Murine modeling of menstruation identifies immune correlates of protection during Chlamydia muridarum challenge.

The menstrual cycle influences the risk of acquiring sexually transmitted infections (STIs), including Chlamydia trachomatis (C. trachomatis), although the underlying immune contributions are poorly defined. A mouse model simulating the immune-mediated process of menstruation could provide valuable insights into tissue-specific determinants of protection against chlamydial infection within the cervicovaginal and uterine mucosae comprising the female reproductive tract (FRT). Here, we used the pseudopregnancy approach in naïve C57Bl/6 mice and performed vaginal challenge with Chlamydia muridarum (C. muridarum) at decidualization, endometrial tissue remodeling, or uterine repair. This strategy identified that the time frame comprising uterine repair correlated with robust infection and greater bacterial burden as compared with mice on hormonal contraception, while challenges during endometrial remodeling were least likely to result in a productive infection. By comparing the infection site at early time points following chlamydial challenge, we found that a greater abundance of innate effector populations and proinflammatory signaling, including IFNγ correlated with protection. FRT immune profiling in uninfected mice over pseudopregnancy or in pig-tailed macaques over the menstrual cycle identified NK cell infiltration into the cervicovaginal tissues and lumen over the course of endometrial remodeling. Notably, NK cell depletion over this time frame reversed protection, with mice now productively infected with C. muridarum following challenge. This study shows that the pseudopregnancy murine menstruation model recapitulates immune changes in the FRT as a result of endometrial remodeling and identifies NK cell localization at the FRT as essential for immune protection against primary C. muridarum infection.

Improved efficacy of a next-generation ERT in murine Pompe disease.

Pompe disease is a rare inherited disorder of lysosomal glycogen metabolism due to acid α-glucosidase (GAA) deficiency. Enzyme replacement therapy (ERT) using alglucosidase alfa, a recombinant human GAA (rhGAA), is the only approved treatment for Pompe disease. Although alglucosidase alfa has provided clinical benefits, its poor targeting to key disease-relevant skeletal muscles results in suboptimal efficacy. We are developing an rhGAA, ATB200 (Amicus proprietary rhGAA), with high levels of mannose-6-phosphate that are required for efficient cellular uptake and lysosomal trafficking. When administered in combination with the pharmacological chaperone AT2221 (miglustat), which stabilizes the enzyme and improves its pharmacokinetic properties, ATB200/AT2221 was substantially more potent than alglucosidase alfa in a mouse model of Pompe disease. The new investigational therapy is more effective at reversing the primary abnormality - intralysosomal glycogen accumulation - in multiple muscles. Furthermore, unlike the current standard of care, ATB200/AT2221 dramatically reduces autophagic buildup, a major secondary defect in the diseased muscles. The reversal of lysosomal and autophagic pathologies leads to improved muscle function. These data demonstrate the superiority of ATB200/AT2221 over the currently approved ERT in the murine model.

Mutations in HIV-1 reverse transcriptase affect the errors made in a single cycle of viral replication.

UNLABELLED: The genetic variation in HIV-1 in patients is due to the high rate of viral replication, the high viral load, and the errors made during viral replication. Some of the mutations in reverse transcriptase (RT) that alter the deoxynucleoside triphosphate (dNTP)-binding pocket, including those that confer resistance to nucleoside/nucleotide analogs, affect dNTP selection during replication. The effects of mutations in RT on the spectrum (nature, position, and frequency) of errors made in vivo are poorly understood. We previously determined the mutation rate and the frequency of different types of mutations and identified hot spots for mutations in a lacZα (the α complementing region of lacZ) reporter gene carried by an HIV-1 vector that replicates using wild-type RT. We show here that four mutations (Y115F, M184V, M184I, and Q151M) in the dNTP-binding pocket of RT that had relatively small effects on the overall HIV-1 mutation rate (less than 3-fold compared to the wild type) significantly increased mutations at some specific positions in the lacZα reporter gene. We also show that changes in a sequence that flanks the reporter gene can affect the mutations that arise in the reporter. These data show that changes either in HIV-1 RT or in the sequence of the nucleic acid template can affect the spectrum of mutations made during viral replication. This could, by implication, affect the generation of drug-resistant mutants and immunological-escape mutants in patients. IMPORTANCE: RT is the viral enzyme that converts the RNA genome of HIV into DNA. Errors made during replication allow the virus to escape from the host's immune system and to develop resistance to the available anti-HIV drugs. We show that four different mutations in RT which are known to be associated with resistance to anti-RT drugs modestly increased the overall frequency of errors made during viral replication. However, the increased errors were not uniformly distributed; the additional errors occurred at a small number of positions (hot spots). Moreover, some of the RT mutations preferentially affected the nature of the errors that were made (some RT mutations caused an increase in insertion and deletion errors; others caused an increase in substitution errors). We also show that sequence changes in a region adjacent to a target gene can affect the errors made within the target gene.

