Structural and functional characterization of MBS301, an afucosylated bispecific anti-HER2 antibody

MBS301, a glyco-engineered bispecific anti-human epidermal growth factor receptor 2 (HER2) antibody with a typical IgG1 monoclonal antibody structure, was developed through dual-cell expression and in vitro assembling process. MBS301 consists of two half antibodies engineered from trastuzumab and pertuzumab, respectively. Integrity and purity profiles of MB301 indicated that the heterodimerization of the two half antibodies was successful. The high and similar melting temperatures (Tm1,72.0°C and Tm2, 84.8°C) of MBS301 compared with those of its parental monoclonal antibodies trastuzumab and pertuzumab (in-house made T-mab and P-mab, respectively) revealed its structural compactness. With computer-modeling experiments and Biacore binding and competition kinetics studies, the binding stoichiometry between MBS301 and HER2-ECD was determined to be 1:1 and the two arms of MBS301 were shown to bind to domains II and IV of HER2-ECD antigen simultaneously. MBS301 displayed synergistic bioactivities as the combination of T-mab and P-mab in vitro in multiple cancer cell lines and in vivo in xenograft mouse model studies, and showed more effective activity than T-mab or P-mab used individually. Moreover, fucose-knockout dramatically increased MBS301’s binding affinity to low affinity FcγRIIIa allotype 158F (K_D = 2.35 × 10^?7M) to near the high affinity level of allotype V158 (K_D = 1.17 × 10^?7M). This resulted in far more effective ADCC activity of MBS301 than the combination of T-mab and P-mab in killing HER2-positive cancer cells. Hence, a novel fully afucosylated anti-HER2 bispecific antibody with improved antitumor activities was generated and shown to have the potential to be used for treating HER2-positive but trastuzumab-resistant solid tumors.     

Conformational flexibility of the ErbB2 ectodomain and trastuzumab antibody complex as revealed by molecular dynamics and principal component analysis

Human epidermal growth factor receptor 2 (ErbB2) is a transmembrane oncoprotein that is over expressed in breast cancer. A successful therapeutic treatment is a monoclonal antibody called trastuzumab which interacts with the ErbB2 extracellular domain (ErbB2-ECD). A better understanding of the detailed structure of the receptor-antibody interaction is indeed of prime interest for the design of more effective anticancer therapies. In order to discuss the flexibility of the complex ErbB2-ECD/trastuzumab, we present, in this study, a multi-nanosecond molecular dynamics simulation (MD) together with an analysis of fluctuations, through a principal component analysis (PCA) of this system. Previous to this step and in order to validate the simulations, we have performed a detailed analysis of the variable antibody domain interactions with the extracellular domain IV of ErbB2. This structure has been statically elucidated by x-ray studies. Indeed, the simulation results are in excellent agreement with the available experimental information during the full trajectory. The PCA shows eigenvector fluctuations resulting in a hinge motion in which domain II and C_H domains approach each other. This move is likely stabilized by the formation of H-bonds and salt bridge interactions between residues of the dimerization arm in the domain II and trastuzumab residues located in the C_H domain. Finally, we discuss the flexibility of the MD/PCA model in relation with the static x-ray structure. A movement of the antibody toward the dimerization domain of the ErbB2 receptor is reported for the first time. This finding could have important consequences on the biological action of the monoclonal antibody.

Dual Fatty Acid Synthase and HER2 Signaling Blockade Shows Marked Antitumor Activity against Breast Cancer Models Resistant to Anti-HER2 Drugs

