Biology subjects

Fyfe, P. K.

Publications and source records attributed to Fyfe, P. K..

4 recordsLinked to original sources

bioRxiv2024

Characterisation of RNA guanine-7 methyltransferase (RNMT) using a small molecule approach

The maturation of the RNA cap involving guanosine N-7 methylation, catalyzed by the HsRNMT (RNA guanine-7 methyltransferase)-RAM (RNA guanine-N7 methyltransferase activating subunit) complex, is currently under investigation as a novel strategy to combat PIK3CA mutant breast cancer. However, the development of effective drugs is hindered by a limited understanding of the enzymes mechanism and a lack of small molecule inhibitors. Following the elucidation of the HsRNMT-RAM molecular mechanism, we report the biophysical characterization of two small molecule hits. Biophysics, biochemistry and structural biology confirm that both compounds bind competitively with cap and bind effectively to HsRNMT-RAM in the presence of the co-product SAM, with a binding affinity (KD) of approximately 1 M. This stabilisation of the enzyme-product complex results in uncompetitive inhibition. Finally, we describe the properties of the cap pocket and provided suggestions for further development of the tool compounds.

biochemistry↗

bioRxiv2023

Application of an NMR/crystallography fragment screening platform for the assessment and rapid discovery of new HIV-CA binding fragments

Identification and assessment of novel targets is essential to combat drug resistance in the treatment of HIV/AIDS. HIV Capsid (HIV-CA), the protein playing a major role in both the early and late stages of the viral life cycle, has emerged as an important target. We have applied an NMR fragment screening platform and identified molecules that bind to the N-terminal domain (NTD) of HIV-CA at a site close to the interface with the C-terminal domain (CTD). Using X-ray crystallography, we have been able to obtain crystal structures to identify the binding mode of these compounds. This allowed for rapid progression of the initial, weak binding, fragment starting points to compounds 37 and 38, which have 19F-pK values of 5.3 and 5.4 respectively. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=56 SRC="FIGDIR/small/569544v2_ufig1.gif" ALT="Figure 1"> View larger version (6K): org.highwire.dtl.DTLVardef@1c7043corg.highwire.dtl.DTLVardef@e73044org.highwire.dtl.DTLVardef@11c1d9org.highwire.dtl.DTLVardef@1cd99e7_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2022

Structural insights into the assembly and activation of IL-27 signalling complex

Interleukin 27 (IL-27) is a heterodimeric cytokine that elicits potent immuno-suppressive responses. Comprised of EBI3 and p28 subunits, IL-27 binds GP130 and IL-27R receptor chains to activate the JAK/STAT signalling cascade. However, how these receptors recognize IL-27 and form a complex capable of phosphorylating JAK proteins remains unclear. Here, we used cryo electron microscopy (cryoEM) to solve the structure of the IL-27 receptor recognition complex. Our data show how IL-27 serves as a bridge connecting IL-27R with GP130 to initiate signalling. While both receptors weakly bind the p28 component of the heterodimeric cytokine, EBI3 stabilizes the complex by binding a positively charged surface of IL-27R. We find that assembly of the IL-27 receptor recognition complex is distinct from both IL-12 and IL-6 cytokine families and provides a mechanistic blueprint for tuning IL-27 pleiotropic actions.

immunology↗

bioRxiv2021

Competitive binding of STATs to receptor phospho-Tyr motifs accounts for altered cytokine responses in autoimmune disorders

Cytokines elicit pleiotropic and non-redundant activities despite strong overlap in their usage of receptors, JAKs and STATs molecules. We use IL-6 and IL-27 to ask how two cytokines activating the same signaling pathway have different biological roles. We found that IL-27 induces more sustained STAT1 phosphorylation than IL-6, with the two cytokines inducing comparable levels of STAT3 phosphorylation. Mathematical and statistical modelling of IL-6 and IL-27 signaling identified STAT3 binding to GP130, and STAT1 binding to IL-27R, as the main dynamical processes contributing to sustained pSTAT1 by IL-27. Mutation of Tyr613 on IL-27R decreased IL-27-induced STAT1 phosphorylation by 80% but had limited effect on STAT3 phosphorylation. Strong receptor/STAT coupling by IL-27 initiated a unique gene expression program, which required sustained STAT1 phosphorylation and IRF1 expression and was enriched in classical Interferon Stimulated Genes. Interestingly, the STAT/receptor coupling exhibited by IL-6/IL-27 was altered in patients with Systemic lupus erythematosus (SLE). IL-6/IL-27 induced a more potent STAT1 activation in SLE patients than in healthy controls, which correlated with higher STAT1 expression in these patients. Partial inhibition of JAK activation by sub-saturating doses of Tofacitinib specifically lowered the levels of STAT1 activation by IL-6. Our data show that receptor and STATs concentrations critically contribute to shape cytokine responses and generate functional pleiotropy in health and disease.

bioinformatics↗