Mapping of discontinuous conformational epitopes by amide hydrogen/deuterium exchange mass spectrometry and computational docking.

Understanding antigen-antibody interactions at the sub-molecular level is of particular interest for scientific, regulatory, and intellectual property reasons, especially with increasing demand for monoclonal antibody therapeutic agents. Although various techniques are available for the determination of an epitope, there is no widely applicable, high-resolution, and reliable method available. Here, a combination approach using amide hydrogen/deuterium exchange coupled with proteolysis and mass spectrometry (HDX-MS) and computational docking was applied to investigate antigen-antibody interactions. HDX-MS is a widely applicable, medium-resolution, medium-throughput technology that can be applied to epitope identification. First, the epitopes of cytochrome c-E8, IL-13-CNTO607, and IL-17A-CAT-2200 interactions identified using the HDX-MS method were compared with those identified by X-ray co-crystal structures. The identified epitopes are in good agreement with those identified using high-resolution X-ray crystallography. Second, the HDX-MS data were used as constraints for computational docking. More specifically, the non-epitope residues of an antigen identified using HDX-MS were designated as binding ineligible during computational docking. This approach, termed HDX-DOCK, gave more tightly clustered docking poses than stand-alone docking for all antigen-antibody interactions examined and improved docking results significantly for the cytochrome c-E8 interaction.

Conformation-dependent ligand regulation of ATP hydrolysis by human KSP: activation of basal hydrolysis and inhibition of microtubule-stimulated hydrolysis by a single, small molecule modulator.

Human kinesin spindle protein (KSP)/hsEg5, a member of the kinesin-5 family, is essential for mitotic spindle assembly in dividing human cells and is required for cell cycle progression through mitosis. Inhibition of the ATPase activity of KSP leads to cell cycle arrest during mitosis and subsequent cell death. Ispinesib (SB-715992), a potent and selective inhibitor of KSP, is currently in phase II clinical trials for the treatment of multiple tumor types. Mutations that attenuate Ispinesib binding to KSP in vitro have been identified, highlighting the need for inhibitors that target different binding sites and inhibit KSP activity by novel mechanisms. We report here a small-molecule modulator, KSPA-1, that activates KSP-catalyzed ATP hydrolysis in the absence of microtubules yet inhibits microtubule-stimulated ATP hydrolysis by KSP. KSPA-1 inhibits cell proliferation and induces monopolar-spindle formation in tumor cells. Results from kinetic analyses, microtubule (MT) binding competition assays, and hydrogen/deuterium-exchange studies show that KSPA-1 does not compete directly for microtubule binding. Rather, this compound acts by driving a conformational change in the KSP motor domain and disrupts productive ATP turnover stimulated by MT. These findings provide a novel mechanism for targeting KSP and perhaps other mitotic kinesins.

Isofagomine induced stabilization of glucocerebrosidase.

Structurally destabilizing mutations in acid beta-glucosidase (GCase) can result in Gaucher disease (GD). The iminosugar isofagomine (IFG), a competitive inhibitor and a potential pharmacological chaperone of GCase, is currently undergoing clinical evaluation for the treatment of GD. An X-ray crystallographic study of the GCase-IFG complex revealed a hydrogen bonding network between IFG and certain active site residues. It was suggested that this network may translate into greater global stability. Here it is demonstrated that IFG does increase the global stability of wild-type GCase, shifting its melting curve by approximately 15 degrees C and that it enhances mutant GCase activity in pre-treated N370S/N370S and F213I/L444P patient fibroblasts. Additionally, amide hydrogen/deuterium exchange mass spectroscopy (H/D-Ex) was employed to identify regions within GCase that undergo stabilization upon IFG-binding. H/D-Ex data indicate that the binding of IFG not only restricts the local protein dynamics of the active site, but also propagates this effect into surrounding regions.

Identification of pharmacological chaperones for Gaucher disease and characterization of their effects on beta-glucocerebrosidase by hydrogen/deuterium exchange mass spectrometry.

Point mutations in beta-glucocerebrosidase (GCase) can result in a deficiency of both GCase activity and protein in lysosomes thereby causing Gaucher Disease (GD). Enzyme inhibitors such as isofagomine, acting as pharmacological chaperones (PCs), increase these levels by binding and stabilizing the native form of the enzyme in the endoplasmic reticulum (ER), and allow increased lysosomal transport of the enzyme. A high-throughput screen of the 50,000-compound Maybridge library identified two, non-carbohydrate-based inhibitory molecules, a 2,4-diamino-5-substituted quinazoline (IC(50) 5 microM) and a 5-substituted pyridinyl-2-furamide (IC(50) 8 microM). They raised the levels of functional GCase 1.5-2.5-fold in N370S or F213I GD fibroblasts. Immunofluorescence confirmed that treated GD fibroblasts had decreased levels of GCase in their ER and increased levels in lysosomes. Changes in protein dynamics, monitored by hydrogen/deuterium-exchange mass spectrometry, identified a domain III active-site loop (residues 243-249) as being significantly stabilized upon binding of isofagomine or either of these two new compounds; this suggests a common mechanism for PC enhancement of intracellular transport.