Blocking the enzyme Fatty Acid Synthase (FASN) leads to apoptosis of HER2-positive breast carcinoma cells. The hypothesis is that blocking FASN, in combination with anti-HER2 signaling agents, would be an effective antitumor strategy in preclinical HER2+ breast cancer models of trastuzumab and lapatinib resistance. We developed and molecularly characterized in vitro HER2+ models of resistance to trastuzumab (SKTR), lapatinib (SKLR) and both (SKLTR). The cellular interactions of combining anti-FASN polyphenolic compounds (EGCG and the synthetic G28UCM) with anti-HER2 signaling drugs (trastuzumab plus pertuzumab and temsirolimus) were analyzed. Tumor growth inhibition after treatment with EGCG, pertuzumab, temsirolimus or the combination was evaluated in two in vivo orthoxenopatients: one derived from a HER2+ patient and another from a patient who relapsed on trastuzumab and lapatinib-based therapy. SKTR, SKLR and SKLTR showed hyperactivation of EGFR and p-ERK1/2 and PI3KCA mutations. Dual-resistant cells (SKLTR) also showed hyperactivation of HER4 and recovered levels of p-AKT compared with mono-resistant cells. mTOR, p-mTOR and FASN expression remained stable in SKTR, SKLR and SKLTR. In vitro, anti-FASN compounds plus pertuzumab showed synergistic interactions in lapatinib- and dual- resistant cells and improved the results of pertuzumab plus trastuzumab co-treatment. FASN inhibitors combined with temsirolimus displayed the strongest synergistic interactions in resistant cells. In vivo, both orthoxenopatients showed strong response to the antitumor activity of the combination of EGCG with pertuzumab or temsirolimus, without signs of toxicity. We showed that the simultaneous blockade of FASN and HER2 pathways is effective in cells and in breast cancer models refractory to anti-HER2 therapies.     

Multi-parametric analysis reveals enhanced G2-phase arrest of an optimized anti-HER2 antibody to inhibit breast cancer

Objectives

To find a “me-better” antibody by epitope-specific antibody optimization and multi-parametric analysis.

Results

Using epitope-specific library based on the commercial drug, Pertuzumab/2C4, we screened a novel human anti-HER2 antibody, MIL5, which has slightly higher affinity than the drug. MIL5 and 2C4 share the same epitope to bind HER2; however, MIL5 bound to HER2 His235–His245 more tightly than 2C4, which could be the main reason of its enhanced affinity. In vivo experiments also showed MIL5 had stronger anti-cancer activity than 2C4; however, the classical flow cytometry assays to detect cell apoptosis or cycling did not show convincing evidence of the advantages of MIL5. Thus we introduced the multi-parameter in-cell analysis method to evaluate the superiority of MIL5 to 2C4 in arresting cancer cells in G2-phase to inhibit cell growth and/or proliferation.

Conclusion

Multi-parametric method confirmed stronger arrest of G2 by MIL5 to show better anti-cancer function both in vitro and in vivo than 2C4.

     

Insights into HER2 signaling from step-by-step optimization of anti-HER2 antibodies

HER2, a ligand-free tyrosine kinase receptor of the HER family, is frequently overexpressed in breast cancer. The anti-HER2 antibody trastuzumab has shown significant clinical benefits in metastatic breast cancer; however, resistance to trastuzumab is common. The development of monoclonal antibodies that have complementary mechanisms of action results in a more comprehensive blockade of ErbB2 signaling, especially HER2/HER3 signaling. Use of such antibodies may have clinical benefits if these antibodies can become widely accepted. Here, we describe a novel anti-HER2 antibody, hHERmAb-F0178C1, which was isolated from a screen of a phage display library. A step-by-step optimization method was employed to maximize the inhibitory effect of this anti-HER2 antibody. Crystallographic analysis was used to determine the three-dimensional structure to 3.5 Å resolution, confirming that the epitope of this antibody is in domain III of HER2. Moreover, this novel anti-HER2 antibody exhibits superior efficacy in blocking HER2/HER3 heterodimerization and signaling, and its use in combination with pertuzumab has a synergistic effect. Characterization of this antibody revealed the important role of a ligand binding site within domain III of HER2. The results of this study clearly indicate the unique potential of hHERmAb-F0178C1, and its complementary inhibition effect on HER2/HER3 signaling warrants its consideration as a promising clinical treatment.     

