Structural and functional insights into the activation of the dual incision activity of UvrC, a key player in bacterial NER.

Bacterial nucleotide excision repair (NER), mediated by the UvrA, UvrB and UvrC proteins is a multistep, ATP-dependent process, that is responsible for the removal of a very wide range of chemically and structurally diverse DNA lesions. DNA damage removal is performed by UvrC, an enzyme possessing a dual endonuclease activity, capable of incising the DNA on either side of the damaged site to release a short single-stranded DNA fragment containing the lesion. Using biochemical and biophysical approaches, we have probed the oligomeric state, UvrB- and DNA-binding abilities and incision activities of wild-type and mutant constructs of UvrC from the radiation resistant bacterium, Deinococcus radiodurans. Moreover, by combining the power of new structure prediction algorithms and experimental crystallographic data, we have assembled the first model of a complete UvrC, revealing several unexpected structural motifs and in particular, a central inactive RNase H domain acting as a platform for the surrounding domains. In this configuration, UvrC is maintained in a 'closed' inactive state that needs to undergo a major rearrangement to adopt an 'open' active state capable of performing the dual incision reaction. Taken together, this study provides important insight into the mechanism of recruitment and activation of UvrC during NER.

European Medicines Agency extension of indication to include the combination immunotherapy cancer drug treatment with nivolumab (Opdivo) and ipilimumab (Yervoy) for adults with intermediate/poor-risk advanced renal cell carcinoma.

On the 15 November 2018, the Committee for Medicinal Products for Human Use adopted an extension to an existing indication for the use of nivolumab (Opdivo) in combination with ipilimumab (Yervoy) for the first-line treatment of adult patients with intermediate/poor-risk advanced renal cell carcinoma (RCC). The approval was based on results from the Pivotal CA209214 study, a randomised, open-label, phase III study, comparing nivolumab +ipilimumab with sunitinib in subjects≥18 years of age with previously untreated advanced RCC (not amenable for surgery or radiotherapy) or metastatic RCC, with a clear-cell component. A total of 1096 patients were randomised in the trial, of which 847 patients had intermediate/poor-risk RCC and received either nivolumab (n=425) in combination with ipilimumab administered every 3 weeks for 4 doses followed by nivolumab monotherapy 3 mg/kg every 2 weeks or sunitinib (n=422) administered orally for 4 weeks followed by 2 weeks off, every cycle. A statistically significant difference in overall survival (OS) was observed in the nivolumab + ipilimumab group compared with the sunitinib group in intermediate/poor-risk subjects (HR 0.63 (99.8% CI 0.44 to 0.89); stratified log-rank 2-sided p-value<0.0001). The median OS was not reached for the nivolumab + ipilimumab group and was 25.95 months for the sunitinib group. The OS rates were 89.5% and 86.2% at 6 months, and 80.1% and 72.1% at 12 months in the nivolumab +ipilimumab and the sunitinib groups, respectively. K-M curves separated after approximately 3 months, favouring nivolumab + ipilimumab. This was not mirrored in the favourable-risk patients where no statistically significant difference was observed between nivolumab + ipilimumab and sunitinib in favourable-risk patients (HR 1.45 (descriptive 99.8% CI 0.51 to 4.12), p =0.2715).

The European Medicines Agency Review of Gilteritinib (Xospata) for the Treatment of Adult Patients with Relapsed or Refractory Acute Myeloid Leukemia with an FLT3 Mutation.

