STUDY OF STRUCTURAL ORGANIZATION IN DNA-DEPENDED RNA-POLYMERASE OF ESCHERICHIA COLI BY AFFINE MODIFICATION METHODS

The aim is to study the topology of the active enzyme centre in the initiator and elongated trascription complexes and also structure of the DNA- and RNA-connecting centres of RNA-polymerase in the elongated complexes. The new data about structure of the active enzyme centre have been obtained, a whole number of the regions of two the most large subunits involved into the formation of the nucleotide-, DNA- and RNA-connecting pockets of RNA-polymerase has been revealed. A number of the new methods for mapping of the obtained sutures between RNA and protein, DNA and protein has been developed. The procedure for revealing residuals participating in the co-ordination of the magnesium ions in the active enzyme centre on the segregation of the subunits as the parts of the triple complex in presence of the ferrum ions and dithiotreitol has been proposed. The obtained results can be used at planning of the experiments on the directed mutagenesis of the subunits in the RNA-polymerase and also for affine modification of other types of the nucleotide-, DNA- and RNA-connecting enzymesAvailable from VNTIC / VNTIC - Scientific & Technical Information Centre of RussiaSIGLERURussian Federatio

SYK inhibition with fostamatinib in Waldenström's macroglobulinemia: In vitro cell based assays.

e19047 Background: Waldenström's macroglobulinemia (WM) is characterized by accumulation of IgM secreting neoplastic B cells in the bone marrow (BM). B-cell antigen receptor (BCR) signaling plays a pivotal role in the regulation of integrin-mediated retention of lymphoma cells in the BM. Signaling through the BCR leads to activation of spleen tyrosine kinase (SYK) and downstream kinases which play a role in lymphoma pathobiology. The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib targets BCR-controlled signaling and integrin-mediated adhesion in WM cells, but patients with WM may become refractory to ibrutinib treatment. As SYK signaling is upstream of BTK, we explored the effect of the SYK inhibitor R406 (the active metabolite of fostamatinib) on BCR mediated signaling and cell adhesion in WM. Methods: MWCL-1 cells were treated with R406, CAL101 (PI3 kinase inhibitor) or ibrutinib at concentrations up to 10µM and stimulated with anti-IgM antibodies or PMA. The cells were transferred to plates coated with fibronectin to assess cell adhesion. For the proliferation assays, MWCL-1 cells or primary B cells were pretreated with kinase inhibitors, followed by stimulation of primary B cells with anti-IgM. Results: Anti-IgM or PMA alone stimulated the adhesion of MWCL-1 on fibronectin. The addition of R406 had a comparable effect to ibrutinib on inhibition of IgM mediated cell adhesion (IC50 = 0.32µM), consistent with the effect of both compounds on BCR mediated signaling. Neither compound affected PMA induced cell adhesion, indicating that the effect on IgM mediated cell adhesion was not due to toxicity. Lack of off-target toxicity was further confirmed in the MWCL-1 proliferation assays where the compounds showed less potency than in BCR-mediated assays. Comparable inhibition of adhesion and proliferation was seen with CAL101. Conclusions: Preliminary preclinical studies with R406 show that SYK is involved in BCR mediated signaling and cell adhesion in WM. CAL101 mediated inhibition of adhesion further confirmed the role of SYK, which is upstream of PI3K. R406 was as effective as ibrutinib in blocking WM cell adhesion. Further studies of SYK inhibition with fostamatinib as a potential treatment option for WM are warranted. </jats:p

Roles for SR proteins and hnRNP A1 in the regulation of c-src exon N1.

The splicing of the c-src exon N1 is controlled by an intricate combination of positive and negative RNA elements. Most previous work on these sequences focused on intronic elements found upstream and downstream of exon N1. However, it was demonstrated that the 5' half of the N1 exon itself acts as a splicing enhancer in vivo. Here we examine the function of this regulatory element in vitro. We show that a mutation in this sequence decreases splicing of the N1 exon in vitro. Proteins binding to this element were identified as hnRNP A1, hnRNP H, hnRNP F, and SF2/ASF by site-specific cross-linking and immunoprecipitation. The binding of these proteins to the RNA was eliminated by a mutation in the exonic element. The activities of hnRNP A1 and SF2/ASF on N1 splicing were examined by adding purified protein to in vitro splicing reactions. SF2/ASF and another SR protein, SC35, are both able to stimulate splicing of c-src pre-mRNA. However, splicing activation by SF2/ASF is dependent on the N1 exon enhancer element whereas activation by SC35 is not. In contrast to SF2/ASF and in agreement with other systems, hnRNP A1 repressed c-src splicing in vitro. The negative activity of hnRNP A1 on splicing was compared with that of PTB, a protein previously demonstrated to repress splicing in this system. Both proteins repress exon N1 splicing, and both counteract the enhancing activity of the SR proteins. Removal of the PTB binding sites upstream of N1 prevents PTB-mediated repression but does not affect A1-mediated repression. Thus, hnRNP A1 and PTB use different mechanisms to repress c-src splicing. Our results link the activity of these well-known exonic splicing regulators, SF2/ASF and hnRNP A1, to the splicing of an exon primarily controlled by intronic factors

