作者: Murphy, Natasha P ; Owen, Darerca ; Revell, Jefferson ; Tetley, George J N ; Ivanova-Berndt, Gabriela ; Cooley, R Neil ; Mott, Helen R ; Bonetto, Stephane

Aberrant Ras signaling drives 30% of cancers, and inhibition of the Rho family small GTPase signaling has been shown to combat Ras-driven cancers. Here, we present the discovery of a 16-mer cyclic peptide that binds to Cdc42 with nanomolar affinity. Affinity maturation of this sequence has produced a panel of derived candidates with increased affinity and modulated specificity for other closely-related small GTPases. The structure of the tightest binding peptide was solved by NMR, and its binding site on Cdc42 was determined. Addition of a cell-penetrating sequence allowed the peptides to access the cell interior and engage with their target(s), modulating signaling pathways. In Ras-driven cancer cell models, the peptides have an inhibitory effect on proliferation and show suppression of both invasion and motility. As such, they represent promising candidates for Rho-family small GTPase inhibitors and therapeutics targeting Ras-driven cancers. Our data add to the growing literature demonstrating that peptides are establishing their place in the biologics arm of drug discovery.

2017-10-20·ACS Chemical Biology

Exploiting Overlapping Advantages of In Vitro and In Cellulo Selection Systems to Isolate a Novel High-Affinity cJun Antagonist.

Article

作者: Mason, Jody M ; Ullman, Christopher G ; Frigotto, Laura ; Baxter, Daniel

We have combined two peptide library-screening systems, exploiting the benefits offered by both to select novel antagonistic agents of cJun. CIS display is an in vitro cell-free system that allows very large libraries (≤10¹⁴) to be interrogated. However, affinity-based screening conditions can poorly reflect those relevant to therapeutic application, particularly for difficult intracellular targets, and can lead to false positives. In contrast, an in cellulo screening system such as the Protein-fragment Complementation Assay (PCA) selects peptides with high target affinity while additionally profiling for target specificity, protease resistance, solubility, and lack of toxicity in a more relevant context. A disadvantage is the necessity to transform cells, limiting library sizes that can be screened to ≤10⁶. However, by combining both cell-free and cell-based systems, we isolated a peptide (CPW) from a ∼10¹⁰ member library, which forms a highly stable interaction with cJun (T_m = 63 °C, K_d = 750 nM, ΔG = -8.2 kcal/mol) using the oncogenic transcriptional regulator Activator Protein-1 (AP-1) as our exemplar target. In contrast, CIS display alone selected a peptide with low affinity for cJun (T_m = 34 °C, K_d = 25 μM, ΔG = -6.2 kcal/mol), highlighting the benefit of CIS → PCA. Furthermore, increased library size with CIS → PCA vs PCA alone allows the freedom to introduce noncanonical options, such as interfacial aromatics, and solvent exposed options that may allow the molecule to explore alternative structures and interact with greater affinity and efficacy with the target. CIS → PCA therefore offers significant potential as a peptide-library screening platform by synergistically combining the relative attributes of both assays to generate therapeutically interesting compounds that may otherwise not be identified.

2017-07-01·The Journal of biological chemistry2区 · 生物学

A dock and coalesce mechanism driven by hydrophobic interactions governs Cdc42 binding with its effector protein ACK

2区 · 生物学

Article

作者: Owen, Darerca ; Mott, Helen R ; Tetley, George J N ; Cooley, R Neil

Cdc42 is a Rho-family small G protein that has been widely studied for its role in controlling the actin cytoskeleton and plays a part in several potentially oncogenic signaling networks. Similar to most other small G proteins, Cdc42 binds to many downstream effector proteins to elicit its cellular effects. These effector proteins all engage the same face of Cdc42, the conformation of which is governed by the activation state of the G protein. Previously, the importance of individual residues in conferring binding affinity has been explored for residues within Cdc42 for three of its Cdc42/Rac interactive binding (CRIB) effectors, activated Cdc42 kinase (ACK), p21-activated kinase (PAK), and Wiskott-Aldrich syndrome protein (WASP). Here, in a complementary study, we have used our structure of Cdc42 bound to ACK via an intrinsically disordered ACK region to guide an analysis of the Cdc42 interface on ACK, creating a panel of mutant proteins with which we can now describe the complete energetic landscape of the Cdc42-binding site on ACK. Our data suggest that the binding affinity of ACK relies on several conserved residues that are critical for stabilizing the quaternary structure. These residues are centered on the CRIB region, with the complete binding region anchored at each end by hydrophobic interactions. These findings suggest that ACK adopts a dock and coalesce binding mechanism with Cdc42. In contrast to other CRIB-family effectors and indeed other intrinsically disordered proteins, hydrophobic residues likely drive Cdc42-ACK binding.

项与 Isogenica Ltd. 相关的新闻（医药）

2022-05-31

·wmbio.co

Wellmarker Bio signs MoU with Isogenica for the development of multiple therapeutic assets

Wellmarker Bio (WMBIO) and Isogenica today announced the signing of a Memorandum of Understanding (MoU). Under the terms of the MoU, the two companies will collaborate in the research and development of antibody-based cancer immunotherapy, Protein-Protein Interaction (PPI) Inhibitors, biomarkers and peptide libraries; with joint funding, including UK or other government grants they will collaborate to create a portfolio of preclinical programs from early target identification through to candidate nomination. The collaboration will combine each company’s expertise and experience in recombinant protein technologies, through the design and construction of highly diverse synthetic libraries and the use of novel in vitro polypeptide display systems. Isogenica will apply its proprietary CIS display technology for the discovery of novel therapeutics to progress Wellmarker Bio’s pipeline in cancer and other indications. “Isogenica’s collaboration with WMBIO focusing on individualised, effective and affordable therapies for patients with high unmet medical need is an exciting opportunity to accelerate development of novel targeted therapeutics,” said Dr Bill Eldridge, CEO of Isogenica. “Pairing Isogenica’s expertise in synthetic libraries and protein engineering with WMBIO commitment to developing first-in-class anti-cancer drugs based on predictive biomarkers promises to be an efficient and cost-effective means to develop individualised therapies.” Professor Dong-Hoon Jin, CEO of WMBIO, commented, “Wellmarker Bio is very excited to be collaborating with Isogenica and look forward to developing a host of effective and affordable anti–cancer drugs based on Isogenica's platform." About Isogenica Isogenica is a peptide and antibody discovery and development company. Isogenica’s CIS Display technology, an in vitro biotherapeutic discovery technology with significant advantages over more traditional techniques in terms of library size and versatility, enables the full diversity of the company’s massive peptide and antibody libraries to be exploited. With this technology, the company has a unique and proven engine for biotherapeutic discovery and development. Working in collaboration with biopharmaceutical partners over the last decade the company has developed a deep pipeline including two clinical stage assets and more than ten partnered pre-clinical and discovery stage programmes. Further information, visit: www.isogenica.com Please click on the link below for the full release. https://www.isogenica.com/wellmarkerbio-mou/

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