On October 24, 2019, a marketing authorization valid through the European Union (EU) was issued for gilteritinib monotherapy for adult patients who have relapsed or refractory acute myeloid leukemia (AML) with an Fms-like tyrosine kinase 3 (FLT3) mutation. Gilteritinib inhibits FLT3 receptor signaling and proliferation in cells exogenously expressing FLT3 including FLT3 internal tandem duplication (ITD), FLT3 D835Y, and FLT3 ITD D835Y, and it induced apoptosis in leukemic cells expressing FLT3 ITD. The recommended starting dose of gilteritinib is 120 mg (three 40 mg tablets) once daily. Gilteritinib was evaluated in one, phase III, open-label, multicenter, randomized study of gilteritinib (n = 247, gilteritinib arm) versus salvage chemotherapy (n = 124, salvage chemotherapy arm) in patients with relapsed or refractory AML with FLT3 mutation. Overall survival (OS) was statistically significantly different between the two groups with a median OS of 9.3 months in the gilteritinib arm compared with 5.6 months for salvage chemotherapy (hazard ratio, 0.637; 95% confidence interval, 0.490-0.830; p = .0004 one-sided log-rank test). The most common adverse reactions with gilteritinib treatment were blood creatine phosphokinase increase, alanine aminotransferase increase, aspartate aminotransferase increase, blood alkaline phosphatase increase, diarrhea, fatigue, nausea, constipation, cough, peripheral edema, dyspnea, dizziness, hypotension, pain in extremity, asthenia, arthralgia, and myalgia. The objective of this article is to summarize the scientific review of the application leading to regulatory approval in the EU. IMPLICATIONS FOR PRACTICE: Xospata was approved in the European Union as monotherapy for the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) with an Fms-like tyrosine kinase 3 (FLT3) mutation. Gilteritinib resulted in a clinically meaningful and statistically significant improvement of overall survival compared with salvage chemotherapy. At the time of the marketing authorization of gilteritinib, there were no approved standard therapies specifically for adult patients diagnosed with relapsed or refractory AML with FLT3 mutation. In terms of safety, the overall accepted safety profile was considered manageable.

Re-boost immunizations with the peptide-based therapeutic HIV vaccine, Vacc-4x, restores geometric mean viral load set-point during treatment interruption.

BACKGROUND: Vacc-4x, a therapeutic HIV vaccine candidate has previously induced a significant reduction in viral load (VL) set-point compared to placebo upon interruption of combination anti-retroviral therapy (ART) (2007/1 study). This study, (2012/1), explored the potential to maintain Vacc-4x effect by re-boosting eligible 2007/1 study participants. METHODS: Participant inclusion required 2007/1 participants to have completed all Vacc-4x immunizations and interrupted ART for up to 26 weeks. At weeks (wk)0 and 2, participants received intradermal (i.d.) Vacc-4x booster immunizations (1.2mg) on ART with GM-CSF (60µg) i.d. as a local adjuvant. ART was interrupted for up to 16 weeks (wk12-wk28). Participants were then followed on ART until wk36. VL set-point, total proviral DNA (pvDNA) and immunogenicity assessed by IFN-γ ELISPOT, T-cell proliferation and delayed type hypersensitivity (DTH) reactions were compared to participants' values in the 2007/1 study where available. RESULTS: This open, multicenter, clinical study enrolled 33 participants from 9 clinical trial sites in the US and Europe. In the per-protocol (PP) population, the VL set-point geometric mean (GM) 18162 copies/mL was not significantly changed compared to the 2007/1 study (GM VL 22035 copies/mL), (p = 0.453, n = 18). For participants with available preART VL values, the VL set-point (GM 26279 copies/mL) remained significantly lower than the preART VL set-point (GM 74048 copies/mL, p = 0.021, n = 13). A statistically significant reduction in pvDNA (49%) from baseline to wk4 was observed (p = 0.03, n = 26). DTH responses (wk4) increased significantly from baseline (p = 0.006, n = 30) and compared to the 2007/1 study (p = 0.022, n = 29) whilst the proportion of participants with ELISPOT and T-cell proliferation responses was similar between the two studies. CONCLUSIONS: Vacc-4x booster immunizations safely maintained the mean VL set-point at that established following primary Vacc-4x therapeutic immunization. The reduction in pvDNA during ART supports the potential for Vacc-4x immunization to reduce HIV reservoirs and thereby contribute to combination HIV cure strategies.

Postvaccination C-Reactive Protein and C5/gp41732-744 Antibody Level Fold-Changes Over Baseline Are Independent Predictors of Therapeutic HIV Vaccine Effect in a Phase 2 Clinical Study of Vacc-4x.