Insights into HER2 signaling from step-by-step optimization of anti-HER2 antibodies

HER2, a ligand-free tyrosine kinase receptor of the HER family, is frequently overexpressed in breast cancer. The anti-HER2 antibody trastuzumab has shown significant clinical benefits in metastatic breast cancer; however, resistance to trastuzumab is common. The development of monoclonal antibodies that have complementary mechanisms of action results in a more comprehensive blockade of ErbB2 signaling, especially HER2/HER3 signaling. Use of such antibodies may have clinical benefits if these antibodies can become widely accepted. Here, we describe a novel anti-HER2 antibody, hHERmAb-F0178C1, which was isolated from a screen of a phage display library. A step-by-step optimization method was employed to maximize the inhibitory effect of this anti-HER2 antibody. Crystallographic analysis was used to determine the three-dimensional structure to 3.5 Å resolution, confirming that the epitope of this antibody is in domain III of HER2. Moreover, this novel anti-HER2 antibody exhibits superior efficacy in blocking HER2/HER3 heterodimerization and signaling, and its use in combination with pertuzumab has a synergistic effect. Characterization of this antibody revealed the important role of a ligand binding site within domain III of HER2. The results of this study clearly indicate the unique potential of hHERmAb-F0178C1, and its complementary inhibition effect on HER2/HER3 signaling warrants its consideration as a promising clinical treatment.     

Glycoengineered Pichia produced anti-HER2 is comparable to trastuzumab in preclinical study

Mammalian cell culture systems are used predominantly for the production of therapeutic monoclonal antibody (mAb) products. A number of alternative platforms, such as Pichia engineered with a humanized N-linked glycosylation pathway, have recently been developed for the production of mAbs. The glycosylation profiles of mAbs produced in glycoengineered Pichia are similar to those of mAbs produced in mammalian systems. This report presents for the first time the comprehensive characterization of an anti-human epidermal growth factor receptor 2 (HER2) mAb produced in glycoengineered Pichia, and a study comparing the anti-HER2 from Pichia, which had an amino acid sequence identical to trastuzumab, with trastuzumab. The comparative study covered a full spectrum of preclinical evaluation, including bioanalytical characterization, in vitro biological functions, in vivo anti-tumor efficacy and pharmacokinetics in both mice and non-human primates. Cell signaling and proliferation assays showed that anti-HER2 from Pichia had antagonist activities comparable to trastuzumab. However, Pichia-produced material showed a 5-fold increase in binding affinity to FcγIIIA and significantly enhanced antibody dependent cell-mediated cytotoxicity (ADCC) activity, presumably due to the lack of fucose on N-glycans. In a breast cancer xenograft mouse model, anti-HER2 was comparable to trastuzumab in tumor growth inhibition. Furthermore, comparable pharmacokinetic profiles were observed for anti-HER2 and trastuzumab in both mice and cynomolgus monkeys. We conclude that glycoengineered Pichia provides an alternative production platform for therapeutic mAbs and may be of particular interest for production of antibodies for which ADCC is part of the clinical mechanism of action.Key words: glycoengineered Pichia, anti-HER2, trastuzumab, xenograft, PK, ADCC     

Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system

We describe protein synthesis, folding and assembly of antibody fragments and full-length aglycosylated antibodies using an Escherichia coli-based open cell-free synthesis (OCFS) system. We use DNA template design and high throughput screening at microliter scale to rapidly optimize production of single-chain Fv (scFv) and Fab antibody fragments that bind to human IL-23 and IL-13α1R, respectively. In addition we demonstrate production of aglycosylated immunoglobulin G (IgG1) trastuzumab. These antibodies are produced rapidly over several hours in batch mode in standard bioreactors with linear scalable yields of hundreds of milligrams/L over a 1 million-fold change in scales up to pilot scale production. We demonstrate protein expression optimization of translation initiation region (TIR) libraries from gene synthesized linear DNA templates, optimization of the temporal assembly of a Fab from independent heavy chain and light chain plasmids and optimized expression of fully assembled trastuzumab that is equivalent to mammalian expressed material in biophysical and affinity based assays. These results illustrate how the open nature of the cell-free system can be used as a seamless antibody engineering platform from discovery to preclinical development of aglycosylated monoclonal antibodies and antibody fragments as potential therapeutics.Key words: cell-free protein synthesis, Fab antibody, aglycosylated antibodies, HER2, trastuzumab     

Effects of protein binding on a lipid bilayer containing local anesthetic articaine, and the potential of mean force calculation: a molecular dynamics simulation approach

Articaine, as a local anesthetic drug has been simulated in neutral and charged forms, and its interaction with the dimyristoylphosphatidylcholine (DMPC) lipid bilayer membrane is investigated by molecular dynamics simulation using GROMACS software. In order to obtain the optimum location of the drug molecules, as they penetrate into the membrane, umbrella sampling is applied and the free energy is calculated. The effect of protein binding to DMPC membrane on the process of drug diffusion through the membrane is considered. Five simulation systems are designed and by applying the potential of mean force, the molecular dynamics simulation on the system is performed. In light of the obtained results, the electrostatic potential, variation of lipid bilayer’s order parameter and the diffusion coefficient of drug are discussed.

