A novel method for in silico assessment of Methionine oxidation risk in monoclonal antibodies: Improvement over the 2-shell model.

Over the past decade, therapeutic monoclonal antibodies (mAbs) have established their role as valuable agents in the treatment of various diseases ranging from cancers to infectious, cardiovascular and autoimmune diseases. Reactive groups of the amino acids within these proteins make them susceptible to many kinds of chemical modifications during manufacturing, storage and in vivo circulation. Among these reactions, the oxidation of methionine residues to their sulfoxide form is a commonly observed chemical modification in mAbs. When the oxidized methionine is in the complementarity-determining region (CDR), this modification can affect antigen binding and thus abrogate biological activity. For these reasons, it is essential to identify oxidation liabilities during the antibody discovery and development phases. Here, we present an in silico method, based on protein modeling and molecular dynamics simulations, to predict the oxidation-liable residues in the variable region of therapeutic antibodies. Previous studies have used the 2-shell water coordination number descriptor (WCN) to identify methionine residues susceptible to oxidation. Although the WCN descriptor successfully predicted oxidation liabilities when the residue was solvent exposed, the method was much less accurate for partially buried methionine residues. Consequently, we introduce a new descriptor, WCN-OH, that improves the accuracy of prediction of methionine oxidation susceptibility by extending the theoretical framework of the water coordination number to incorporate the effects of polar amino acids side chains in close proximity to the methionine of interest.

Accurate Prediction of Protein Thermodynamic Stability Changes upon Residue Mutation using Free Energy Perturbation.

This work describes the application of a physics-based computational approach to predict the relative thermodynamic stability of protein variants, and evaluates the quantitative accuracy of those predictions compared to experimental data obtained from a diverse set of protein systems assayed at variable pH conditions. Physical stability is a key determinant of the clinical and commercial success of biological therapeutics, vaccines, diagnostics, enzymes and other protein-based products. Although experimental techniques for measuring the impact of amino acid residue mutation on the stability of proteins exist, they tend to be time consuming and costly, hence the need for accurate prediction methods. In contrast to many of the commonly available computational methods for stability prediction, the Free Energy Perturbation approach applied in this paper explicitly accounts for solvent effects and samples conformational dynamics using a rigorous molecular dynamics simulation process. On the entire validation dataset, consisting of 328 single point mutations spread across 14 distinct protein structures, our results show good overall correlation with experiment with an R2 of 0.65 and a low mean unsigned error of 0.95 kcal/mol. Application of the FEP approach in conjunction with experimental assessment techniques offers opportunities to lower the time and expense of product development and reduce the risk of costly late-stage failures.

Immunodominance in T cell responses elicited against different domains of detoxified pneumolysin PlyD1.

Detoxified pneumolysin, PlyD1, is a protein vaccine candidate that induces protection against infections with Streptococcus pneumoniae in mouse models. Despite extensive knowledge on antibody responses against PlyD1, limited information is available about PlyD1 induced T cell recognition. Here we interrogated epitope breadth and functional characteristics of the T cell response to PlyD1 in two mouse strains. BALB/c (H-2d) and C57BL/6 (H-2b) mice were vaccinated with Al(OH)3-adjuvanted or non-adjuvanted PlyD1, or placebo, on day 0, 21 and 42 and were sacrificed at day 56 for collection of sera and spleens. Vaccination with adjuvanted and non-adjuvanted PlyD1 induced anti-pneumolysin IgG antibodies with neutralizing capacity in both mouse strains. Adjuvantation of PlyD1 enhanced the serological responses in both strains. In vitro restimulation of splenocytes with PlyD1 and 18-mer synthetic peptides derived from pneumolysin revealed specific proliferative and cytokine responses. For both mouse strains, one immunodominant and three subdominant natural epitopes were identified. Overlap between H-2d and H-2b restricted T cell epitopes was limited, yet similarities were found between epitopes processed in mice and predicted to be immunogenic in humans. H-2d restricted T cell epitopes were localized in pneumolysin domains 2 and 3, whereas H-2b epitopes were scattered over the protein. Cytokine responses show mostly a Th2 profile, with low levels of Th1 cytokines, in both mouse strains. In conclusion, PlyD1 evokes T cell responses in mice directed against multiple epitope regions, that is dependent on Major Histocompatibility Complex (MHC) background. These results are important to understand human PlyD1 T cell immunogenicity, to guide cell mediated immunity studies in the context of vaccine development.

