Impact of deglycosylation and thermal stress on conformational stability of a full length murine igG2a monoclonal antibody: Observations from molecular dynamics simulations

With the rise of antibody based therapeutics as successful medicines, there is an emerging need to understand the fundamental antibody conformational dynamics and its implications towards stability of these medicines. Both deglycosylation and thermal stress have been shown to cause conformational destabilization and aggregation in monoclonal antibodies. Here, we study instabilities caused by deglycosylation and by elevated temperature (400 K) by performing molecular dynamic simulations on a full length murine IgG2a mAb whose crystal structure is available in the Protein Data bank. C^α‐atom root mean square deviation and backbone root mean square fluctuation calculations show that deglycosylation perturbs quaternary and tertiary structures in the C_H2 domains. In contrast, thermal stress pervades throughout the antibody structure and both Fabs and Fc regions are destabilized. The thermal stress applied in this study was not sufficient to cause large scale unfolding within the simulation time and most amino acid residues showed similar average solvent accessible surface area and secondary structural conformations in all trajectories. C_H3 domains were the most successful at resisting the conformational destabilization. The simulations helped identify aggregation prone regions, which may initiate cross‐β motif formation upon deglycosylation and upon applying thermal stress. Deglycosylation leads to increased backbone fluctuations and solvent exposure of a highly conserved APR located in the edge β‐strand A of the C_H2 domains. Aggregation upon thermal stress is most likely initiated by two APRs that overlap with the complementarity determining regions. This study has important implications for rational design of antibody based therapeutics that are resistant towards aggregation. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Structural basis of Bacillus anthracis MoxXT disruption and the modulation of MoxT ribonuclease activity by rationally designed peptides

Bacillus anthracis MoxXT is a Type II proteic Toxin–Antitoxin (TA) module wherein MoxT is a ribonuclease that cleaves RNA specifically while MoxX interacts with MoxT and inhibits its activity. Disruption of the TA interaction has been proposed as a novel antibacterial strategy. Peptides, either based on antitoxin sequence or rationally designed, have previously been reported to disrupt the MoxXT interaction but cause a decrease in MoxT ribonuclease activity. In the present study, we report the crystal structure of MoxT, and the effect of several peptides in disrupting the MoxXT interaction as well as augmentation of MoxT ribonuclease activity by binding to MoxT in vitro. Docking studies on the peptides were carried out in order to explain the observed structure activity relationships. The peptides with ribonuclease augmentation activity possess a distinct structure and are proposed to bind to a distinct site on MoxT. The docking of the active peptides with MoxT showed that they possess an aromatic group that occupies a conserved hydrophobic pocket. Additionally, the peptides inducing high ribonuclease activity were anchored by a negatively charged group near a cluster of positively charged residues present near the pocket. Our study provides a structural basis and rationale for the observed properties of the peptides and may aid the development of small molecules to disrupt the TA interaction.     

Enthalpie and Entropie Contributions in the Transesterification of Sucrose: Computational Study of Lipases and Subtilisin

Abstract

Transesterification of sucrose with fatty acids catalyzed by subtilisin Carlsberg occurs with regioselectivity that is different from that in lipases. Thermomyces lanuginosus lipase (TlL) and Candida antarctica lipase B (CALB) catalyze synthesis at positions 6 and 6′, with differing abilities, while subtilisin catalysis leads to the l′-acylated sucrose. The catalytic machinery in lipases is approximately mirrored in subtilisins but different pocket morphologies including size, shape, and rearrangement of the catalytic elements underlies the differing regioselectivities. The thermodynamic consequences of these differences on the above reactions have been explored systematically using computational methods, determining the free energies of interaction of the putative transition-state adducts. Analysis of the conformers with the lowest transition state energies (protein-ligand interactions and vibrational entropy contributions) indicates that enthalpic factors control specificities in lipases while entropic factors are more important in subtilisin.     

Alcohol Consumption Negates Estrogen-mediated Myocardial Repair in Ovariectomized Mice by Inhibiting Endothelial Progenitor Cell Mobilization and Function

