Identification of lead molecules for the development of antivirals targeting the Ebola virus matrix protein VP40

Viruses of the genus Ebola virus cause severe fevers with unusually high case fatality rates, and as of today, no licensed antiviral drugs are available. The Ebola virus matrix protein VP40 plays a major role during budding of new viral particles and is also involved in the regulation of viral genome replication and transcription. VP40 exists in three different homo-oligomeric forms, namely dimers, octamers and polymeric filaments. Dimers are transported towards the plasma membrane where the interaction with lipids triggers the formation of VP40-filaments. Filaments represent a flexible row of dimers which enable budding of viral particles and line the inner layer of the new virions. Upon binding of cellular RNA, VP40 dimers turn into ring-shaped octamers that exert an inhibitory effect on viral RNA synthesis. For the present work, the resolution of the VP40 crystal structure was improved and a structure-guided drug design was employed with the aim to impair VP40’s essential homo-oligomerization. The study investigated VP40 of two Ebola virus strains Zaire (zVP40) and Sudan (sVP40). Residues L117 and W95 are so-called hot spot amino acids of the dimeric and octameric interface of sVP40 that were characterized by mutational analyses. As expected, both sVP40 wildtype (WT) and W95A formed dimers nearly exclusively whereas sVP40 L117A formed mainly monomers when expressed in E. coli. Surprisingly though, the structure of both sVP40∆43 mutant oligomers was similar to sVP40 WT. As a dimeric crystal packing was surprising for monomeric sVP40 L117A, crystallographic artefacts were considered which prompted the structural analysis of the mutants in solution. Using hydrogen-deuterium exchange mass spectrometry and thermal shift assays, it could be shown that both mutants exhibited increased fluidity and decreased stability in solution and it could be confirmed that sVP40 L117A was indeed a monomer. In cellulo, the ability of sVP40 L117A to form virus-like particles (VLPs) and inhibit viral genome replication and transcription was completely abolished, whereas sVP40 W95A exhibited a gain of function as this protein released more VP40-VLPs into the cellular supernatant and also showed a stronger inhibition of viral RNA synthesis. This data suggests that targeting homo-oligomerization is a promising strategy to impair VP40 functionality, but demands interdisciplinary methods, especially regarding structure determination. Based on a high-resolution crystal structure of sVP40∆43 WT, the structure of the C-terminus of sVP40 could be analyzed. The C-terminus contains the only two cysteines of the molecule which were oxidized and formed a disulfide bridge in the crystal. When VP40 was expressed in mammalian cells and released into the supernatant, the cysteines were also oxidized by post-translational modifications such as glutathionylation and nitrosylation. In vitro, VP40 could be reduced again by human thioredoxin. While the overall structure and oligomeric state of sVP40 was preserved, mutation of the cysteins resulted in altered phenotypes with regard to VP40’s ability to regulate viral RNA synthesis and to induce budding and particle formation. In an attempt to disrupt homo-oligomerization directly, interface-mimicking peptides were designed and tested in both functional cell culture and biochemical assays. While their binding to VP40 could be demonstrated, the peptides were unable to influence VP40’s functions or self-assembly. Further, the dimeric interface of VP40 should be targeted with small molecules. To this end, disulfide tethering was established as an alternative approach using a sVP40 variant with a cysteine residue near the dimeric interface (N67C). This method exploits the formation of a covalent disulfide bridge between the introduced cysteine at position 67 and thiol-containing fragments. Upon incubation under reducing conditions, only fragments with additional interactions to VP40 were bound favourably and could be detected via intact protein mass spectrometry, yielding several fragment hits. While no structural information of one of the sVP40 N67C-fragment complexes could be determined to assess binding mode and location, this strategy proved to be highly successful in identifying promising lead-like molecules. Using a library of 96 preselected fragments and crystal soaking, salicylic acid (SA) was identified as a crystallographic binder of VP40. The binding to VP40 could be confirmed in solution. As expected, the weak binding resulted in only minor effects on VP40’s function in RNA synthesis and budding. The characterization of residues of VP40 involved in the interaction with SA (L158 and R214) suggested that the binding pocket between the N- and the C-terminal domain is a highly vulnerable target site as mutation of these residues resulted in a decreased ability to regulate viral genome replication and transcription for sVP40 R214A as well as decreased budding for both sVP40 L158A and R214A. This prompted the testing of SA derivatives and the identification of four further crystallographic binders (3-amino-SA; 4-fluoro-SA, 4-fluoro-2-hydroxybenzamide and 5-amino-SA). Further structure-guided drug design led to the design, synthesis and testing of LL060, a compound that was also able to impair the formation of VP40-VLPs. In summary, a drug design process from scratch to target the function of Ebola virus VP40 is described. Here, we characterized and validated VP40 homo-oligomerization as a target and identified several lead-like molecules originating from a site-directed ligand discovery screening. The highly promising lead compound LL060, which was identified via crystal soaking represents the starting point for the development of a potent Ebola virus inhibitor