Therapeutic vaccination has the potential to contribute to functional HIV cure strategies. However, to show functional HIV cure, study participants must be taken off combination antiretroviral therapy (cART). The availability of suitable biomarkers that can predict viral load (VL) or CD4 count outcomes following therapeutic HIV vaccination would reduce the risks associated with cART interruption in such studies. This report sought to determine baseline and postvaccination biomarker predictors of vaccine effect (VE) on VL and CD4 counts following cART interruption in a double-blind, randomized phase 2 study of the peptide-based therapeutic HIV vaccine, Vacc-4x (n = 93), versus placebo (n = 43). Antibody responses to a novel envelope glycoprotein antigen, C5/gp41732-744, and three safety marker measurements [C-reactive protein (CRP), white blood cell, and lactate dehydrogenase] were considered. Interaction tests in univariate and multivariate linear regression models were used to estimate the effect of biomarkers on VE, defined as the VL or CD4 count difference in Vacc-4x versus placebo groups. The reported q-values (considered significant for hypothesis-generating purposes if ≤0.2) accounted for multiple comparisons using the false discovery rate method. Data were analyzed from all available 58 Vacc-4x and 25 placebo recipients before cART resumption. Lower postvaccination fold-change over baseline of CRP concentration (interaction p- (q-) value = 0.005 (0.11) for VL) and higher fold-change of anti-C5/gp41732-744 antibody levels (0.005 (0.11) for VL and 0.009 (0.20) for CD4) were associated with Vacc-4x benefit. These findings suggest potential roles for inflammation and immune activation markers in predicting therapeutic HIV VE.

A novel peptide-based vaccine candidate with protective efficacy against influenza A in a mouse model.

Current influenza vaccines mainly induce antibody responses to the variable hemagglutinin proteins of the virus strains included in the vaccine. Instead, a broadly protective influenza vaccine should aim at inducing antibody- and/or cell-mediated immunity against conserved viral proteins. Vacc-FLU is a peptide based vaccine combining conserved B and T cell epitopes. Peptide selection was done using a proprietary peptide design platform technology focusing on responses to human leukocyte antigen (HLA)-restricted epitopes. Immunization of wild-type mice and mice transgenic for HLA-A2.1 with the peptide mix successfully induced both humoral and cell mediated immune responses. Partial protection from severe weight loss upon challenge was observed in both mouse strains but was stronger and observed at lower vaccine doses in transgenic mice. Our results show that the Vacc-FLU peptide mix is capable of inducing IFNγ-producing T cells and antibody-producing B cells which can protect from severe disease symptoms upon infection.

Cell-Mediated Immune Predictors of Vaccine Effect on Viral Load and CD4 Count in a Phase 2 Therapeutic HIV-1 Vaccine Clinical Trial.

BACKGROUND: In a placebo-controlled trial of the peptide-based therapeutic HIV-1 p24Gag vaccine candidate Vacc-4x, participants on combination antiretroviral therapy (cART) received six immunizations over 18weeks, followed by analytical treatment interruption (ATI) between weeks 28 and 52. Cell-mediated immune responses were investigated as predictors of Vacc-4x effect (VE) on viral load (VL) and CD4 count during ATI. METHODS: All analyses of week 28 responses and fold-changes relative to baseline considered per-protocol participants (Vacc-4x:placebo=72:32) resuming cART after week 40. Linear regression models with interaction tests were used. VE was estimated as the Vacc-4x-placebo difference in log10-transformed VL (VEVL) or CD4 count (VECD4). FINDINGS: A lower fold-change of CD4+ T-cell proliferation was associated with VECD4 at week 48 (p=0.036, multiplicity adjusted q=0.036) and week 52 (p=0.040, q=0.080). A higher fold-change of IFN-γ in proliferation supernatants was associated with VEVL at week 44 (p=0.047, q=0.07). A higher fold-change of TNF-α was associated with VEVL at week 44 (p=0.045, q=0.070), week 48 (p=0.028, q=0.070), and week 52 (p=0.037, q=0.074). A higher fold-change of IL-6 was associated with VEVL at week 48 (p=0.017, q=0.036). TNF-α levels (>median) were associated with VECD4 at week 48 (p=0.009, q=0.009). INTERPRETATION: These exploratory analyses highlight the potential value of investigating biomarkers in T-cell proliferation supernatants for VE in clinical studies.

