Evaluation of a Covalent Library of Diverse Warheads (CovLib) Binding to JNK3, USP7, or p53

Theresa Klett,1,* Martin Schwer,1,* Larissa N Ernst,1,* Marc U Engelhardt,1 Simon J Jaag,2 Benedikt Masberg,2 Cornelius Knappe,2 Michael Lämmerhofer,2 Matthias Gehringer,3,4 Frank M Boeckler1,5 1Laboratory for Molecular Design & Pharmaceutical Biophysics, Institute of Pharmaceutical Sciences, Department of Pharmacy and Biochemistry, Eberhard Karls Universität Tübingen, Tübingen, 72076, Germany; 2Pharmaceutical (Bio-) Analysis, Institute of Pharmaceutical Sciences, Department of Pharmacy and Biochemistry, Eberhard Karls Universität Tübingen, Tübingen, 72076, Germany; 3Pharmaceutical Chemistry, Institute of Pharmaceutical Sciences, Department of Pharmacy and Biochemistry, Eberhard Karls Universität Tübingen, Tübingen, 72076, Germany; 4Medicinal Chemistry, Institute for Biomedical Engineering, Eberhard Karls Universität Tübingen, Tübingen, 72076, Germany; 5Interfaculty Institute for Biomedical Informatics (IBMI), Eberhard Karls Universität Tübingen, Tübingen, 72076, Germany*These authors contributed equally to this workCorrespondence: Frank M Boeckler, Laboratory for Molecular Design & Pharmaceutical Biophysics, Institute of Pharmaceutical Sciences, Department of Pharmacy and Biochemistry, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 8 (Haus B), Tübingen, D – 72076, Germany, Tel +49 7071 29 74567, Email [email protected]: Over the last few years, covalent fragment-based drug discovery has gained significant importance. Thus, striving for more warhead diversity, we conceived a library consisting of 20 covalently reacting compounds. Our covalent fragment library (CovLib) contains four different warhead classes, including five α-cyanoacacrylamides/acrylates (CA), three epoxides (EO), four vinyl sulfones (VS), and eight electron-deficient heteroarenes with a leaving group (SNAr/SN).Methods: After predicting the theoretical solubility of the fragments by LogP and LogS during the selection process, we determined their experimental solubility using a turbidimetric solubility assay. The reactivities of the different compounds were measured in a high-throughput 5,5’-dithiobis-(2-nitrobenzoic acid) DTNB assay, followed by a (glutathione) GSH stability assay. We employed the CovLib in a (differential scanning fluorimetry) DSF-based screening against different targets: c-Jun N-terminal kinase 3 (JNK3), ubiquitin-specific protease 7 (USP7), and the tumor suppressor p53. Finally, the covalent binding was confirmed by intact protein mass spectrometry (MS).Results: In general, the purchased fragments turned out to be sufficiently soluble. Additionally, they covered a broad spectrum of reactivity. All investigated α-cyanoacrylamides/acrylates and all structurally confirmed epoxides turned out to be less reactive compounds, possibly due to steric hindrance and reversibility (for α-cyanoacrylamides/acrylates). The SNAr and vinyl sulfone fragments are either highly reactive or stable. DSF measurements with the different targets JNK3, USP7, and p53 identified reactive fragment hits causing a shift in the melting temperatures of the proteins. MS confirmed the covalent binding mode of all these fragments to USP7 and p53, while additionally identifying the SNAr-type electrophile SN002 as a mildly reactive covalent hit for p53.Conclusion: The screening and target evaluation of the CovLib revealed first interesting hits. The highly cysteine-reactive fragments VS004, SN001, SN006, and SN007 covalently modify several target proteins and showed distinct shifts in the melting temperatures up to +5.1 °C and − 9.1 °C. Keywords: covalent fragment-based drug discovery, differential scanning fluorimetry, 5,5’-dithiobis-(2-nitrobenzoic acid), intact protein mass spectrometry, glutathion