Decompressive craniectomy plus best medical treatment versus best medical treatment alone for spontaneous severe deep supratentorial intracerebral haemorrhage:a randomised controlled clinical trial

BACKGROUND: It is unknown whether decompressive craniectomy improves clinical outcome for people with spontaneous severe deep intracerebral haemorrhage. The SWITCH trial aimed to assess whether decompressive craniectomy plus best medical treatment in these patients improves outcome at 6 months compared to best medical treatment alone.METHODS: In this multicentre, randomised, open-label, assessor-blinded trial conducted in 42 stroke centres in Austria, Belgium, Finland, France, Germany, the Netherlands, Spain, Sweden, and Switzerland, adults (18-75 years) with a severe intracerebral haemorrhage involving the basal ganglia or thalamus were randomly assigned to receive either decompressive craniectomy plus best medical treatment or best medical treatment alone. The primary outcome was a score of 5-6 on the modified Rankin Scale (mRS) at 180 days, analysed in the intention-to-treat population. This trial is registered with ClincalTrials.gov, NCT02258919, and is completed.FINDINGS: SWITCH had to be stopped early due to lack of funding. Between Oct 6, 2014, and April 4, 2023, 201 individuals were randomly assigned and 197 gave delayed informed consent (96 decompressive craniectomy plus best medical treatment, 101 best medical treatment). 63 (32%) were women and 134 (68%) men, the median age was 61 years (IQR 51-68), and the median haematoma volume 57 mL (IQR 44-74). 42 (44%) of 95 participants assigned to decompressive craniectomy plus best medical treatment and 55 (58%) assigned to best medical treatment alone had an mRS of 5-6 at 180 days (adjusted risk ratio [aRR] 0·77, 95% CI 0·59 to 1·01, adjusted risk difference [aRD] -13%, 95% CI -26 to 0, p=0·057). In the per-protocol analysis, 36 (47%) of 77 participants in the decompressive craniectomy plus best medical treatment group and 44 (60%) of 73 in the best medical treatment alone group had an mRS of 5-6 (aRR 0·76, 95% CI 0·58 to 1·00, aRD -15%, 95% CI -28 to 0). Severe adverse events occurred in 42 (41%) of 103 participants receiving decompressive craniectomy plus best medical treatment and 41 (44%) of 94 receiving best medical treatment.INTERPRETATION: SWITCH provides weak evidence that decompressive craniectomy plus best medical treatment might be superior to best medical treatment alone in people with severe deep intracerebral haemorrhage. The results do not apply to intracerebral haemorrhage in other locations, and survival is associated with severe disability in both groups.FUNDING: Swiss National Science Foundation, Swiss Heart Foundation, Inselspital Stiftung, and Boehringer Ingelheim.</p

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Structural Analysis of <i>Plasmodium falciparum</i> Hexokinase Provides Novel Information about Catalysis Due to a <i>Plasmodium</i>-Specific Insertion

The protozoan parasite Plasmodium falciparum is the causative pathogen of the most severe form of malaria, for which novel strategies for treatment are urgently required. The primary energy supply for intraerythrocytic stages of Plasmodium is the production of ATP via glycolysis. Due to the parasite’s strong dependence on this pathway and the significant structural differences of its glycolytic enzymes compared to its human counterpart, glycolysis is considered a potential drug target. In this study, we provide the first three-dimensional protein structure of P. falciparum hexokinase (PfHK) containing novel information about the mechanisms of PfHK. We identified for the first time a Plasmodium-specific insertion that lines the active site. Moreover, we propose that this insertion plays a role in ATP binding. Residues of the insertion further seem to affect the tetrameric interface and therefore suggest a special way of communication among the different monomers. In addition, we confirmed that PfHK is targeted and affected by oxidative posttranslational modifications (oxPTMs). Both S-glutathionylation and S-nitrosation revealed an inhibitory effect on the enzymatic activity of PfHK

Effects of inorganic nitrate and vitamin C co-supplementation on blood pressure and vascular function in younger and older healthy adults: A randomised double-blind crossover trial

Background: Vitamin C and inorganic nitrate have been linked to enhanced nitric oxide (NO) production and reduced oxidative stress. Vitamin C may also enhance the conversion of nitrite into NO. Aims: We investigated the potential acute effects of vitamin C and inorganic nitrate co-supplementation on blood pressure (BP) and peripheral vascular function. The secondary aim was to investigate whether age modified the effects of vitamin C and inorganic nitrate on these vascular outcomes. Methods: Ten younger (age 18–40 y) and ten older (age 55–70 y) healthy participants were enrolled in a randomised double-blind crossover clinical trial. Participants ingested a solution of potassium nitrate (7 mg/kg body weight) and/or vitamin C (20 mg/kg body weight) or their placebos. Acute changes in resting BP and vascular function (post-occlusion reactive hyperemia [PORH], peripheral pulse wave velocity [PWV]) were monitored over a 3-h period. Results: Vitamin C supplementation reduced PWV significantly (vitamin C: −0.70 ± 0.31 m/s; vitamin C placebo: +0.43 ± 0.30 m/s; P = 0.007). There were significant interactions between age and vitamin C for systolic, diastolic, and mean arterial BP (P = 0.02, P = 0.03, P = 0.02, respectively), with systolic, diastolic and mean BP decreasing in older participants and diastolic BP increasing in younger participants following vitamin C administration. Nitrate supplementation did not influence BP (systolic: P = 0.81; diastolic: P = 0.24; mean BP: P = 0.87) or vascular function (PORH: P = 0.05; PWV: P = 0.44) significantly in both younger and older participants. However, combined supplementation with nitrate and vitamin C reduced mean arterial BP (−2.6 mmHg, P = 0.03) and decreased PWV in older participants (PWV: −2.0 m/s, P = 0.02). Conclusions: The co-administration of a single dose of inorganic nitrate and vitamin C lowered diastolic BP and improved PVW in older participants. Vitamin C supplementation improved PWV in both age groups but decreased systolic and mean BP in older participants only