Prodrugs for masking bitter taste of antibacterial drugs—a computational approach

DFT calculations for the acid-catalyzed hydrolysis of several maleamic acid amide derivatives revealed that the reaction rate-limiting step is determined on the nature of the amine leaving group. Further, it was established that when the amine leaving group was a secondary amine, acyclovir or cefuroxime moiety the tetrahedral intermediate formation was the rate-limiting step such as in the cases of acyclovir ProD 1- ProD 4 and cefuroxime ProD 1- ProD 4. In addition, the linear correlation between the calculated and experimental rates provided a credible basis for designing prodrugs for masking bitter taste of the corresponding parental drugs which have the potential to release the parent drug in a sustained release fashion. For example, based on the DFT calculated rates the predicted t_1/2 (a time needed for 50 % of the reactant to be hydrolyzed to products) for cefuroxime prodrugs, cefuroxime ProD 1- ProD 4, were 12 min, 18 min, 200 min and 123 min, respectively.

Structural and energetic insights into sequence-specific interaction in DNA–drug recognition: development of affinity predictor and analysis of binding selectivity

Although the molecular mechanism and thermodynamic profile of a wide variety of chemical agents have been examined intensively in the past decades in terms of specific recognition of their protein receptors, to date the physicochemical nature of DNA–drug recognition and association still remains largely unexplored. The present study focused on understanding the structural basis, energetic landscape, and biological implications underlying the binding of small-molecule ligands to their cognate or non-cognate DNA receptors. First, a new method to capture the structural features of DNA–drug complex architecture was proposed and then used to correlate the extracted features with binding affinity of the complexes. By employing this method, a statistical regression-based predictor was developed to quantitatively evaluate the interaction potency of drug compounds with DNA in a fast and reliable manner. Subsequently, we use the predictor to examine the binding behavior of a number of structure-available, affinity-known DNA–drug complexes as well as a large pool of randomly generated DNA decoys in complex with the same drugs. It was found that (1) as compared with protein–DNA recognition, small-molecule agents have relatively low specificity in selecting their cognate DNA targets from the background of numerous random decoys; (2) the abundance of A–T base pairs in the DNA core motif exhibits a significant positive correlation with the affinity of drug ligand binding to the DNA receptor; and (3) high affinity seems not to be closely related to high selectivity for a DNA-targeting drug, although high-affinity drug entities have a greater possibility of being ranked computationally as top binders. We hope that this work will provide a preliminary insight into the molecular origin of sequence-specific interactions in DNA–drug recognition.

Scytonin,a novel cyanobacterial photoprotective pigment: calculations of Raman spectroscopic biosignatures

Experimental studies have shown that chemical vapor decomposition method by using C₂H₂/C₂H₄ as carbon source could dramatically decrease the defects in prepared CNT. However, the inherent mechanism with regards to reduction of defects is quite unclear. In the present paper, density functional theory is used to study the healing process of CNT with divacancy defect by C₂H₂/C₂H₄ molecule. The healing processes undergo three evolution steps: (i) the chemisorption of the first C₂H₂/C₂H₄ molecule on defective CNT; (ii) the insertion of C atoms from C₂H₂/C₂H₄ molecule into defective CNT; (iii) the removal of the H atoms on CNT, forming perfect CNT. The estimated adsorption energy barrier of C₂H₂/C₂H₄ molecules on defective CNT is within the range from 1.10 to 1.63 eV, and the eventual formation of CNT is strongly exothermic (4.40/4.54 eV in (8, 0) CNT). In light of the unique conditions of CNT synthesis, i.e., high temperature in a closed container, such healing processes could most likely take place. Therefore, we propose that during CNT synthesis procedures, both C₂H₂ and C₂H₄ could act as a carbon source and the defect healer.

