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"Marchand, Bruno"
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Moving on: Is Existenzminimum Still Relevant?
In the inter-war period, progressive architects confronted the building of mass housing with an analogy with rational and functional workplaces. At the 2nd CIAM (Congrès Internationaux d’Architecture Moderne), held in Frankfurt in 1929, this was tested against the formulation of space standards for a vital minimum, in order to increase the quantity of housing and reduce construction costs. This approach presumed the search for optimal living conditions and hygiene. The analogy with the world of work is particularly striking in the case of design of kitchens, removable furniture and storage spaces to maximize the use of space. In rational—and above all minimum—housing, the size of the rooms mainly depends on the size of the furniture. In this perspective, today in Switzerland new housing projects face the same issues, caused by a housing shortage that has plagued the country in the last decades. This suggests that Existenzminimum is still current for contemporary design.
Journal Article
Structural basis of HIV inhibition by translocation-defective RT inhibitor 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA)
4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is the most potent nucleoside analog inhibitor of HIV reverse transcriptase (RT). It retains a 3′-OH yet acts as a chain-terminating agent by diminishing translocation from the pretranslocation nucleotide-binding site (N site) to the posttranslocation primer-binding site (P site). Also, facile misincorporation of EFdA-monophosphate (MP) results in difficult-to-extend mismatched primers. To understand the high potency and unusual inhibition mechanism of EFdA, we solved RT crystal structures (resolutions from 2.4 to 2.9 Å) that include inhibition intermediates (i) before inhibitor incorporation (catalytic complex, RT/DNA/EFdA-triphosphate), (ii) after incorporation of EFdA-MP followed by dT-MP (RT/DNAEFdA-MPP•dT-MPN
), or (iii) after incorporation of two EFdA-MPs (RT/DNAEFdA-MPP•EFdA-MPN
); (iv) the latter was also solved with EFdA-MP mismatched at the N site (RT/DNAEFdA-MPP•EFdA-MP*N
). We report that the inhibition mechanism and potency of EFdA stem from interactions of its 4′-ethynyl at a previously unexploited conserved hydrophobic pocket in the polymerase active site. The high resolution of the catalytic complex structure revealed a network of ordered water molecules at the polymerase active site that stabilize enzyme interactions with nucleotide and DNA substrates. Finally, decreased translocation results from favorable interactions of primer-terminating EFdA-MP at the pretranslocation site and unfavorable posttranslocation interactions that lead to observed localized primer distortions.
Journal Article
Mechanism of Nucleic Acid Unwinding by SARS-CoV Helicase
The non-structural protein 13 (nsp13) of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a helicase that separates double-stranded RNA (dsRNA) or DNA (dsDNA) with a 5' → 3' polarity, using the energy of nucleotide hydrolysis. We determined the minimal mechanism of helicase function by nsp13. We showed a clear unwinding lag with increasing length of the double-stranded region of the nucleic acid, suggesting the presence of intermediates in the unwinding process. To elucidate the nature of the intermediates we carried out transient kinetic analysis of the nsp13 helicase activity. We demonstrated that the enzyme unwinds nucleic acid in discrete steps of 9.3 base-pairs (bp) each, with a catalytic rate of 30 steps per second. Therefore the net unwinding rate is ~280 base-pairs per second. We also showed that nsp12, the SARS-CoV RNA-dependent RNA polymerase (RdRp), enhances (2-fold) the catalytic efficiency of nsp13 by increasing the step size of nucleic acid (RNA/RNA or DNA/DNA) unwinding. This effect is specific for SARS-CoV nsp12, as no change in nsp13 activity was observed when foot-and-mouth-disease virus RdRp was used in place of nsp12. Our data provide experimental evidence that nsp13 and nsp12 can function in a concerted manner to improve the efficiency of viral replication and enhance our understanding of nsp13 function during SARS-CoV RNA synthesis.
Journal Article
Atelier Descombes Rampini: architects of public spaces
Descombes Rampini's projects are prime examples of the skillful, spatial combination of architecture and landscape architecture. In texts, drawings, models, and photographs, this monograph presents more than 45 completed projects and unbuilt designs from the last fifteen years. The publication covers all scales, ranging from spatial planning and urban planning to individual engineering structures.