We have shown previously that estrogen (estradiol, E2) supplementation enhances voluntary alcohol consumption in ovariectomized female rodents and that increased alcohol consumption impairs ischemic hind limb vascular repair. However, the effect of E2-induced alcohol consumption on post-infarct myocardial repair and on the phenotypic/functional properties of endothelial progenitor cells (EPCs) is not known. Additionally, the molecular signaling of alcohol-estrogen interactions remains to be elucidated. This study examined the effect of E2-induced increases in ethanol consumption on post-infarct myocardial function/repair. Ovariectomized female mice, implanted with 17β-E2 or placebo pellets were given access to alcohol for 6 weeks and subjected to acute myocardial infarction. Left ventricular functions were consistently depressed in mice consuming ethanol compared with those receiving only E2. Alcohol-consuming mice also displayed significantly increased infarct size and reduced capillary density. Ethanol consumption also reduced E2-induced mobilization and homing of EPCs to injured myocardium compared with the E2-alone group. In vitro, exposure of EPCs to ethanol suppressed E2-induced proliferation, survival, and migration and markedly altered E2-induced estrogen receptor-dependent cell survival signaling and gene expression. Furthermore, ethanol-mediated suppression of EPC biology was endothelial nitric oxide synthase-dependent because endothelial nitric oxide synthase-null mice displayed an exaggerated response to post-acute myocardial infarction left ventricular functions. These data suggest that E2 modulation of alcohol consumption, and the ensuing EPC dysfunction, may negatively compete with the beneficial effects of estrogen on post-infarct myocardial repair.     

A liquid chromatography–tandem mass spectrometry assay for d-Ala-d-Lac: A key intermediate for vancomycin resistance in vancomycin-resistant enterococci

Vancomycin exerts its antibacterial activity by binding to d-Ala-d-Ala in bacterial cell wall precursors. Vancomycin resistance in vancomycin-resistant enterococci (VRE) is due to an alternative cell wall biosynthesis pathway in which d-Ala-d-Ala is replaced, most commonly by d-Ala-d-Lac. In this study, we extend our recently developed Marfey’s derivatization-based liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for l-Ala, d-Ala, and d-Ala-d-Ala to d-Ala-d-Lac and apply it to the quantitation of these metabolites in VRE. The first step in this effort was the development of an effective washing method for removing medium components from VRE cells. Mar-d-Ala-d-Lac was well resolved chromatographically from Mar-d-Ala-d-Ala, a prerequisite for MS/MS quantitation of d-Ala-d-Ala and d-Ala-d-Lac. Mar-d-Ala-d-Lac gave similar detection parameters, sensitivity, and linearity as Mar-d-Ala-d-Ala. l-Ala, d-Ala, d-Ala-d-Ala, and d-Ala-d-Lac levels in VRE were then determined in the presence of variable vancomycin levels. Exposure to vancomycin resulted in a dramatic reduction of d-Ala-d-Ala, with a response midpoint at approximately 0.06 μg/ml vancomycin and with a broad response profile up to 128 μg/ml vancomycin. In contrast, d-Ala-d-Lac was present in the absence of vancomycin, with its level constant up to 128 μg/ml vancomycin. This method will be useful for the discovery, characterization, and refinement of new agents targeting vancomycin resistance in VRE.     

Minocycline,levodopa and MnTMPyP induced changes in the mitochondrial proteome profile of MPTP and maneb and paraquat mice models of Parkinson's disease

Mitochondrial dysfunction is the foremost perpetrator of the nigrostriatal dopaminergic neurodegeneration leading to Parkinson's disease (PD). However, the roles played by majority of the mitochondrial proteins in PD pathogenesis have not yet been deciphered. The present study investigated the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and combined maneb and paraquat on the mitochondrial proteome of the nigrostriatal tissues in the presence or absence of minocycline, levodopa and manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP). The differentially expressed proteins were identified and proteome profiles were correlated with the pathological and biochemical anomalies induced by MPTP and maneb and paraquat. MPTP altered the expression of twelve while combined maneb and paraquat altered the expression of fourteen proteins. Minocycline, levodopa and MnTMPyP, respectively, restored the expression of three, seven and eight proteins in MPTP and seven, eight and eight proteins in maneb- and paraquat-treated groups. Although levodopa and MnTMPyP rescued from MPTP- and maneb- and paraquat-mediated increase in the microglial activation and decrease in manganese-superoxide dismutase expression and complex I activity, dopamine content and number of dopaminergic neurons, minocycline defended mainly against maneb- and paraquat-mediated alterations. The results demonstrate that MPTP and combined maneb and paraquat induce mitochondrial dysfunction and microglial activation and alter the expression of a bunch of mitochondrial proteins leading to the nigrostriatal dopaminergic neurodegeneration and minocycline, levodopa or MnTMPyP variably offset scores of such changes.     

