131 research outputs found
Small molecule and peptide inhibitors of the pro-survival protein Mcl-1
The ability of protein–protein interactions to regulate cellular processes in both beneficial and detrimental ways has made them obvious drug targets. The Bcl-2 family of proteins undergo a series of protein–protein interactions which regulate the intrinsic cell-death pathway. The pro-survival members of the Bcl-2 family, including Bcl-2, Bcl-xL, and Mcl-1, are commonly overexpressed in a number of human cancers. Effective modulators of members of the Bcl-2 family have been developed and are undergoing clinical trials, but the efficient modulation of Mcl-1 is still not represented in the clinic. In addition, Mcl-1 is a major cause of resistance to radio- and chemotherapies, including inhibitors that target other Bcl-2 family members. Subsequently, the inhibition of Mcl-1 has become of significant interest to the scientific community. This review covers the progress made to date in modulating the activity of Mcl-1, by both stapled peptides and small molecules. The development of peptides as drug candidates, and the advancement of experimental and computational techniques used to discover small molecules are also highlighted
Self-Reactivity and the Expression of Memory Markers Vary Independently in MRL-Mp+/+ and MRL-Mp-lpr/lpr Mice
MRL-Mp-lpr/lpr mice contain phenotypically abnormal populations of T cells, and
exhibit an SLE-like autoimmune disease in which autoantibodies are a prominent
feature. We analyzed the phenotype and T-cell receptor Vß expression pattern in CD4+ T
cells of this mutant mouse strain to detect abnormalities that could explain the
autoimmunity. The CD4+ T cells contain two distinct abnormal populations. One of
these expresses B220 and HSA, and in these and other respects closely resembles the
accumulating CD4–CD8– population. The other expresses a high level of CD44 (Pgp-1),
and a high level of the 16A epitope of CD45, and so resembles post-activation T cells.
Both of these cell types are exclusive to MRL-Mp-lpr/lpr. We also identified V ß5- and
V ß11-positive CD4+ T cells, in both MRL-Mp-lpr/lpr and MRL-Mp-+/+ mice. We
conclude that autoimmune T cells can be detected in these mice, but that they are not the
cause of the accumulation of abnormal CD4+ and CD4–CD8–cells
Rac1 plays a role in CXCL12 but not CCL3-induced chemotaxis and Rac1 GEF inhibitor NSC23766 has off target effects on CXCR4
Cell migration towards a chemotactic stimulus relies on the re-arrangement of the cytoskeleton, which is triggered by activation of small G proteins RhoA, Rac1 and Cdc42, and leads to formation of lamellopodia and actin polymerisation amongst other effects. Here we show that Rac1 is important for CXCR4 induced chemotaxis but not for CCR1/CCR5 induced chemotaxis. For CXCL12-induced migration via CXCR4, breast cancer MCF-7 cells are reliant on Rac1, similarly to THP-1 monocytes and Jurkat T-cells. For CCL3-induced migration via CCR1 and/or CCR5, Rac1 signalling does not regulate cell migration in either suspension or adherent cells. We have confirmed the involvement of Rac1 with the use of a specific Rac1 blocking peptide. We also used a Rac1 inhibitor EHT 1864 and a Rac1-GEF inhibitor NSC23766 to probe the importance of Rac1 in chemotaxis. Both inhibitors did not block CCL3-induced chemotaxis, but they were able to block CXCL12-induced chemotaxis. This confirms that Rac1 activation is not essential for CCL3-induced migration, however NSC23766 might have secondary effects on CXCR4. This small molecule exhibits agonistic features in internalisation and cAMP assays, whereas it acts as an antagonist for CXCR4 in migration and calcium release assays. Our findings strongly suggest that Rac1 activation is not necessary for CCL3 signalling, and reveal that NSC23766 could be a novel CXCR4 receptor ligand
Design and development of stapled transmembrane peptides that disrupt the activity of G-protein coupled receptor oligomers