eBook
Combinations of an acetyl CoA carboxylase inhibitor with hepatic lipid modulating agents do not augment antifibrotic efficacy in preclinical models of NASH and fibrosis
Dysregulated hepatocyte lipid metabolism is a hallmark of hepatic lipotoxicity and contributes to the pathogenesis of nonalcoholic steatohepatitis (NASH). Acetyl CoA carboxylase (ACC) inhibitors decrease hepatocyte lipotoxicity by inhibiting de novo lipogenesis and concomitantly increasing fatty acid oxidation (FAO), and firsocostat, a liver‐targeted inhibitor of ACC1/2, is under evaluation clinically in patients with NASH. ACC inhibition is associated with improvements in indices of NASH and reduced liver triglyceride (TG) content, but also increased circulating TG in subjects with NASH and preclinical rodent models. Here we evaluated whether enhancing hepatocyte FAO by combining ACC inhibitors with peroxisomal proliferator‐activated receptor (PPAR) or thyroid hormone receptor beta (THRβ) agonists could drive greater liver TG reduction and NASH/antifibrotic efficacy, while ameliorating ACC inhibitor–induced hypertriglyceridemia. In high‐fat diet–fed dyslipidemic rats, the addition of PPAR agonists fenofibrate (Feno), elafibranor (Ela), lanifibranor (Lani), seladelpar (Sela) or saroglitazar (Saro), or the THRb agonist resmetirom (Res), to an analogue of firsocostat (ACCi) prevented ACCi‐induced hypertriglyceridemia. However, only PPARα agonists (Feno and Ela) and Res provided additional liver TG reduction. In the choline‐deficient high‐fat diet rat model of advanced liver fibrosis, neither PPARα (Feno) nor THRβ (Res) agonism augmented the antifibrotic efficacy of ACCi. Conclusion: These data suggest that combination therapies targeting hepatocyte lipid metabolism may have beneficial effects on liver TG reduction; however, they may not be sufficient to drive fibrosis regression. Here we evaluated whether combinations of an acetyl CoA carboxylase inhibitor with agents that modulate hepatocyte lipotoxicity would synergistically decrease liver triglyceride and inhibit fibrosis progression in rodent models. Our data clearly demonstrate that some combinations have synergistically lower liver TG without inhibiting fibrosis progression.
Journal Article
Hypersusceptibility mechanism of Tenofovir-resistant HIV to EFdA
Background
The K65R substitution in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is the major resistance mutation selected in patients treated with first-line antiretroviral tenofovir disoproxil fumarate (TDF). 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), is the most potent nucleoside analog RT inhibitor (NRTI) that unlike all approved NRTIs retains a 3'-hydroxyl group and has remarkable potency against wild-type (WT) and drug-resistant HIVs. EFdA acts primarily as a chain terminator by blocking translocation following its incorporation into the nascent DNA chain. EFdA is in preclinical development and its effect on clinically relevant drug resistant HIV strains is critically important for the design of optimal regimens prior to initiation of clinical trials.
Results
Here we report that the K65R RT mutation causes hypersusceptibility to EFdA. Specifically, in single replication cycle experiments we found that EFdA blocks WT HIV ten times more efficiently than TDF. Under the same conditions K65R HIV was inhibited over 70 times more efficiently by EFdA than TDF. We determined the molecular mechanism of this hypersensitivity using enzymatic studies with WT and K65R RT. This substitution causes minor changes in the efficiency of EFdA incorporation with respect to the natural dATP substrate and also in the efficiency of RT translocation following incorporation of the inhibitor into the nascent DNA. However, a significant decrease in the excision efficiency of EFdA-MP from the 3’ primer terminus appears to be the primary cause of increased susceptibility to the inhibitor. Notably, the effects of the mutation are DNA-sequence dependent.
Conclusion
We have elucidated the mechanism of K65R HIV hypersusceptibility to EFdA. Our findings highlight the potential of EFdA to improve combination strategies against TDF-resistant HIV-1 strains.
Journal Article
Inhibitors of Foot and Mouth Disease Virus Targeting a Novel Pocket of the RNA-Dependent RNA Polymerase
Background: Foot-and-Mouth Disease Virus (FMDV) is a picornavirus that infects cloven-hoofed animals and leads to severe losses in livestock production. In the case of an FMD outbreak, emergency vaccination requires at least 7 days to trigger an effective immune response. There are currently no approved inhibitors for the treatment or prevention of FMDV infections. Methodology/Principal Findings: Using a luciferase-based assay we screened a library of compounds and identified seven novel inhibitors of 3Dpol, the RNA-dependent RNA polymerase of FMDV. The compounds inhibited specifically 3Dpol (IC50s from 2-17 µM) and not other viral or bacterial polymerases. Enzyme kinetic studies on the inhibition mechanism by compounds 5D9 and 7F8 showed that they are non-competitive inhibitors with respect to NTP and nucleic acid substrates. Molecular modeling and docking studies into the 3Dpol structure revealed an inhibitor binding pocket proximal to, but distinct from the 3Dpol catalytic site. Residues surrounding this pocket are conserved among all 60 FMDV subtypes. Site directed mutagenesis of two residues located at either side of the pocket caused distinct resistance to the compounds, demonstrating that they indeed bind at this site. Several compounds inhibited viral replication with 5D9 suppressing virus production in FMDV-infected cells with EC50 = 12 µM and EC90 = 20 µM). Significance: We identified several non-competitive inhibitors of FMDV 3Dpol that target a novel binding pocket, which can be used for future structure-based drug design studies. Such studies can lead to the discovery of even more potent antivirals that could provide alternative or supplementary options to contain future outbreaks of FMD.
Journal Article