The therapeutic HIV Env C5/gp41 vaccine candidate Vacc-C5 induces specific T cell regulation in a phase I/II clinical study.

BACKGROUND: Levels of non-neutralising antibodies (AB) to the C5 domain of HIV Env gp120 are inversely related to progression of HIV infection. In this phase I/II clinical study we investigated safety of Vacc-C5, a peptide-based therapeutic vaccine candidate corresponding to C5/gp41732-744 as well as the effects on pre-existing AB levels to C5/gp41732-744, immune activation and T cell responses including exploratory assessments of Vacc-C5-induced T cell regulation. Our hypothesis was that exposure of the C5 peptide motif may have detrimental effects due to several of its HLA-like features and that enhancement of non-neutralising anti-C5 AB by vaccination could reduce C5 exposure and thereby chronic immune activation. METHODS: Thirty-six HIV patients on effective antiretroviral therapy were randomised to one of three dose levels of Vacc-C5 administered intramuscularly with Alhydrogel or intradermally with GM-CSF as adjuvant through initial immunisation and two booster periods over 26 weeks. Vacc-C5-specific AB were measured by ELISA and T cell responses by both IFN-γ ELISPOT and proliferative assays analysed by flow cytometry. Immune regulation was assessed by functional blockade of the two inhibitory cytokines IL-10 and TGF-ß in parallel cultures. Non-parametric statistical tests were applied. RESULTS: Vacc-C5 was found safe and well tolerated in all patients. Only marginal changes in humoral and cellular responses were induced, without any effect on immune activation. Overall, anti-Vacc-C5 AB levels seemed to decrease compared to pre-existing levels. Whereas Vacc-C5-specific CD8+ T cell proliferative responses increased after the first booster period (p = 0.020; CD4+, p = 0.057), they were reduced after the second. In contrast, Vacc-C5-induced T cell regulation increased after completed vaccination (p ≤ 0.027) and was lower at baseline in the few AB responders identified (p = 0.027). CONCLUSIONS: The therapeutic HIV vaccine candidate Vacc-C5 safely induced only marginal immune responses, whereas Vacc-C5-induced T cell regulation markedly increased. Our data support further attention on immune regulation during therapeutic HIV vaccination studies. TRIAL REGISTRATION: NCT01627678 .

Correlation of Antibody Responses to a Peptide Antigen gp120-C5501-512/gp41732-744 with HIV Disease Progression.

Antibodies to the carboxy-terminal constant (C5) region 5 of the HIV-1 envelope glycoprotein gp120 have previously been associated with slow disease progression. This is one of the regions on gp120 that interact with the transmembrane glycoprotein, gp41, anchoring it to the viral and infected cell membrane. This study analyzed humoral responses to a novel heterodimeric peptide construct comprising the C5501-512 region and a compatible region on gp41732-744. Antibody levels to C5501-512/gp41732-744 were associated with slow disease progression in a treatment naive historical longitudinal cohort from Norway (n = 32; p = .00001). Elevated anti-C5501-512/gp41732-744 antibody levels correlated with moderate viral load (VL) (50-10,000 copies/ml) in a cohort, including natural viral suppressors (NVS) in the Unites States (n = 58; p = .002). Analysis of HIV-positive sera from treatment naive patients in Estonia (n = 300) showed an inverse correlation between anti-C5501-512/gp41732-744 antibodies and VL when comparing VL 2,000-10,000 copies/ml with VL >10,000 (p = .050). Further mapping using peptide inhibition of antibody binding revealed that responses to the C5501-506 subdomain correlated with preserved CD4 counts (n = 55; p = .0012) irrespective of VL in this cohort. The C5 region encompassing C5501-506 shows sequence similarity to the shared epitope (SE) of certain HLA-DR associated with immune dysfunction. Partial antigenic cross-reactivity between SE and C5 is indicated by partial inhibition of NVS antibody binding using SE 15-mer peptide (median 65% inhibition), the C5501-506 6-mer peptide (79% inhibition), and binding of rheumatoid arthritis patient sera to both SE and C5 peptide sequences. The potential influence of these observations on HIV-1 pathogenesis remains to be determined.

