Prognostic potential of creatinine and Cystatin C in COVID-19 - a retrospective cohort study from Karolinska University Hospital.

Acute kidney injury (AKI) is common in COVID-19 and is diagnosed using relative serum creatinine increase. Estimated GFR (eGFR) is a more accurate measure of glomerular filtration due to compensation for age and sex. Serum Cystatin-C, less affected by non-renal factors than creatinine, may further improve renal function estimation and add prognostic information. Our aim is to investigate the importance of a calculated eGFR in relation to creatinine as well as the value of Cystatin-C in patients with severe COVID-19. This study is a retrospective cohort study investigating levels and trends of routine laboratory parameters combined with clinical data from 286 consecutive patients with severe COVID-19 from Karolinska University Hospital. AKI developed in 38% of the patients and 15% were treated with hemodialysis. Mortality in the AKI group was 42% compared to 5% in the non-AKI group. At admission, eGFR, but not creatinine, was significantly associated with AKI development, need of intubation and mortality. Moreover, discrepant results between eGFR creatinine (eGFRCR) and eGFR Cystatin-C (eGFRCYS) was common in the ICU patients compared to non-ICU patients and related to outcome. In addition, we found that daily median Cystatin-C levels during the hospital stay were correlated to neutrophil count. eGFRCR was found to be an overall better prognostic marker than creatinine regarding AKI development and prognosis in severe COVID-19. Fulfillment of Shrunken pore syndrome criteria indicated a higher mortality risk. Cystatin-C may be related to neutrophil count, which could be a clue to the discrepant eGFR results.

Development of a novel therapeutic vaccine carrier that sustains high antibody titers against several targets simultaneously.

With the aim to improve the efficacy of therapeutic vaccines that target self-antigens, we have developed a novel fusion protein vaccine on the basis of the C-terminal multimerizing end of the variable lymphocyte receptor B (VLRB), the Ig equivalent in jawless fishes. Recombinant vaccines were produced in Escherichia coli by fusing the VLRB sequence to 4 different cancer-associated target molecules. The anti-self-immune response generated in mice that were vaccinated with VLRB vaccines was compared with the response in mice that received vaccines that contained bacterial thioredoxin (TRX), previously identified as an efficient carrier. The anti-self-Abs were analyzed with respect to titers, binding properties, and duration of response. VLRB-vaccinated mice displayed a 2- to 10-fold increase in anti-self-Ab titers and a substantial decrease in Abs against the foreign part of the fusion protein compared with the response in TRX-vaccinated mice (P < 0.01). VLRB-generated Ab response had duration similar to the corresponding TRX-generated Abs, but displayed a higher diversity in binding characteristics. Of importance, VLRB vaccines could sustain an immune response against several targets simultaneously. VLRB vaccines fulfill several key criteria for an efficient therapeutic vaccine that targets self-antigens as a result of its small size, its multimerizing capacity, and nonexposed foreign sequences in the fusion protein.-Saupe, F., Reichel, M., Huijbers, E. J. M., Femel, J., Markgren, P.-O., Andersson, C. E., Deindl, S., Danielson, U. H., Hellman, L. T., Olsson, A.-K. Development of a novel therapeutic vaccine carrier that sustains high antibody titers against several targets simultaneously.

2-thioxanthines are mechanism-based inactivators of myeloperoxidase that block oxidative stress during inflammation.

Myeloperoxidase (MPO) is a prime candidate for promoting oxidative stress during inflammation. This abundant enzyme of neutrophils uses hydrogen peroxide to oxidize chloride to highly reactive and toxic chlorine bleach. We have identified 2-thioxanthines as potent mechanism-based inactivators of MPO. Mass spectrometry and x-ray crystal structures revealed that these inhibitors become covalently attached to the heme prosthetic groups of the enzyme. We propose a mechanism whereby 2-thioxanthines are oxidized, and their incipient free radicals react with the heme groups of the enzyme before they can exit the active site. 2-Thioxanthines inhibited MPO in plasma and decreased protein chlorination in a mouse model of peritonitis. They slowed but did not prevent neutrophils from killing bacteria and were poor inhibitors of thyroid peroxidase. Our study shows that MPO is susceptible to the free radicals it generates, and this Achilles' heel of the enzyme can be exploited to block oxidative stress during inflammation.