The TPR Domain in the Host Cyp40-like Cyclophilin Binds to the Viral Replication Protein and Inhibits the Assembly of the Tombusviral Replicase

Replication of plus-stranded RNA viruses is greatly affected by numerous host-coded proteins acting either as susceptibility or resistance factors. Previous genome-wide screens and global proteomics approaches with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host revealed the involvement of cyclophilins, which are a large family of host prolyl isomerases, in TBSV replication. In this paper, we identified those members of the large cyclophilin family that interacted with the viral replication proteins and inhibited TBSV replication. Further characterization of the most effective cyclophilin, the Cyp40-like Cpr7p, revealed that it strongly inhibits many steps during TBSV replication in a cell-free replication assay. These steps include viral RNA recruitment inhibited via binding of Cpr7p to the RNA-binding region of the viral replication protein; the assembly of the viral replicase complex and viral RNA synthesis. Since the TPR (tetratricopeptide repeats) domain, but not the catalytic domain of Cpr7p is needed for the inhibitory effect on TBSV replication, it seems that the chaperone activity of Cpr7p provides the negative regulatory function. We also show that three Cyp40-like proteins from plants can inhibit TBSV replication in vitro and Cpr7p is also effective against Nodamura virus, an insect pathogen. Overall, the current work revealed a role for Cyp40-like proteins and their TPR domains as regulators of RNA virus replication

Optimal Social Insurance and Health Inequality

This paper integrates into public economics a biologically founded, stochastic process of individual ageing. The novel approach enables us to quantitatively characterize the optimal joint design of health and retirement policy behind the veil of ignorance for today and in response to future medical progress. Calibrating our model to Germany, we find that future progress in medical technology calls for a potentially drastic increase in health spending that typically should be accompanied by a lower pension savings rate and a higher retirement age. Interestingly, medical progress and higher health spending are in conflict with the goal to reduce health inequality

Flexible pension take-up in social security

This paper studies the redistribution and welfare effects of increasing the flexibility of individual pension take-up. We use an overlapping-generations model with Beveridgean pay-as-you-go pensions and heterogeneous individuals who differ in ability and lifespan. We find that introducing flexible pension take-up can induce a Pareto improvement when the initial pension scheme contains within-cohort redistribution and induces early retirement. Such a Pareto improving reform entails the application of uniform actuarial adjustment of pension entitlements based on average lifespan. Introducing actuarial non-neutrality that stimulates later retirement further improves such a flexibility reform

A Unique Role for the Host ESCRT Proteins in Replication of Tomato bushy stunt virus

Plus-stranded RNA viruses replicate in infected cells by assembling viral replicase complexes consisting of viral- and host-coded proteins. Previous genome-wide screens with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host revealed the involvement of seven ESCRT (endosomal sorting complexes required for transport) proteins in viral replication. In this paper, we show that the expression of dominant negative Vps23p, Vps24p, Snf7p, and Vps4p ESCRT factors inhibited virus replication in the plant host, suggesting that tombusviruses co-opt selected ESCRT proteins for the assembly of the viral replicase complex. We also show that TBSV p33 replication protein interacts with Vps23p ESCRT-I and Bro1p accessory ESCRT factors. The interaction with p33 leads to the recruitment of Vps23p to the peroxisomes, the sites of TBSV replication. The viral replicase showed reduced activity and the minus-stranded viral RNA in the replicase became more accessible to ribonuclease when derived from vps23Δ or vps24Δ yeast, suggesting that the protection of the viral RNA is compromised within the replicase complex assembled in the absence of ESCRT proteins. The recruitment of ESCRT proteins is needed for the precise assembly of the replicase complex, which might help the virus evade recognition by the host defense surveillance system and/or prevent viral RNA destruction by the gene silencing machinery