Membrane proteins can associate into larger complexes. Examples include receptor tyrosine complexes, ion channels, transporters and G-protein coupled receptors (GPCRs). For the latter, there is abundant evidence indicating that GPCRs, assemble into complexes, through both homo or heterodimerization. However, the tools for studying and disrupting these complexes, GPCR or otherwise, are limited. Here we have developed stabilized interference peptides for this purpose. We have previously reported that tetrahydrocannabinol-mediated cognitive impairment arises from homo- or hetero-oligomerization between the GPCRs cannabinoid receptor type 1 (CB1R) and 5-hydroxytryptamine 2A (5-HT2AR) receptors. Here, to disrupt this interaction through targeting CB1–5-HT2A receptor heteromers in HEK293 cells and using an array of biochemical techniques, including calcium and cAMP measurements, bimolecular fluorescence complementation assays, and CD-based helicity assessments, we developed a NanoLuc binary technology (NanoBiT)-based reporter assay to screen a small library of aryl-carbon–stapled transmembrane mimicking peptides produced by solid-phase peptide synthesis. We found that these stapling peptides have increased α-helicity and improved proteolytic resistance without any loss of disrupting activity in vitro, suggesting that this approach may also have utility in vivo. In summary, our results provide proof of concept for using NanoBiT to study membrane protein complexes and for stabilizing disrupting peptides to target such membrane complexes through hydrocarbon-mediated stapling. We propose that these peptides could be developed to target previously un-druggable GPCR heteromers
Reliability and validity of the Actiwatch and Clouclip for measuring illumination in real‐world conditions
Reliability and validity of the Actiwatch and Clouclip for measuring illumination in real‐world conditions
Orexin-Corticotropin-Releasing Factor Receptor Heteromers in the Ventral Tegmental Area as Targets for Cocaine
Release of the neuropeptides corticotropin-releasing factor (CRF) and orexin-A in the ventral tegmental area (VTA) play an important role in stress-induced cocaine-seeking behavior. We provide evidence for pharmacologically significant interactions between CRF and orexin-A that depend on oligomerization of CRF1 receptor (CRF1R) and orexin OX1 receptors (OX1R). CRF1R–OX1R heteromers are the conduits of a negative crosstalk between orexin-A and CRF as demonstrated in transfected cells and rat VTA, in which they significantly modulate dendritic dopamine release. The cocaine target σ1 receptor (σ1R) also associates with the CRF1R–OX1R heteromer. Cocaine binding to the σ1R–CRF1R–OX1R complex promotes a long-term disruption of the orexin-A–CRF negative crosstalk. Through this mechanism, cocaine sensitizes VTA cells to the excitatory effects of both CRF and orexin-A, thus providing a mechanism by which stress induces cocaine seeking
The malaria parasite egress protease SUB1 is a calcium-dependent redox switch subtilisin.
Malaria is caused by a protozoan parasite that replicates within an intraerythrocytic parasitophorous vacuole. Release (egress) of malaria merozoites from the host erythrocyte is a highly regulated and calcium-dependent event that is critical for disease progression. Minutes before egress, an essential parasite serine protease called SUB1 is discharged into the parasitophorous vacuole, where it proteolytically processes a subset of parasite proteins that play indispensable roles in egress and invasion. Here we report the first crystallographic structure of Plasmodium falciparum SUB1 at 2.25 Å, in complex with its cognate prodomain. The structure highlights the basis of the calcium dependence of SUB1, as well as its unusual requirement for interactions with substrate residues on both prime and non-prime sides of the scissile bond. Importantly, the structure also reveals the presence of a solvent-exposed redox-sensitive disulphide bridge, unique among the subtilisin family, that likely acts as a regulator of protease activity in the parasite
Identification of a new p53/MDM2 inhibitor motif inspired by studies of chlorofusin
Previous studies on the natural product chlorofusin have shown that the full peptide and azaphilone structure are required for inhibition of the interaction between MDM2 and p53. In the current work, we utilized the cyclic peptide as a template and introduced an azidonorvaline amino acid in place of the ornithine/azaphilone of the natural product and carried out click chemistry with the resulting peptide. From this small library the first ever non-azaphilone containing chlorofusin analogue with MDM2/p53 activity was identified. Further studies then suggested that the simple structure of the Fmoc-norvaline amino acid that had undergone a click reaction was also able to inhibit MDM2/p53 interaction. This is an example where studies of a natural product have led to the serendipitous identification of a new small molecule inhibitor of a protein-protein interaction
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