Neural Stem Cells as a Novel Platform for Tumor-Specific Delivery of Therapeutic Antibodies

Background

Recombinant monoclonal antibodies have emerged as important tools for cancer therapy. Despite the promise shown by antibody-based therapies, the large molecular size of antibodies limits their ability to efficiently penetrate solid tumors and precludes efficient crossing of the blood-brain-barrier into the central nervous system (CNS). Consequently, poorly vascularized solid tumors and CNS metastases cannot be effectively treated by intravenously-injected antibodies. The inherent tumor-tropic properties of human neural stem cells (NSCs) can potentially be harnessed to overcome these obstacles and significantly improve cancer immunotherapy. Intravenously-delivered NSCs preferentially migrate to primary and metastatic tumor sites within and outside the CNS. Therefore, we hypothesized that NSCs could serve as an ideal cellular delivery platform for targeting antibodies to malignant tumors.

Methods and Findings

As proof-of-concept, we selected Herceptin™ (trastuzumab), a monoclonal antibody widely used to treat HER2-overexpressing breast cancer. HER2 overexpression in breast cancer is highly correlated with CNS metastases, which are inaccessible to trastuzumab therapy. Therefore, NSC-mediated delivery of trastuzumab may improve its therapeutic efficacy. Here we report, for the first time, that human NSCs can be genetically modified to secrete anti-HER2 immunoglobulin molecules. These NSC-secreted antibodies assemble properly, possess tumor cell-binding affinity and specificity, and can effectively inhibit the proliferation of HER2-overexpressing breast cancer cells in vitro. We also demonstrate that immunoglobulin-secreting NSCs exhibit preferential tropism to tumor cells in vivo, and can deliver antibodies to human breast cancer xenografts in mice.

Conclusions

Taken together, these results suggest that NSCs modified to secrete HER2-targeting antibodies constitute a promising novel platform for targeted cancer immunotherapy. Specifically, this NSC-mediated antibody delivery system has the potential to significantly improve clinical outcome for patients with HER2-overexpressing breast cancer.     

Quantitative Proteomics with siRNA Screening Identifies Novel Mechanisms of Trastuzumab Resistance in HER2 Amplified Breast Cancers