Decompressive craniectomy plus best medical treatment versus best medical treatment alone for spontaneous severe deep supratentorial intracerebral haemorrhage: a randomised controlled clinical trial.

BACKGROUND It is unknown whether decompressive craniectomy improves clinical outcome for people with spontaneous severe deep intracerebral haemorrhage. The SWITCH trial aimed to assess whether decompressive craniectomy plus best medical treatment in these patients improves outcome at 6 months compared to best medical treatment alone. METHODS In this multicentre, randomised, open-label, assessor-blinded trial conducted in 42 stroke centres in Austria, Belgium, Finland, France, Germany, the Netherlands, Spain, Sweden, and Switzerland, adults (18-75 years) with a severe intracerebral haemorrhage involving the basal ganglia or thalamus were randomly assigned to receive either decompressive craniectomy plus best medical treatment or best medical treatment alone. The primary outcome was a score of 5-6 on the modified Rankin Scale (mRS) at 180 days, analysed in the intention-to-treat population. This trial is registered with ClincalTrials.gov, NCT02258919, and is completed. FINDINGS SWITCH had to be stopped early due to lack of funding. Between Oct 6, 2014, and April 4, 2023, 201 individuals were randomly assigned and 197 gave delayed informed consent (96 decompressive craniectomy plus best medical treatment, 101 best medical treatment). 63 (32%) were women and 134 (68%) men, the median age was 61 years (IQR 51-68), and the median haematoma volume 57 mL (IQR 44-74). 42 (44%) of 95 participants assigned to decompressive craniectomy plus best medical treatment and 55 (58%) assigned to best medical treatment alone had an mRS of 5-6 at 180 days (adjusted risk ratio [aRR] 0·77, 95% CI 0·59 to 1·01, adjusted risk difference [aRD] -13%, 95% CI -26 to 0, p=0·057). In the per-protocol analysis, 36 (47%) of 77 participants in the decompressive craniectomy plus best medical treatment group and 44 (60%) of 73 in the best medical treatment alone group had an mRS of 5-6 (aRR 0·76, 95% CI 0·58 to 1·00, aRD -15%, 95% CI -28 to 0). Severe adverse events occurred in 42 (41%) of 103 participants receiving decompressive craniectomy plus best medical treatment and 41 (44%) of 94 receiving best medical treatment. INTERPRETATION SWITCH provides weak evidence that decompressive craniectomy plus best medical treatment might be superior to best medical treatment alone in people with severe deep intracerebral haemorrhage. The results do not apply to intracerebral haemorrhage in other locations, and survival is associated with severe disability in both groups. FUNDING Swiss National Science Foundation, Swiss Heart Foundation, Inselspital Stiftung, and Boehringer Ingelheim

Searching for VHE gamma-ray emission associated with IceCube neutrino alerts using FACT, H.E.S.S., MAGIC, and VERITAS

The realtime follow-up of neutrino events is a promising approach to search for astrophysical neutrino sources. It has so far provided compelling evidence for a neutrino point source: the flaring gamma-ray blazar TXS 0506+056 observed in coincidence with the high-energy neutrino IceCube-170922A detected by IceCube. The detection of very-high-energy gamma rays (VHE, E&gt;100GeV E &gt; 100 G e V ) from this source helped establish the coincidence and constrained the modeling of the blazar emission at the time of the IceCube event. The four major imaging atmospheric Cherenkov telescope arrays (IACTs) - FACT, H.E.S.S., MAGIC, and VERITAS - operate an active follow-up program of target-of-opportunity observations of neutrino alerts sent by IceCube. This program has two main components. One are the observations of known gamma-ray sources around which a cluster of candidate neutrino events has been identified by IceCube (Gamma-ray Follow-Up, GFU). Second one is the follow-up of single high-energy neutrino candidate events of potential astrophysical origin such as IceCube-170922A. GFU has been recently upgraded by IceCube in collaboration with the IACT groups. We present here recent results from the IACT follow-up programs of IceCube neutrino alerts and a description of the upgraded IceCube GFU system