepared by a low temperature route exhibits a change in the d-band center of Pd and Zn which in turn enhances the selectivity towards ethylene. TPD, XPS and a range of catalysis experiments provided in-depth insights into the reaction mechanism, indicating the key role of particle size, surface area, Pd-Zn interactions, and the capping agent.

Automated Design of Macrocycles for Therapeutic Applications: From Small Molecules to Peptides and Proteins.

Macrocycles and cyclic peptides are increasingly attractive therapeutic modalities as they often have improved affinity, are able to bind to extended protein surfaces, and otherwise have favorable properties. Macrocyclization of a known binder may stabilize its bioactive conformation and improve its metabolic stability, cell permeability, and in certain cases oral bioavailability. Herein, we present implementation and application of an approach that automatically generates, evaluates, and proposes cyclizations utilizing a library of well-established chemical reactions and reagents. Using the three-dimensional (3D) conformation of the linear molecule in complex with a target protein as the starting point, this approach identifies attachment points, generates linkers, evaluates their geometric compatibility, and ranks the resulting molecules with respect to their predicted conformational stability and interactions with the target protein. As we show here with prospective and retrospective case studies, this procedure can be applied for the macrocyclization of small molecules and peptides and even PROteolysis TArgeting Chimeras (PROTACs) and proteins.

Peptide Optimization at the Drug Discovery-Development Interface: Tailoring of Physicochemical Properties Toward Specific Formulation Requirements.

Physicochemical properties of peptides need to be compatible with the manufacturing process and formulation requirements to ensure developability toward the commercial drug product. This aspect is often disregarded and only evaluated late in discovery, imposing a high risk for delays in development, increased costs, and finally for the project in general. Here, we report a case study of early physicochemical peptide characterization and optimization of dual glucagon-like peptide 1/glucagon receptor agonists toward specific formulation requirements. Aggregation issues which were observed at acidic pH in the presence of phenolic preservatives could be eliminated by modification of the peptide sequence, and chemical stability issues were significantly improved by addition of stabilizing formulation excipients. We describe structural, analytical, and biophysical characterization in different compositions to analyze the effect of pH and formulation excipients on physical and chemical stability. Molecular models have been generated to rationalize peptide stability behavior based on computed physicochemical descriptors and interactions with excipients. To conclude these studies, a general roadmap is proposed how to assess and optimize early physicochemical peptide properties in a sophisticated way by combining experimental and in silico profiling to provide stable peptide drugs under relevant formulation conditions at the end of discovery.

1-Aminocyclopropane-1-carboxylic acid deaminase producing beneficial rhizobacteria ameliorate the biomass characters of Panicum maximum Jacq. by mitigating drought and salt stress.

1-Aminocyclopropane-1-carboxylic acid (ACC) is a precursor molecule of ethylene whose concentration is elevated in the plant subjected to biotic and abiotic stress. Several soil microorganisms are reported to produce ACC deaminase (ACCd) which degrades ACC thereby reducing stress ethylene in host plants. This study is aimed to apply ACCd producing beneficial rhizobacteria to improve biochemical parameters and cell wall properties of Panicum maximum exposed to salt and drought stress, focusing on bioethanol production. Thirty-seven ACCd producing bacteria isolated from rhizospheric soil of field grown P. maximum and 13 were shortlisted based on their beneficial traits (root colonization, production of indole acetic acid, siderophore, hydrogen cyanide, phosphate solubilization, biofilm formation, tolerance to salt and Polyethylene glycol) and a total score obtained. All shortlisted bacteria were found significant in enhancing the plant growth, water conservation, membrane stability, biocompatible solutes and protein, phenolic contents and photosynthetic pigments in plants grown under stress conditions. Cell wall composition (Cellulose, Hemicellulose and Lignin) of the treated plants grown under stress conditions recorded a significant improvement over their respective controls and found equivalent to the plants grown under normal circumstances. Biomass from bacterial treatment recorded higher total reducing sugars upon pre-treatment and hydrolysis, and theoretical bioethanol yield.

Hydration effects on the efficacy of the Epidermal growth factor receptor kinase inhibitor afatinib.

Small molecules targeting the EGFR tyrosine kinase domain have been used with some success at treating patients with non-small cell lung cancer driven by activating mutations in the kinase domain. The initial class of inhibitors displaced ATP noncovalently but were rendered ineffective due to the development of resistance mutations in the kinase domain. These were overcome by the development of covalent inhibitors such as afatinib which also bind in the ATP pocket. However pooled analysis of two recent clinical trials LUX-3 and LUX-6 demonstrated an unprecedented overall survival benefit of afatinib over chemotherapy for the EGFR 19del , but not the EGFR L858R . In the current study we use modelling and simulations to show that structural constraints in EGFR 19del deletion result in significantly attenuated flexibilities in the binding pocket resulting in strong hydrogen and halogen bonds with afatinib in the EGFR 19del ; these constraints are modulated by buried water and result in the differential affinities of afatinib for the different mutants. SNP analysis of residues surrounding the buried water points to the likelihood of further differential effects of afatinib and provides a compelling case for investigating the effects of the SNPs towards further stratification of patients for ensuring the most effective use of afatinib.