Clinical Trial to Evaluate an Approved ITP Therapy Targeting Spleen Tyrosine Kinase (SYK) for Prevention and Treatment of COVID-19 Related Complications

Fostamatinib is a potent SYK inhibitor that targets activation of both the innate and acquired immune systems caused by tissue damage or pathogen signals via C-type lectin receptors (CLR) as well as by antibody-antigen immune complexes via Fc receptors (Figure 1). SYK is also a key modulator of pathways involved in thrombosis formation. Fostamatinib is FDA-approved for chronic immune thrombocytopenia (ITP) and has been evaluated in over 4000 patients for treatment of allergic, autoimmune, and neoplastic disorders. Fostamatinib is well tolerated, with a consistent safety profile across a number of diseases. In rheumatoid arthritis patients, fostamatinib reduced the plasma levels of IL-6.1 The active metabolite (R406) was protective in mouse models of LPS-induced acute lung injury (ALI)2 and LPS- or antibody-induced acute kidney injury (AKI).3 SYK inhibition decreased the incidence of thrombosis in mouse models of thromboembolism,4 and the incidence of thromboembolic events was decreased in patients treated with fostamatinib compared with other treatments for ITP. These immunomodulatory effects suggest that SYK inhibition represents a new therapeutic strategy for the treatment of COVID-19, which led to design of a clinical study of fostamatinib in hospitalized COVID-19 patients. COVID-19 patients are at risk for a potentially fatal acute respiratory distress syndrome (ARDS), cytokine storm, severe systemic capillary leak syndrome, thromboembolic events, and multi-organ dysfunction/failure. SYK is a master regulator of signal transduction pathways implicated in these COVID-19 associated complications, which involves hyperactivation of both innate and acquired immune systems. Targeting SYK could prevent the cytokine storm, downstream activation of endothelial cells and platelets, and influx of neutrophils and monocytes into lungs or kidney leading to ALI and AKI, respectively. Methods: A randomized, double-blind, placebo-controlled, multi-center, Phase 3 study will evaluate the efficacy and safety of fostamatinib in adult, hospitalized, high-risk COVID-19 patients. Patients will be treated with fostamatinib 150mg BID for up to 28 days plus standard of care. Improvement in clinical status will be assessed using the 8-point ordinal scale. References 1. Weinblatt ME et al.Arth Rheum2008;58:3309-18 2. Nadeem A et al.Int Immunopharm2019;68:39-47 3. Al-Harbi NO, et al.Biochimie2019;158: 102-10 4. Van Eeuwijk JMM et al.Arterioscler Thromb Vasc Biol2016;36:1247-53 Results and Conclusions: Trial in Progress Disclosures Dummer: Rigel:Current Employment, Current equity holder in publicly-traded company.Markovtsov:Rigel:Current Employment, Current equity holder in publicly-traded company.Tong:Rigel:Current Employment, Current equity holder in publicly-traded company.Masuda:Rigel:Current Employment, Current equity holder in publicly-traded company. </jats:sec

Roles for SR proteins and hnRNP A1 in the regulation of c-src exon N1.

The splicing of the c-src exon N1 is controlled by an intricate combination of positive and negative RNA elements. Most previous work on these sequences focused on intronic elements found upstream and downstream of exon N1. However, it was demonstrated that the 5' half of the N1 exon itself acts as a splicing enhancer in vivo. Here we examine the function of this regulatory element in vitro. We show that a mutation in this sequence decreases splicing of the N1 exon in vitro. Proteins binding to this element were identified as hnRNP A1, hnRNP H, hnRNP F, and SF2/ASF by site-specific cross-linking and immunoprecipitation. The binding of these proteins to the RNA was eliminated by a mutation in the exonic element. The activities of hnRNP A1 and SF2/ASF on N1 splicing were examined by adding purified protein to in vitro splicing reactions. SF2/ASF and another SR protein, SC35, are both able to stimulate splicing of c-src pre-mRNA. However, splicing activation by SF2/ASF is dependent on the N1 exon enhancer element whereas activation by SC35 is not. In contrast to SF2/ASF and in agreement with other systems, hnRNP A1 repressed c-src splicing in vitro. The negative activity of hnRNP A1 on splicing was compared with that of PTB, a protein previously demonstrated to repress splicing in this system. Both proteins repress exon N1 splicing, and both counteract the enhancing activity of the SR proteins. Removal of the PTB binding sites upstream of N1 prevents PTB-mediated repression but does not affect A1-mediated repression. Thus, hnRNP A1 and PTB use different mechanisms to repress c-src splicing. Our results link the activity of these well-known exonic splicing regulators, SF2/ASF and hnRNP A1, to the splicing of an exon primarily controlled by intronic factors