Hydrogen/deuterium-exchange (H/D-Ex) of PPARgamma LBD in the presence of various modulators.

A nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARgamma), is a ligand-dependent transcription factor involved in glucose homeostasis and adipocyte differentiation. PPARgamma is the molecular target of various natural and synthetic molecules, including anti-diabetic agents such as rosiglitazone. Amide hydrogen/deuterium-exchange (H/D-Ex), coupled with proteolysis and mass spectrometry, was applied to study the dynamics of the PPARgamma ligand binding domain (LBD) with or without molecules that modulate PPARgamma activity. The H/D-Ex patterns of ligand-free PPARgamma LBD show that the ligand binding pocket of LBD is significantly more dynamic than the rest of the LBD. Presumably, the binding pocket is intrinsically disordered in order to accommodate different ligands. The presence of two full agonists (rosiglitazone and GW1929), a partial agonist (nTZDpa), and a covalent antagonist (GW9662), changed the dynamics/conformation of PPARgamma LBD and slowed the H/D exchange rate in various regions of the protein. The full agonists slowed the H/D exchange more globally and to a greater extent than the partial agonist or the antagonist, indicating that the full agonist stabilizes the PPARgamma LBD more than the partial agonist or the antagonist. One interesting observation is that the two full agonists significantly stabilized helix 12 while the partial agonist and the antagonist did not perturb the H/D exchange of this region. The results showed that the change in protein dynamics induced by ligand binding may be an important factor for the activation of genes and that H/D-Ex is a useful method for analyzing the biological activity of drug leads.

Epitope mapping by amide hydrogen/deuterium exchange coupled with immobilization of antibody, on-line proteolysis, liquid chromatography and mass spectrometry.

The epitope of horse cytochrome c against monoclonal antibody E8 was determined using amide hydrogen/deuterium (H/D) exchange combined with immobilized antibody, on-line pepsin proteolysis, liquid chromatography (LC), and mass spectrometry (MS). The results were generally in good agreement with contact residues identified by an X-ray co-crystal structure of the E8-cytochrome c complex and results obtained by H/D exchange with nuclear magnetic resonance (NMR) spectrometry. The H/D exchange reaction of cytochrome c was carried out in the presence or absence of immobilized E8 antibody. Regions that gained less deuterium in the presence of the antibody than in its absence are defined as the epitope by the H/D exchange MS method. Control experiments were carefully designed to help identify the epitope with high confidence.

Structure of IL-17A in complex with a potent, fully human neutralizing antibody.

IL-17A is a pro-inflammatory cytokine produced by the newly identified Th17 subset of T-cells. We have isolated a human monoclonal antibody to IL-17A (CAT-2200) that can potently neutralize the effects of recombinant and native human IL-17A. We determined the crystal structure of IL-17A in complex with the CAT-2200 Fab at 2.6 A resolution in order to provide a definitive characterization of the epitope and paratope regions. Approximately a third of the IL-17A dimer is disordered in this crystal structure. The disorder occurs in both independent copies of the complex in the asymmetric unit and does not appear to be influenced by crystal packing. The complex contains one IL-17A dimer sandwiched between two CAT-2200 Fab fragments. The IL-17A is a disulfide-linked homodimer that is similar in structure to IL-17F, adopting a cystine-knot fold. The structure is not inconsistent with the previous prediction of a receptor binding cavity on IL-17 family members. The epitope recognized by CAT-2200 is shown to involve 12 amino acid residues from the quaternary structure of IL-17A, with each Fab contacting both monomers in the dimer. All complementarity-determining regions (CDRs) in the Fab contribute to a total of 16 amino acid residues in the antibody paratope. In vitro affinity optimization was used to generate CAT-2200 from a parental lead antibody using random mutagenesis of CDR3 loops. This resulted in seven amino acid changes (three in VL-CDR3 and four in VH-CDR3) and gave an approximate 30-fold increase in potency in a cell-based neutralization assay. Two of the seven amino acids form part of the CAT-2200 paratope. The observed interaction site between CAT-2200 and IL-17A is consistent with data from hydrogen/deuterium exchange mass spectrometry and mutagenesis approaches.

Specificity of immobilized porcine pepsin in H/D exchange compatible conditions.

Statistical analysis of data from 39 proteins (13 766 amino acid residues) digested with immobilized porcine pepsin under conditions compatible with hydrogen/deuterium (H/D) exchange (<1 degrees C, <30 s) was performed to examine pepsin cleavage specificity. The cleavage of pepsin was most influenced by the amino acid residue at position P1. Phe and Leu are favored residues each with a cleavage probability greater than 40%. His, Lys, Arg, or Pro residues prohibit cleavage when found at the P1 position. Pro also cannot be at position P2 (cleavage probability <0.3%). Occupation of the P3 position by His, Lys, or Arg, or occupation of the P2' position by Pro, also leads to very little cleavage (cleavage probability <1.7%). The average cleavage probability over the entire data set was 13.6%, which is slightly lower than the value previously obtained by Powers et al. (14.8%). This is due, in part, to the larger protein sizes used in the current study. While the specificity of pepsin was similar to that previously observed, higher selectivity was observed in the present study due to less experimental variation in the conditions used to generate our database.