Toward Understanding the Molecular Recognition of Albumin by p53-Activating Stapled Peptide ATSP-7041.

Reactivation of tumor-suppressing activity of p53 protein by targeting its negative regulator MDM2/MDMX has been pursued as a potential anticancer strategy. A promising dual inhibitor of MDM2/MDMX that has been developed and is currently in clinical trials is the stapled peptide ATSP-7041. The activity of this molecule is reported to be modulated in the presence of serum. Albumin is the most abundant protein in serum and is known to bind reversibly to several molecules. To study this interaction, we develop a protocol combining molecular modeling, docking, and simulations. Exhaustive docking of the peptide with representative simulated structures of human serum albumin led to the identification of probable binding sites on the surface of the protein, including both known canonical and novel binding sites. Sequence differences at putative peptide-binding sites in human and mouse albumin result in differing interaction energies with the peptide and enable us to rationalize the observed differences in vivo. In general, the findings should help in guiding the design of features in such peptides that may affect their distribution and cell permeability, opening a new window in structure-guided design strategies.

Understanding the molecular basis of substrate binding specificity of PTB domains.

Protein-protein interactions mediated by phosphotyrosine binding (PTB) domains play a crucial role in various cellular processes. In order to understand the structural basis of substrate recognition by PTB domains, multiple explicit solvent atomistic simulations of 100ns duration have been carried out on 6 PTB-peptide complexes with known binding affinities. MM/PBSA binding energy values calculated from these MD trajectories and residue based statistical pair potential score show good correlation with the experimental dissociation constants. Our analysis also shows that the modeled structures of PTB domains can be used to develop less compute intensive residue level statistical pair potential based approaches for predicting interaction partners of PTB domains.

Structure-based multiscale approach for identification of interaction partners of PDZ domains.

PDZ domains are peptide recognition modules which mediate specific protein-protein interactions and are known to have a complex specificity landscape. We have developed a novel structure-based multiscale approach which identifies crucial specificity determining residues (SDRs) of PDZ domains from explicit solvent molecular dynamics (MD) simulations on PDZ-peptide complexes and uses these SDRs in combination with knowledge-based scoring functions for proteomewide identification of their interaction partners. Multiple explicit solvent simulations ranging from 5 to 50 ns duration have been carried out on 28 PDZ-peptide complexes with known binding affinities. MM/PBSA binding energy values calculated from these simulations show a correlation coefficient of 0.755 with the experimental binding affinities. On the basis of the SDRs of PDZ domains identified by MD simulations, we have developed a simple scoring scheme for evaluating binding energies for PDZ-peptide complexes using residue based statistical pair potentials. This multiscale approach has been benchmarked on a mouse PDZ proteome array data set by calculating the binding energies for 217 different substrate peptides in binding pockets of 64 different mouse PDZ domains. Receiver operating characteristic (ROC) curve analysis indicates that, the area under curve (AUC) values for binder vs nonbinder classification by our structure based method is 0.780. Our structure based method does not require experimental PDZ-peptide binding data for training.

An in silico analysis of the binding modes and binding affinities of small molecule modulators of PDZ-peptide interactions.

Inhibitors of PDZ-peptide interactions have important implications in a variety of biological processes including treatment of cancer and Parkinson's disease. Even though experimental studies have reported characterization of peptidomimetic inhibitors of PDZ-peptide interactions, the binding modes for most of them have not been characterized by structural studies. In this study we have attempted to understand the structural basis of the small molecule-PDZ interactions by in silico analysis of the binding modes and binding affinities of a set of 38 small molecules with known K(i) or K(d) values for PDZ2 and PDZ3 domains of PSD-95 protein. These two PDZ domains show differential selectivity for these compounds despite having a high degree of sequence similarity and almost identical peptide binding pockets. Optimum binding modes for these ligands for PDZ2 and PDZ3 domains were identified by using a novel combination of semi-flexible docking and explicit solvent molecular dynamics (MD) simulations. Analysis of the binding modes revealed most of the peptidomimectic ligands which had high K(i) or K(d) moved away from the peptide binding pocket, while ligands with high binding affinities remained in the peptide binding pocket. The differential specificities of the PDZ2 and PDZ3 domains primarily arise from differences in the conformation of the loop connecting ßB and ßC strands, because this loop interacts with the N-terminal chemical moieties of the ligands. We have also computed the MM/PBSA binding free energy values for these 38 compounds with both the PDZ domains from multiple 5 ns MD trajectories on each complex i.e. a total of 228 MD trajectories of 5 ns length each. Interestingly, computational binding free energies show good agreement with experimental binding free energies with a correlation coefficient of approximately 0.6. Thus our study demonstrates that combined use of docking and MD simulations can help in identification of potent inhibitors of PDZ-peptide complexes.