A case for preART-adjusted endpoints in HIV therapeutic vaccine trials.

BACKGROUND: In a randomized, double-blind, placebo-controlled phase 2 clinical trial of Vacc-4x, a peptide-based therapeutic HIV-1 p24(Gag) vaccine candidate, 135 HIV-infected participants (vaccine:placebo=92:43) received a series of six immunizations while on combination antiretroviral therapy (cART). At week 28, all participants underwent an analytical treatment interruption (ATI) for up to 24 weeks. preART VL appeared to be higher among Vacc-4x recipients. Based on a previous analysis, during ATI viral load (VL) appeared to be lower in Vacc-4x recipients, but no difference in CD4 level was observed between Vacc-4x and placebo groups. We propose fold-change-based endpoints and report comparative analyses accounting for imbalanced preART VL and missing data. METHODS: All analyses included per-protocol (PP) participants who received the full immunization and underwent ATI. Linear regression models were used to identify predictors of study endpoints and to estimate the vaccine effect based on fold changes in CD4 counts or VL over preART values at week 40 or at set-point (geometric mean over weeks 48 and 52 values). We adjusted for potential baseline factors and used a multiple imputation approach to account for missing endpoints due to cART resumption or dropout. P-values were adjusted for multiple comparisons using q-values. RESULTS: preART VL and CD4 count were significant predictors of study endpoints. The vaccine recipients had a higher fold change in week 40 CD4 counts (vaccine vs. placebo mean fold-change difference=0.08; p=0.02; q=0.03), a higher fold change in CD4 count set-point (0.06; p=0.06; q=0.07), a lower fold change in week 40 VL (-0.47; p=0.03; q=0.05), and a lower fold change in VL set-point (-0.50; p=0.02; q=0.03). CONCLUSIONS: These exploratory analyses consistently suggested that Vacc-4x provided positive effects on both CD4 counts and VL. Future HIV therapeutic vaccine studies may adopt similar preART-adjusted endpoints and missing data imputation methods in vaccine effect evaluations.

Remote Control by Inter-Enzyme Allostery: A Novel Paradigm for Regulation of the Shikimate Pathway.

DAHP synthase and chorismate mutase catalyze key steps in the shikimate biosynthetic pathway en route to aromatic amino acids. In Mycobacterium tuberculosis, chorismate mutase (MtCM; Rv0948c), located at the branch point toward phenylalanine and tyrosine, has poor activity on its own. However, it is efficiently activated by the first enzyme of the pathway, DAHP synthase (MtDS; Rv2178c), through formation of a non-covalent MtCM-MtDS complex. Here, we show how MtDS serves as an allosteric platform for feedback regulation of both enzymes, using X-ray crystallography, small-angle X-ray scattering, size-exclusion chromatography, and multi-angle light scattering. Crystal structures of the fully inhibited MtDS and the allosterically down-regulated MtCM-MtDS complex, solved at 2.8 and 2.7Å, respectively, reveal how effector binding at the internal MtDS subunit interfaces regulates the activity of MtDS and MtCM. While binding of all three metabolic end products to MtDS shuts down the entire pathway, the binding of phenylalanine jointly with tyrosine releases MtCM from the MtCM-MtDS complex, hence suppressing MtCM activation by 'inter-enzyme allostery'. This elegant regulatory principle, invoking a transient allosteric enzyme interaction, seems to be driven by dynamics and is likely a general strategy used by nature.