HER2 is a receptor tyrosine kinase that is overexpressed in 20% to 30% of human breast cancers and which affects patient prognosis and survival. Treatment of HER2-positive breast cancer with the monoclonal antibody trastuzumab (Herceptin) has improved patient survival, but the development of trastuzumab resistance is a major medical problem. Many of the known mechanisms of trastuzumab resistance cause changes in protein phosphorylation patterns, and therefore quantitative proteomics was used to examine phosphotyrosine signaling networks in trastuzumab-resistant cells. The model system used in this study was two pairs of trastuzumab-sensitive and -resistant breast cancer cell lines. Using stable isotope labeling, phosphotyrosine immunoprecipitations, and online TiO₂ chromatography utilizing a dual trap configuration, ∼1700 proteins were quantified. Comparing quantified proteins between the two cell line pairs showed only a small number of common protein ratio changes, demonstrating heterogeneity in phosphotyrosine signaling networks across different trastuzumab-resistant cancers. Proteins showing significant increases in resistant versus sensitive cells were subjected to a focused siRNA screen to evaluate their functional relevance to trastuzumab resistance. The screen revealed proteins related to the Src kinase pathway, such as CDCP1/Trask, embryonal Fyn substrate, and Paxillin. We also identify several novel proteins that increased trastuzumab sensitivity in resistant cells when targeted by siRNAs, including FAM83A and MAPK1. These proteins may present targets for the development of clinical diagnostics or therapeutic strategies to guide the treatment of HER2+ breast cancer patients who develop trastuzumab resistance.HER2 is a member of the epidermal growth factor receptor (EGFR)/ErbB family of receptor tyrosine kinases. Under normal physiologic conditions, HER2 tyrosine kinase signaling is tightly regulated spatially and temporally by the requirement for it to heterodimerize with a ligand bound family member, such as EGFR, HER3/ErbB3, or HER4/ErbB4 (1). However, in 20% to 30% of human breast cancer cases, HER2 gene amplification is present, resulting in a high level of HER2 protein overexpression and unregulated, constitutive HER2 tyrosine kinase signaling (2, 3). HER2 gene amplified breast cancer, also termed HER2-positive breast cancer, carries a poor prognosis, but the development of the HER2 targeted monoclonal antibody trastuzumab (Herceptin) has significantly improved patient survival (2). Despite the clinical effectiveness of trastuzumab, the development of drug resistance significantly increases the risk of patient death. This poses a major medical problem, as most metastatic HER2-positive breast cancer patients develop trastuzumab resistance over the course of their cancer treatment (4). The treatment approach for HER2+ breast cancer patients after trastuzumab resistance develops is mostly a trial-and-error process that subjects the patient to increased toxicity. Therefore, there is a substantial medical need for strategies to overcome trastuzumab resistance.Multiple trastuzumab-resistance mechanisms have been identified, and they alter signaling networks and protein phosphorylation patterns in either a direct or an indirect manner. These mechanisms can be grouped into three categories. The first category is the activation of a parallel signaling network by other tyrosine kinases. These kinases include the receptor tyrosine kinases, EGFR, IGF1R, Her3, Met, EphA2, and Axl, as well as the erythropoietin-receptor-mediated activation of the cytoplasmic tyrosine kinases Jak2 and Src (5–11). The second category is the activation of downstream signaling proteins. Multiple studies have demonstrated activation of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway in trastuzumab resistance, which occurs either via deletion of the PTEN lipid phosphatase or mutation of the PI3K genes (12, 13). Activation of Src family kinases or overexpression of cyclin E, which increases the cyclin E–cyclin-dependent kinase 2 signaling pathway, has also been reported (14). The third category includes mechanisms that maintain HER2 signaling even in the presence of trastuzumab. The production of a truncated isoform of HER2, p95HER2, which lacks the trastuzumab binding site, causes constitutive HER2 signaling (15, 16). Overexpression of the MUC4 sialomucin complex inhibits trastuzumab binding to HER2 and thereby maintains HER2 signaling (17, 18).Given that multiple trastuzumab-resistance mechanisms alter signaling networks and protein phosphorylation patterns, we reasoned that mapping phosphotyrosine signaling networks using quantitative proteomics would be a highly useful strategy for analyzing known mechanisms and identifying novel mechanisms of trastuzumab resistance. Quantitative proteomics and phosphotyrosine enrichment approaches have been extensively used to study the EGFR signal networks (19–23). We and others have used these approaches to map the HER2 signaling network (22, 24, 25). Multiple other tyrosine kinase signaling networks were analyzed using quantitative proteomics, including Ephrin receptor, EphB2 (26–28), platelet-derived growth factor receptor (PDGFR) (21), insulin receptor (29, 30), and the receptor for hepatocyte growth factor, c-MET (31).The goal of this study is to identify, quantify, and functionally screen proteins that might be involved in trastuzumab resistance. We used two pairs of HER2 gene amplified trastuzumab-sensitive (parental, SkBr3 and BT474) and -resistant (SkBr3^R and BT474^R) human breast cancer cell lines as models for trastuzumab resistance. These cell lines and their trastuzumab-resistant derivatives have been extensively characterized and highly cited in the breast cancer literature (32, 33). Using stable isotope labeling of amino acids in cell culture (SILAC),¹ phosphotyrosine immunoprecipitations, and online TiO₂ chromatography with dual trap configuration, we quantified the changes in phosphotyrosine containing proteins and interactors between trastuzumab-sensitive and -resistant cells. Several of the known trastuzumab-resistance mechanisms were identified, which serves as a positive control and validation of our approach, and large protein ratio changes were measured in proteins that had not been previously connected with trastuzumab resistance. We then performed a focused siRNA screen targeting the proteins with significantly increased protein ratios. This screen functionally tested the role of the identified proteins and identifies which proteins might have the largest effect on reversing trastuzumab resistance.     