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.

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.

Structural basis for the role of the K65R mutation in HIV-1 reverse transcriptase polymerization, excision antagonism, and tenofovir resistance.

K65R is a primary reverse transcriptase (RT) mutation selected in human immunodeficiency virus type 1-infected patients taking antiretroviral regimens containing tenofovir disoproxil fumarate or other nucleoside analog RT drugs. We determined the crystal structures of K65R mutant RT cross-linked to double-stranded DNA and in complexes with tenofovir diphosphate (TFV-DP) or dATP. The crystals permit substitution of TFV-DP with dATP at the dNTP-binding site. The guanidinium planes of the arginines K65R and Arg(72) were stacked to form a molecular platform that restricts the conformational adaptability of both of the residues, which explains the negative effects of the K65R mutation on nucleotide incorporation and on excision. Furthermore, the guanidinium planes of K65R and Arg(72) were stacked in two different rotameric conformations in TFV-DP- and dATP-bound structures that may help explain how K65R RT discriminates the drug from substrates. These K65R-mediated effects on RT structure and function help us to visualize the complex interaction with other key nucleotide RT drug resistance mutations, such as M184V, L74V, and thymidine analog resistance mutations.

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.

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.

The RNA polymerase "switch region" is a target for inhibitors.

The alpha-pyrone antibiotic myxopyronin (Myx) inhibits bacterial RNA polymerase (RNAP). Here, through a combination of genetic, biochemical, and structural approaches, we show that Myx interacts with the RNAP "switch region"--the hinge that mediates opening and closing of the RNAP active center cleft--to prevent interaction of RNAP with promoter DNA. We define the contacts between Myx and RNAP and the effects of Myx on RNAP conformation and propose that Myx functions by interfering with opening of the RNAP active-center cleft during transcription initiation. We further show that the structurally related alpha-pyrone antibiotic corallopyronin (Cor) and the structurally unrelated macrocyclic-lactone antibiotic ripostatin (Rip) function analogously to Myx. The RNAP switch region is distant from targets of previously characterized RNAP inhibitors, and, correspondingly, Myx, Cor, and Rip do not exhibit crossresistance with previously characterized RNAP inhibitors. The RNAP switch region is an attractive target for identification of new broad-spectrum antibacterial therapeutic agents.

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.

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.

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.

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.

Inhibition of bacterial RNA polymerase by streptolydigin: stabilization of a straight-bridge-helix active-center conformation.

We define the target, mechanism, and structural basis of inhibition of bacterial RNA polymerase (RNAP) by the tetramic acid antibiotic streptolydigin (Stl). Stl binds to a site adjacent to but not overlapping the RNAP active center and stabilizes an RNAP-active-center conformational state with a straight-bridge helix. The results provide direct support for the proposals that alternative straight-bridge-helix and bent-bridge-helix RNAP-active-center conformations exist and that cycling between straight-bridge-helix and bent-bridge-helix RNAP-active-center conformations is required for RNAP function. The results set bounds on models for RNAP function and suggest strategies for design of novel antibacterial agents.

Nonnucleoside inhibitor binding affects the interactions of the fingers subdomain of human immunodeficiency virus type 1 reverse transcriptase with DNA.

Site-directed photoaffinity cross-linking experiments were performed by using human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with unique cysteine residues at several positions (i.e., positions 65, 67, 70, and 74) in the fingers subdomain of the p66 subunit. Since neither the introduction of the unique cysteine residues into the fingers nor the modification of the SH groups of these residues with photoaffinity cross-linking reagents caused a significant decrease in the enzymatic activities of RT, we were able to use this system to measure distances between specific positions in the fingers domain of RT and double-stranded DNA. HIV-1 RT is quite flexible. There are conformational changes associated with binding of the normal substrates and nonnucleoside RT inhibitors (NNRTIs). Cross-linking was used to monitor intramolecular movements associated with binding of an NNRTI either in the presence or in the absence of an incoming deoxynucleoside triphosphate (dNTP). Binding an incoming dNTP at the polymerase active site decreased the efficiency of cross-linking but caused only modest changes in the preferred positions of cross-linking. This finding suggests that the fingers of p66 are closer to an extended template in the "open" configuration of the enzyme with the fingers away from the active site than in the closed configuration with the fingers in direct contact with the incoming dNTP. NNRTI binding caused increased cross-linking in experiments with diazirine reagents (especially with a diazirine reagent with a longer linker) and a moderate shift in the preferred sites of interaction with the template. Cross-linking occurred closer to the polymerase active site for RTs modified at positions 70 and 74. The effects of NNRTI binding were more pronounced in the absence of a bound dNTP; pretreatment of HIV-1 RT with an NNRTI reduced the effect of dNTP binding. These observations can be explained if the binding of NNRTI causes a decrease in the flexibility in the fingers subdomain of RT-NNRTI complex and a decrease in the distance from the fingers to the template extension.