Exploring the Chemical Space around 8-Mercaptoguanine as a Route to New Inhibitors of the Folate Biosynthesis Enzyme HPPK

As the second essential enzyme of the folate biosynthetic pathway, the potential antimicrobial target, HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase), catalyzes the Mg^2+-dependant transfer of pyrophosphate from the cofactor (ATP) to the substrate, 6-hydroxymethyl-7,8-dihydropterin. Recently, we showed that 8-mercaptoguanine (8-MG) bound at the substrate site (K_D ∼13 µM), inhibited the S. aureus enzyme (SaHPPK) (IC₅₀ ∼ 41 µM), and determined the structure of the SaHPPK/8-MG complex. Here we present the synthesis of a series of guanine derivatives, together with their HPPK binding affinities, as determined by SPR and ITC analysis. The binding mode of the most potent was investigated using 2D NMR spectroscopy and X-ray crystallography. The results indicate, firstly, that the SH group of 8-MG makes a significant contribution to the free energy of binding. Secondly, direct N ⁹ substitution, or tautomerization arising from N ⁷ substitution in some cases, leads to a dramatic reduction in affinity due to loss of a critical N ⁹-H···Val46 hydrogen bond, combined with the limited space available around the N ⁹ position. The water-filled pocket under the N ⁷ position is significantly more tolerant of substitution, with a hydroxyl ethyl 8-MG derivative attached to N ⁷ (compound 21a) exhibiting an affinity for the apo enzyme comparable to the parent compound (K_D ∼ 12 µM). In contrast to 8-MG, however, 21a displays competitive binding with the ATP cofactor, as judged by NMR and SPR analysis. The 1.85 Å X-ray structure of the SaHPPK/21a complex confirms that extension from the N ⁷ position towards the Mg²⁺-binding site, which affords the only tractable route out from the pterin-binding pocket. Promising strategies for the creation of more potent binders might therefore include the introduction of groups capable of interacting with the Mg²⁺ centres or Mg²⁺ -binding residues, as well as the development of bitopic inhibitors featuring 8-MG linked to a moiety targeting the ATP cofactor binding site.     

The relationship between anthropometric indexes of adiposity and vascular function in the FATE cohort

Objective:

Numerous indexes of adiposity have been proposed and are currently in use in clinical practice and research. However, the correlation of these indexes with measures of vascular health remain poorly defined. This study investigated which measure of adiposity is most strongly associated with endothelial function.

Design and Methods:

Data from the Firefighters And Their Endothelium (FATE) study was used. The relationships between three measures of vascular function: flow‐mediated dilation (FMD), hyperemic velocity time integral (VTI), and hyperemic shear stress (HSS), and five measures of adiposity: BMI, waist circumference (WC), waist‐to‐hip ratio (WHR), waist‐to‐height ratio (WHtR), and body adiposity index (BAI) were tested. Univariate comparisons were made, and subsequently models adjusted for traditional risk factors were constructed.

Results:

A total of 1,462 male firefighters (mean age 49 ± 9) without cardiovascular disease comprised the study population. No measure of adiposity correlated with FMD; all five measures of adiposity were negatively correlated with VTI and HSS (P values <0.0001), with WHtR most strongly correlated with VTI, and WC most strongly correlated with HSS (both P < 0.05). In models including all five measures of obesity simultaneously, BMI, WC, and WHtR were all predictive of HSS (all P values <0.05), and BMI and WHR were both predictive of VTI (P values <0.05).

Conclusions:

Anthropometric measures of adiposity may help refine estimations of atherosclerotic burden. BMI was most consistently associated with endothelial dysfunction, but measures of adiposity that reflect distribution of mass were additive.     

Incidence and Prevalence of Tuberculosis among Household Contacts of Pulmonary Tuberculosis Patients in a Peri-Urban Population of South Delhi,India

Background

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the leading causes of mortality and morbidity across all age groups throughout the world, especially in developing countries.

Methodology/Principal Findings

In this study, we have included 432 open index cases with their 1608 household contacts in a prospective cohort study conducted from May 2007 to March 2009. The follow-up period was 2 years. All Index cases were diagnosed on the basis of suggestive signs and symptoms and sputum being AFB positive. Among the 432 index patients, 250 (57.9%) were males and 182 (42.1%) females; with mean age of 34±14.4 yr and 26±11.1 yr, respectively. Out of 1608 household contacts, 866 (53.9%) were males and 742 (46.1%) females; with mean age of 26.5±15.8 and 26.5±16.0 yr, respectively. Of the total 432 households, 304 (70.4%) had ≤4 members and 128 (29.6%) had ≥5 members. The median size of the family was four. Of the 1608 contacts, 1206 were able to provide sputum samples, of whom 83 (6.9%) were found MTB culture positive. Household contacts belonging to adult age group were predominantly (74, 89.2%) infected as compared to the children (9, 10.8%). On screening the contact relationship status with index patients, 52 (62.7%) were first-degree relatives, 18 (34.6%) second-degree relatives and 12 (14.5%) spouses who got infected from their respective index patients. Co-prevalent and incident tuberculosis was found in 52 (4.3%) and 31 (2.6%) contacts, respectively. In incident cases, the diagnosis could be made between 4 to 24 months of follow-up, after their baseline evaluation.

Conclusion

Active household contact investigation is a powerful tool to detect and treat tuberculosis at early stages and the only method to control TB in high-TB-burden countries.