A Co-Opted DEAD-Box RNA Helicase Enhances Tombusvirus Plus-Strand Synthesis

Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. In this paper, we show that an essential translation factor, Ded1p DEAD-box RNA helicase of yeast, directly affects replication of Tomato bushy stunt virus (TBSV). To separate the role of Ded1p in viral protein translation from its putative replication function, we utilized a cell-free TBSV replication assay and recombinant Ded1p. The in vitro data show that Ded1p plays a role in enhancing plus-strand synthesis by the viral replicase. We also find that Ded1p is a component of the tombusvirus replicase complex and Ded1p binds to the 3′-end of the viral minus-stranded RNA. The data obtained with wt and ATPase deficient Ded1p mutants support the model that Ded1p unwinds local structures at the 3′-end of the TBSV (−)RNA, rendering the RNA compatible for initiation of (+)-strand synthesis. Interestingly, we find that Ded1p and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is another host factor for TBSV, play non-overlapping functions to enhance (+)-strand synthesis. Altogether, the two host factors enhance TBSV replication synergistically by interacting with the viral (−)RNA and the replication proteins. In addition, we have developed an in vitro assay for Flock house virus (FHV), a small RNA virus of insects, that also demonstrated positive effect on FHV replicase activity by the added Ded1p helicase. Thus, two small RNA viruses, which do not code for their own helicases, seems to recruit a host RNA helicase to aid their replication in infected cells

Education, Demographics, and the Economy

This paper deals with two issues concerning the effects of population aging on education decisions in the presence of a PAYG pension system: We first analyze the effects of an aging population per se on individual skill choices and continuous education and the production structure. Second, we study the implications of postponed retirement, which is often proposed as a measure to cope with the economic challenges of increased longevity. Our study uses a dynamic general equilibrium framework with overlapping generations and probabilistic aging. Themodel allows for capital-skill complementarity in the production of final output. As a response to population aging, in a small open economy with a fixed interest rate, our first simulation shows that GDP is depressed due to an adverse effect on skill choice and labor supply. We then introduce postponed retirement as a potentially dampening policy measure due to its encouragement of human capital formation. However, since there is less private saving in this scenario, the overall effect on GDP is even worse than in the pure aging scenario

Neutron-induced nucleosynthesis

Neutron--induced nucleosynthesis plays an important role in astrophysical scenarios like in primordial nucleosynthesis in the early universe, in the s--process occurring in Red Giants, and in the $\alpha$--rich freeze--out and r--process taking place in supernovae of type II. A review of the three important aspects of neutron--induced nucleosynthesis is given: astrophysical background, experimental methods and theoretical models for determining reaction cross sections and reaction rates at thermonuclear energies. Three specific examples of neutron capture at thermal and thermonuclear energies are discussed in some detail.Comment: 40 pages (uses kluwer.sty), 2 postscript figures (uses psfig), accepted for publication in Surveys in Geophysics, uuencoded tex-files and postscript-files available at ftp://is1.kph.tuwien.ac.at/pub/ohu/Geo.u

Should Pensions Be Progressive? Yes, at Least in Germany!

Recent reforms that aim at reducing the upcoming burdens of population ageing might seriously harm low income individuals. An increase in old-age poverty and disability will be the result. Under this prospect, the present paper quantitatively characterizes the optimal progressivity of unfunded pension systems in an overlapping generations model with idiosyncratic income, disability and longevity risk as well as endogenous labor supply at the intensive and extensive margin. Focusing on the German pension system, our model features the most recent demographic projections and distinguishes three skill classes with skill-dependent risk profiles. Starting from a baseline path that reflects a purely earnings related pension system, we increase the degree of progressivity and compute the resulting macroeconomic, welfare and efficiency effects. For our most preferred parametrization we find an optimal flat-rate pension share of 40 percent. This indicates that in Germany recent reforms that aim at raising retirement age and cutting benefit levels should be complemented by increases in pension progressivity, since improved insurance provision dominates higher labor supply distortions. In addition, we also find that reductions in the benefit level (i.e. privatization) will only reduce economic efficiency