Structural insights and biological effects of glycogen synthase kinase 3-specific inhibitor AR-A014418.

Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase that has been implicated in pathological conditions such as diabetes and Alzheimer's disease. We report the characterization of a GSK3 inhibitor, AR-A014418, which inhibits GSK3 (IC50 = 104 +/- 27 nM), in an ATP-competitive manner (Ki = 38 nM). AR-A014418 does not significantly inhibit cdk2 or cdk5 (IC50 > 100 microM) or 26 other kinases demonstrating high specificity for GSK3. We report the co-crystallization of AR-A014418 with the GSK3beta protein and provide a description of the interactions within the ATP pocket, as well as an understanding of the structural basis for the selectivity of AR-A014418. AR-A014418 inhibits tau phosphorylation at a GSK3-specific site (Ser-396) in cells stably expressing human four-repeat tau protein. AR-A014418 protects N2A neuroblastoma cells against cell death mediated by inhibition of the phosphatidylinositol 3-kinase/protein kinase B survival pathway. Furthermore, AR-A014418 inhibits neurodegeneration mediated by beta-amyloid peptide in hippocampal slices. AR-A014418 may thus have important applications as a tool to elucidate the role of GSK3 in cellular signaling and possibly in Alzheimer's disease. AR-A014418 is the first compound of a family of specific inhibitors of GSK3 that does not significantly inhibit closely related kinases such as cdk2 or cdk5.

Elucidation of HIV-1 protease resistance by characterization of interaction kinetics between inhibitors and enzyme variants.

The kinetics of the interaction between drug-resistant variants of HIV-1 protease (G48V, V82A, L90M, I84V/L90M, and G48V/V82A/I84V/L90M) and clinically used inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) were determined using biosensor technology. The enzyme variants were immobilized on a biosensor chip and the association and dissociation rate constants (k(on) and k(off)) and affinities (K(D)) for interactions with inhibitors were determined. A unique interaction kinetic profile was observed for each variant/inhibitor combination. Substitution of single amino acids in the protease primarily resulted in reduced affinity through increased k(off) for the inhibitors. For inhibitors characterized by fast association rates to wild-type protease (ritonavir, amprenavir, and indinavir), additional substitutions resulted in a further reduction of affinity by a combination of decreased k(on) and increased k(off). For inhibitors characterized by slow dissociation rates to wild-type enzyme (saquinavir and nelfinavir), the decrease of affinity conferred by additional mutations was attributed to increased k(off) values. Development of resistance thus appears to be associated with a change of the distinctive kinetic parameter contributing to high affinity. Further inhibitor design should focus on improving the "weak point" of the lead compound, that being either k(on) or k(off).

Relationships between structure and interaction kinetics for HIV-1 protease inhibitors.

The interaction between HIV-1 protease and 58 structurally diverse transition-state analogue inhibitors has been analyzed by a surface plasmon resonance based biosensor. Association and dissociation rate constants and affinities were determined and displayed as k(on)-k(off)-K(D) maps. It was shown that different classes of inhibitors fall into distinct clusters in these maps. Significant changes in association and dissociation rates were found as a result of modifying the P1/P1' or P2/P2' side chains of a linear lead compound. Similarly, cyclic urea and cyclic sulfamide inhibitors displayed different kinetic features and the affinities of both classes of cyclic compounds were limited by fast dissociation rates. These results confirm that association and dissociation rates are important features of drug-target interactions and indicate that optimization of inhibitor efficacy may be guided by aiming for high association and low dissociation rates rather than high affinity alone. The present approach thus provides a new tool for structure-interaction kinetic analysis and drug discovery.