Structure-guided antigen engineering yields pneumolysin mutants suitable for vaccination against pneumococcal disease.

Pneumolysin (PLY) is a cholesterol-binding, pore-forming protein toxin. It is an important virulence factor of Streptococcus pneumoniae and a key vaccine target against pneumococcal disease. We report a systematic structure-driven approach that solves a long-standing problem for vaccine development in this field: detoxification of PLY with retention of its antigenic integrity. Using three conformational restraint techniques, we rationally designed variants of PLY that lack hemolytic activity and yet induce neutralizing antibodies against the wild-type toxin. These results represent a key milestone toward a broad-spectrum protein-based pneumococcal vaccine and illustrate the value of structural knowledge in formulating effective strategies for antigen optimization.

The dynamics of the MgATP-driven closure of MalK, the energy-transducing subunit of the maltose ABC transporter.

The nucleotide binding domains (NBDs) are the energy supplying subunits of ATP-binding cassette (ABC) proteins. They power transport by binding and hydrolyzing ATP. Tracing the pathway between different conformational states of the NBDs during ATP binding, hydrolysis, and release has, however, proven difficult. We have used molecular dynamics simulations to study the ATP-driven association of the NBDs of the maltose ABC transporter, MalK, based on the crystal structures of its open and semiopen dimers. When MgATP was introduced into the binding pockets, the semiopen dimer transitioned to a closed conformation, whereas the open dimer evolved to a semiopen state. In the absence of docked MgATP, however, the twin NBDs of both the open and semiopen starting configurations drifted further apart. Both the presence of MgATP and direct cross-interface protein-protein hydrogen bonds, primarily involving the D-loop, quite likely play a key role in initiating closure. The simulations of the MgATP-docked semiopen form indicate that completion of closure is driven mainly by cross-interface contacts between the gamma-phosphate of ATP and residues in the signature motif. Our simulations also give insight into possible interactions of MalK with the regulatory proteins MalT and enzyme IIA(glc).

Computer simulations of ABC transporter components.

Current computer simulation techniques provide robust tools for studying the detailed structure and functional dynamics of proteins, as well as their interaction with each other and with other biomolecules. In this minireview, we provide an illustration of recent progress and future challenges in computer modeling by discussing computational studies of ATP-binding cassette (ABC) transporters. ABC transporters have multiple components that work in a well coordinated fashion to enable active transport across membranes. The mechanism by which members of this superfamily execute transport remains largely unknown. Molecular dynamics simulations initiated from high-resolution crystal structures of several ABC transporters have proven to be useful in the investigation of the nature of conformational coupling events that may drive transport. In addition, fruitful efforts have been made to predict unknown structures of medically relevant ABC transporters, such as P-glycoprotein, using homology-based computational methods. The various techniques described here are also applicable to gaining an atomically detailed understanding of the functional mechanisms of proteins in general.

Computer simulations of membrane proteins.

Computer simulations are rapidly becoming a standard tool to study the structure and dynamics of lipids and membrane proteins. Increasing computer capacity allows unbiased simulations of lipid and membrane-active peptides. With the increasing number of high-resolution structures of membrane proteins, which also enables homology modelling of more structures, a wide range of membrane proteins can now be simulated over time spans that capture essential biological processes. Longer time scales are accessible by special computational methods. We review recent progress in simulations of membrane proteins.

Conformational transitions induced by the binding of MgATP to the vitamin B12 ATP-binding cassette (ABC) transporter BtuCD.

ATP-binding cassette transporters use the free energy of ATP hydrolysis to transport structurally diverse molecules across prokaryotic and eukaryotic membranes. Computer simulation studies of the "real-time" dynamics of the ATP binding process in BtuCD, the vitamin B12 importer from Escherichia coli, demonstrate that the docking of ATP to the catalytic pockets progressively draws the two cytoplasmic nucleotide-binding cassettes toward each other. Movement of the cassettes into closer opposition in turn induces conformational rearrangement of alpha-helices in the transmembrane domain. The shape of the translocation pathway consequently changes in a manner that could aid the vectorial movement of vitamin B12. These results suggest that ATP binding may indeed represent the power stroke in the catalytic mechanism. Moreover, occlusion of ATP at one catalytic site is mechanically coupled to opening of the nucleotide-binding pocket at the second site. We propose that this asymmetry in nucleotide binding behavior at the two catalytic pockets may form the structural basis by which the transporter is able to alternate ATP hydrolysis from one site to the other.