Structural and functional characterization of two unusual endonuclease III enzymes from Deinococcus radiodurans.

While most bacteria possess a single gene encoding the bifunctional DNA glycosylase Endonuclease III (EndoIII) in their genomes, Deinococcus radiodurans possesses three: DR2438 (DrEndoIII1), DR0289 (DrEndoIII2) and DR0982 (DrEndoIII3). Here we have determined the crystal structures of DrEndoIII1 and an N-terminally truncated form of DrEndoIII3 (DrEndoIII3Δ76). We have also generated a homology model of DrEndoIII2 and measured activity of the three enzymes. All three structures consist of two all α-helical domains, one of which exhibits a [4Fe-4S] cluster and the other a HhH-motif, separated by a DNA binding cleft, similar to previously determined structures of endonuclease III from Escherichia coli and Geobacillus stearothermophilus. However, both DrEndoIII1 and DrEndoIII3 possess an extended HhH motif with extra helical features and an altered electrostatic surface potential. In addition, the DNA binding cleft of DrEndoIII3 seems to be less accessible for DNA interactions, while in DrEndoIII1 it seems to be more open. Analysis of the enzyme activities shows that DrEndoIII2 is most similar to the previously studied enzymes, while DrEndoIII1 seems to be more distant with a weaker activity towards substrate DNA containing either thymine glycol or an abasic site. DrEndoIII3 is the most distantly related enzyme and displays no detectable activity towards these substrates even though the suggested catalytic residues are conserved. Based on a comparative structural analysis, we suggest that the altered surface potential, shape of the substrate-binding pockets and specific amino acid substitutions close to the active site and in the DNA interacting loops may underlie the unexpected differences in activity.

Expression, purification and crystallization of two endonuclease III enzymes from Deinococcus radiodurans.

Endonuclease III is a bifunctional DNA glycosylase that removes a wide range of oxidized bases in DNA. Deinococcus radiodurans is an extreme radiation-resistant and desiccation-resistant bacterium and possesses three genes encoding endonuclease III enzymes in its genome: DR2438 (EndoIII-1), DR0289 (EndoIII-2) and DR0982 (EndoIII-3). Here, EndoIII-1 and an N-terminally truncated form of EndoIII-3 (EndoIII-3Δ76) have been expressed, purified and crystallized, and preliminary X-ray crystallographic analyses have been performed to 2.15 and 1.31 Šresolution, respectively. The EndoIII-1 crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 181.38, b = 38.56, c = 37.09 Å, ß = 89.34° and one molecule per asymmetric unit. The EndoIII-3Δ76 crystals also belonged to the monoclinic space group C2, but with unit-cell parameters a = 91.47, b = 40.53, c = 72.47 Å, ß = 102.53° and one molecule per asymmetric unit. The EndoIII-1 structure was determined by molecular replacement, while the truncated EndoIII-3Δ76 structure was determined by single-wavelength anomalous dispersion phasing. Refinement of the structures is in progress.

Electrostatic transition state stabilization rather than reactant destabilization provides the chemical basis for efficient chorismate mutase catalysis.

For more than half a century, transition state theory has provided a useful framework for understanding the origins of enzyme catalysis. As proposed by Pauling, enzymes accelerate chemical reactions by binding transition states tighter than substrates, thereby lowering the activation energy compared with that of the corresponding uncatalyzed process. This paradigm has been challenged for chorismate mutase (CM), a well-characterized metabolic enzyme that catalyzes the rearrangement of chorismate to prephenate. Calculations have predicted the decisive factor in CM catalysis to be ground state destabilization rather than transition state stabilization. Using X-ray crystallography, we show, in contrast, that a sluggish variant of Bacillus subtilis CM, in which a cationic active-site arginine was replaced by a neutral citrulline, is a poor catalyst even though it effectively preorganizes chorismate for the reaction. A series of high-resolution molecular snapshots of the reaction coordinate, including the apo enzyme, and complexes with substrate, transition state analog and product, demonstrate that an active site, which is only complementary in shape to a reactive substrate conformer, is insufficient for effective catalysis. Instead, as with other enzymes, electrostatic stabilization of the CM transition state appears to be crucial for achieving high reaction rates.

