Sven M. Lange

Bio: Sven M. Lange is an academic researcher from University of Dundee. The author has contributed to research in topics: Biology & Medicine. The author has an hindex of 3, co-authored 6 publications receiving 16 citations.

Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies.

Abstract: Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.

Impact of 100 LRRK2 variants linked to Parkinson's disease on kinase activity and microtubule binding

Abstract: Mutations enhancing the kinase activity of LRRK2 cause Parkinson’s disease (PD) and therapies that reduce LRRK2 kinase activity are being tested in clinical trials. Numerous rare variants of unknown clinical significance have been reported, but how the vast majority impact on LRRK2 function is unknown. Here, we investigate 100 LRRK2 variants linked to PD, including previously described pathogenic mutations. We identify 23 LRRK2 variants that robustly stimulate kinase activity, including variants within the N-terminal non-catalytic regions [ARM (E334K, A419V), ANK(R767H), LRR (R1067Q, R1325Q)], as well as variants predicted to destabilise the ROC:CORB interface [ROC (A1442P, V1447M), CORA (R1628P) CORB (S1761R, L1795F)] and COR:COR dimer interface [CORB (R1728H/L)]. Most activating variants decrease LRRK2 biomarker site phosphorylation (pSer935/pSer955/pSer973), consistent with the notion that the active kinase conformation blocks their phosphorylation. We conclude that the impact of variants on kinase activity is best evaluated by deploying a cellular assay of LRRK2-dependent Rab10 substrate phosphorylation, compared to a biochemical kinase assay, as only a minority of activating variants [CORB (Y1699C, R1728H/L, S1761R) and kinase (G2019S, I2020T, T2031S)], enhance in vitro kinase activity of immunoprecipitated LRRK2. Twelve variants including several that activate LRRK2 and have been linked to PD, suppressed microtubule association in the presence of a Type I kinase inhibitor [ARM(M712V), LRR(R1320S), ROC (A1442P, K1468E, S1508R), CORA(A1589S), CORB (Y1699C, R1728H/L) and WD40(R2143M, S2350I, G2385R)]. Our findings will stimulate work to better understand the mechanisms by which variants impact biology and provide rationale for variant carrier inclusion or exclusion in ongoing and future LRRK2 inhibitor clinical trials.

An expanded lexicon for the ubiquitin code

Abstract: Our understanding of the ubiquitin code has greatly evolved from conventional E1, E2 and E3 enzymes that modify Lys residues on specific substrates with a single type of ubiquitin chain to more complex processes that regulate and mediate ubiquitylation. In this Review, we discuss recently discovered endogenous mechanisms and unprecedented pathways by which pathogens rewrite the ubiquitin code to promote infection. These processes include unconventional ubiquitin modifications involving ester linkages with proteins, lipids and sugars, or ubiquitylation through a phosphoribosyl bridge involving Arg42 of ubiquitin. We also introduce the enzymatic pathways that write and reverse these modifications, such as the papain-like proteases of severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Furthermore, structural studies have revealed that the ultimate functions of ubiquitin are mediated not simply by straightforward recognition by ubiquitin-binding domains. Instead, elaborate multivalent interactions between ubiquitylated targets or ubiquitin chains and their readers (for example, the proteasome, the MLL1 complex or DOT1L) can elicit conformational changes that regulate protein degradation or transcription. The newly discovered mechanisms provide opportunities for innovative therapeutic interventions for diseases such as cancer and infectious diseases.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp5 main protease.

Abstract: The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.