Cytotoxic 1,3-diarylidene-2-tetralones and related compounds.

A number of 1,3-arylidene-2-tetralones 1, 2 and 4 were synthesised and demonstrated cytotoxic activity towards murine P388 and L1210 cells as well as human Molt 4/C8 and CEM T-lymphocytes. In general, the related 1-arylidene-2-tetralones 3 possessed lower potencies in these screens than the compounds in series 1 and 4. Approximately, half of the compounds were evaluated against a panel of human tumour cell lines. In this screen, most of the enones were more cytotoxic than the established anticancer agent melphalan and some demonstrated selective toxicity towards leukemic and colon cancer cells. The modes of action of representative compounds include interfering with the biosyntheses of nucleic acids and proteins as well as altering redox potentials. The compounds were well tolerated when administered intraperiteonally to mice. Thus these novel enones are promising prototypic molecules due to their potent cytotoxic properties and lack of significant murine toxicity.

Correlations between cytotoxicity and topography of some 2-arylidenebenzocycloalkanones determined by X-ray crystallography.

Three series of 2-arylidenebenzocycloalkanones 1-3 were prepared in order to compare the topography of the molecules with cytotoxicity. These compounds contain two aryl rings whose spatial relationships to each other were influenced by the size of the alicyclic ring and the nature of the substituents in the arylidene aryl rings. All compounds were evaluated against murine P388 and L1210 cells as well as human Molt 4/C8 and CEM T-lymphocytes. From these results, 1l and 2c,l emerged as useful lead molecules and 1l was shown to significantly inhibit macromolecular DNA, RNA, and protein syntheses in L1210 cells. Various interatomic distances, bond angles, and a torsion angle of 19 representative compounds were determined by X-ray crystallography, and correlations between these data and the cytotoxicity were noted in nearly 40% of the cases examined. Structure-activity relationships revealed that in general, the steric properties of the groups in the arylidene aryl ring, as revealed by measurements of the molar refractivity values, contributed more to bioactivity than the electronic and hydrophobic properties of the aryl substituents. The compounds displayed little murine toxicity, which favors the decision to develop these molecules as cytotoxic and anticancer agents.

Cytotoxic 1,4-bis(2-oxo-1-cycloalkylmethylene)benzenes and related compounds.

A series of 1,4-bis(2-oxo-1-cycloalkylmethylene)benzenes 2a-c and 4 and a related acyclic analogue 6a were synthesised and converted to the corresponding Mannich bases 3a-c, 5 and 6b. Evaluation of these compounds against murine P388 and L1210 cells as well as human Molt 4/C8 and CEM T-lymphocytes revealed that the Mannich bases were more cytotoxic than the corresponding unsaturated ketones. 1,4-bis(3-Dimethylaminomethyl-2-oxo-1-cyclohexylmethylene)benzene dihydrochloride (3a) had lower IC(50) values than melphalan against the four cell lines and was 15 times more potent than this drug when examined against a panel of human tumours.

Cytotoxic N-[4-(3-aryl-3-oxo-1-propenyl)phenylcarbonyl]-3,5-bis(phenylmethylene)-4-piperidones and related compounds.

A series of 4-carboxychalcones 1 were prepared and coupled to 3,5-bis(phenylmethylene)-4-piperidone (2) giving rise to a novel series of N-[4-(3-aryl-3-oxo-1-propenyl)phenylcarbonyl]-3,5-bis(phenylmethylene)-4-piperidones (3). Molecular simplification of the amides 3 led to the formation of the corresponding N-(3-aryl-1-oxo-2-propenyl)-3,5-bis(phenylmethylene)-4-piperidones (4). A cytotoxic evaluation of the compounds in series 1-4 utilized murine P388 and L1210 cells as well as human Molt 4/C8 and CEM T-lymphocytes. In general, the compounds displayed significant toxicity; the IC(50) values of 54% of the enones were less than 10 microM when all four screens were considered and less than 1 microM for all members of series 3 in the P388 assay. Various correlations were established between the potencies of the compounds in series 1, 3 and 4 and the Hammett sigma, Hansch pi and molecular refractivity constants of the aryl substituents. Several torsion angles and interatomic distances of five representative compounds in series 3 and 4 were determined by X-ray crystallography, some of which contributed to the observed bioactivity. The marked cytotoxicity and lack of murine toxicity of most of the compounds described in this study, as well as their selective toxicity towards different tumour cell lines, revealed that development of the enones 2-4 as novel candidate antineoplastic agents should be pursued.