Parkinson disease protein DJ-1 binds metals and protects against metal-induced cytotoxicity.

The progressive loss of motor control due to reduction of dopamine-producing neurons in the substantia nigra pars compacta and decreased striatal dopamine levels are the classically described features of Parkinson disease (PD). Neuronal damage also progresses to other regions of the brain, and additional non-motor dysfunctions are common. Accumulation of environmental toxins, such as pesticides and metals, are suggested risk factors for the development of typical late onset PD, although genetic factors seem to be substantial in early onset cases. Mutations of DJ-1 are known to cause a form of recessive early onset Parkinson disease, highlighting an important functional role for DJ-1 in early disease prevention. This study identifies human DJ-1 as a metal-binding protein able to evidently bind copper as well as toxic mercury ions in vitro. The study further characterizes the cytoprotective function of DJ-1 and PD-mutated variants of DJ-1 with respect to induced metal cytotoxicity. The results show that expression of DJ-1 enhances the cells' protective mechanisms against induced metal toxicity and that this protection is lost for DJ-1 PD mutations A104T and D149A. The study also shows that oxidation site-mutated DJ-1 C106A retains its ability to protect cells. We also show that concomitant addition of dopamine exposure sensitizes cells to metal-induced cytotoxicity. We also confirm that redox-active dopamine adducts enhance metal-catalyzed oxidation of intracellular proteins in vivo by use of live cell imaging of redox-sensitive S3roGFP. The study indicates that even a small genetic alteration can sensitize cells to metal-induced cell death, a finding that may revive the interest in exogenous factors in the etiology of PD.

When inhibitors do not inhibit: critical evaluation of rational drug design targeting chorismate mutase from Mycobacterium tuberculosis.

Tuberculosis (TB) is a devastating disease that claims millions of lives every year. Hindered access or non-compliance to medication, especially in developing countries, led to drug resistance, further aggravating the situation. With current standard therapies in use for over 50 years and only few new candidates in clinical trials, there is an urgent call for new TB drugs. A powerful tool for the development of new medication is structure-guided design, combined with virtual screening or docking studies. Here, we report the results of a drug-design project, which we based on a publication that claimed the structure-guided discovery of several promising and highly active inhibitors targeting the secreted chorismate mutase (*MtCM) from Mycobacterium tuberculosis. We set out to further improve on these compounds and synthesized a series of new derivatives. Thorough evaluation of these molecules in enzymatic assays revealed, to our dismay, that neither the claimed lead compounds, nor any of the synthesized derivatives, show any inhibitory effects against *MtCM.

Crystal structures exploring the origins of the broader specificity of escherichia coli heat-labile enterotoxin compared to cholera toxin.

Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are structurally and functionally related and share the same primary receptor, the GM1 ganglioside. Despite their extensive similarities, these two toxins exhibit distinct ligand specificities, with LT being more promiscuous than CT. Here, we have attempted to rationalize the broader binding specificity of LT and the subtle differences between the binding characteristics of LTs from human and porcine origins (mediated by their B subunit pentamers, hLTB and pLTB, respectively). The analysis is based on two crystal structures of pLTB in complexes with the pentasaccharide of its primary ligand, GM1, and with neolactotetraose, the carbohydrate determinant of a typical secondary ligand of LTs, respectively. Important molecular determinants underlying the different binding specificities of LTB and CTB are found to be contributed by Ser95, Tyr18 and Thr4 (or Ser4 of hLTB), which together prestabilize the binding site by positioning Lys91, Glu51 and the adjacent loop region (50-61) containing Ile58 for ligand binding. Glu7 and Ala1 may also play an important role. Many of these residues are closely connected with a recently identified second binding site, and there appears to be cross-talk between the two sites. Binding to N-acetyllactosamine-terminated receptors is further augmented by Arg13 (present in pLT and some hLT variants), as previously predicted.