Molecular dynamics and free energy studies of chirality specificity effects on aminobenzo[a]quinolizine inhibitors binding to DPP-IV

The aminobenzo[a]quinolizines were investigated as a novel class of DPP-IV inhibitors. The stereochemistry of this class plays an important role in the bioactivity. In this study, the mechanisms of how different configuration of three chiral centers of this class influences the binding affinity were investigated by molecular dynamics simulations, free energy decomposition analysis. The S configuration for chiral center 3* is decisive for isomers to maintain high bioactivity; the chirality effect of chiral center 2* on the binding affinity is largely dependent, while the S configuration for chiral center 2* is preferable to R configuration for the bioactivity gain; the effect of chiral center 11b* on the binding affinity is insignificant. The chirality specificity for three chiral centers is responsible for distinction of two van der Waals contacts with Tyr547 and Phe357, and of H-bonding interactions with Arg125 and Glu206. Particularly, the Arg125 to act as a bridge in the H-bonding network contributes to stable H-bonding interactions of isomer in DPP-IV active site.

Role of gold in a complex cascade reaction involving two electrocyclization steps

Quantum chemical computations (B3LYP/LACVP**) were applied to assess the impact of Au(I) complexation on activation barriers for sequential electrocyclization reactions (one a 1,2-dihydroazete ring-opening and another a pentadienyl cation ring-closure) proposed to occur during a complex reaction cascade that converts alkynes and imines to cyclopentenimines.

A DFT study on the initial stage of thermal degradation of Poly(methyl methacrylate)/carbon nanotube system

DFT calculations, with VWN exchange correlation functional and double numeric basis set, were used to evaluate the energies required for the scission reactions taking place in the initial stage of the thermal degradation of Poly(methyl methacrylate) (PMMA) in the presence of a carbon nanotube (CNT). Side group and main chain scissions were investigated. The results averaged from five configurations of pure PMMA (DP?=?5) were used as references and compared to the results obtained for the five same configurations of PMMA grafted on three carbon nanotubes of similar diameter (1.49 nm). The bond dissociation energies (BDE) of main chain scission evaluated for grafted PMMA was 4 % less endothermic than for pure PMMA. These results seemed independent of the tested chirality (11,11); (12,10) and (16,5) of the carbon nanotubes. Comparisons with the BDE of the weakest bonds due to intrinsic defaults (head to head and unsaturated end chain) were performed.

Insights into the structural determinants for selective inhibition of nitric oxide synthase isoforms

Selective inhibition of the nitric oxide synthase isoforms (NOS) is a promising approach for the treatment of various disorders. However, given the high active site conservation among all NOS isoforms, the design of selective inhibitors is a challenging task. Analysis of the X-ray crystal structures of the NOS isoforms complexed with known inhibitors most often gives no clues about the structural determinants behind the selective inhibition since the inhibitors share the same binding conformation. Aimed at a better understanding of the structural factors responsible for selective inhibition of NOS isoforms we have performed MD simulations for iNOS, nNOS and eNOS complexed with N^ω-NO₂-L-Arg (1), and with the aminopyridine derivatives 2 and 3. The slightly better selectivity of 1 for nNOS may be assigned to the presence of extra charge–charge interactions due to its “extended” conformation. While the high affinity of 2 for iNOS can be explained by the formation of an iNOS-specific subpocket upon binding, the lack of affinity for eNOS is associated to a conformational change in Glu363. The strong van der Waals and electrostatic interactions between 3 and the active site of nNOS are most likely responsible for its higher affinity for this isoform. Owing to the elongated and narrow binding pocket of iNOS, the correct positioning of 3 over the heme group is difficult, which may account for its lower affinity toward this isoform. Brought together, our results might help to rationalize the design of selective NOS inhibitors.

Sequence selectivity of azinomycin B in DNA alkylation and cross-linking: a QM/MM study

Azinomycin B—a well-known antitumor drug—forms cross-links with DNA through alkylation of purine bases and blocks tumor cell growth. This reaction has been modeled using the ONIOM (B3LYP/6-31?+?g(d):UFF) method to understand the mechanism and sequence selectivity. ONIOM results have been checked for reliability by comparing them with full quantum mechanics calculations for selected paths. Calculations reveal that, among the purine bases, guanine is more reactive and is alkylated by aziridine ring through the C10 position, followed by alkylation of the epoxide ring through the C21 position of Azinomycin B. While the mono alkylation is controlled kinetically, bis-alkylation is controlled thermodynamically. Solvent effects were included using polarized-continuum-model calculations and no significant change from gas phase results was observed.