A novel noncovalent complex of chorismate mutase and DAHP synthase from Mycobacterium tuberculosis: protein purification, crystallization and X-ray diffraction analysis.

Chorismate mutase catalyzes a key step in the shikimate-biosynthetic pathway and hence is an essential enzyme in bacteria, plants and fungi. Mycobacterium tuberculosis contains two chorismate mutases, a secreted and an intracellular one, the latter of which (MtCM; Rv0948c; 90 amino-acid residues; 10 kDa) is the subject of this work. Here are reported the gene expression, purification and crystallization of MtCM alone and of its complex with another shikimate-pathway enzyme, DAHP synthase (MtDS; Rv2178c; 472 amino-acid residues; 52 kDa), which has been shown to enhance the catalytic efficiency of MtCM. The MtCM-MtDS complex represents the first noncovalent enzyme complex from the common shikimate pathway to be structurally characterized. Soaking experiments with a transition-state analogue are also reported. The crystals of MtCM and the MtCM-MtDS complex diffracted to 1.6 and 2.1 A resolution, respectively.

Crystal structure of an anti-ganglioside antibody, and modelling of the functional mimicry of its NeuGc-GM3 antigen by an anti-idiotypic antibody.

N-Glycolylated (NeuGc) gangliosides are tumor-specific antigens and as such represent attractive targets for cancer immunotherapy. The chimeric antibody chP3 selectively recognizes a broad variety of NeuGc gangliosides, showing no cross-reactivity to the highly similar N-acetylated (NeuAc) gangliosides that are common cellular antigens in humans. Here, we report the crystal structure of the chP3 Fab and its computer-docking model with the trisaccharide NeuGcalpha3Galbeta4Glcbeta, which represents the carbohydrate moiety of the tumor-antigen NeuGc-GM3. The interaction involves only the heavy chain of the chP3 antibody. The modelled complex is consistent with all available experimental data and shows good surface complementarity. The negatively charged sialic acid residue NeuGc is buried in a pocket flanked by two arginine residues, VH Arg31 and VH Arg100A. We have further investigated the interaction of chP3 with its anti-idiotypic antibody, 1E10 (also known as Racotumomab), currently in clinical trials as a cancer vaccine. While many of the chP3 residues predicted to interact with the NeuGc ganglioside also feature prominently in the modelled complex of chP3 and 1E10, we do not observe structural mimicry. Rather, we suspect that the anti-idiotype 1E10 may serve as an imprint of the structural characteristics of the chP3 idiotype and, consequently, give rise to antibodies with P3-like properties upon immunization.

Structure and function of a complex between chorismate mutase and DAHP synthase: efficiency boost for the junior partner.

Chorismate mutase catalyzes a key step in the shikimate biosynthetic pathway towards phenylalanine and tyrosine. Curiously, the intracellular chorismate mutase of Mycobacterium tuberculosis (MtCM; Rv0948c) has poor activity and lacks prominent active-site residues. However, its catalytic efficiency increases >100-fold on addition of DAHP synthase (MtDS; Rv2178c), another shikimate-pathway enzyme. The 2.35 A crystal structure of the MtCM-MtDS complex bound to a transition-state analogue shows a central core formed by four MtDS subunits sandwiched between two MtCM dimers. Structural comparisons imply catalytic activation to be a consequence of the repositioning of MtCM active-site residues on binding to MtDS. The mutagenesis of the C-terminal extrusion of MtCM establishes conserved residues as part of the activation machinery. The chorismate-mutase activity of the complex, but not of MtCM alone, is inhibited synergistically by phenylalanine and tyrosine. The complex formation thus endows the shikimate pathway of M. tuberculosis with an important regulatory feature. Experimental evidence suggests that such non-covalent enzyme complexes comprising an AroQ(delta) subclass chorismate mutase like MtCM are abundant in the bacterial order Actinomycetales.