Proteolysis-targeting chimera molecules (PROTACs) are an emerging class of therapeutics[1-3] with examples progressing to clinical trials in oncology[4]. PROTACs are relatively large bifunctional molecules comprising of a targeting moiety that binds to a target protein, a flexible linker, and an E3 ubiquitin ligase ligand. The targeting moiety binds to the target protein and the E3 ligase ligand binds an E3 ligase, then the E3 ligase recruits E2 ubiquitin-conjugating enzyme, which transfers ubiquitin (Ub) to the target protein. This ubiquitination is repeated, which labels the target protein for proteasomal degradation, in which polyubiquitinated proteins are cleaved into short polypeptides, thus reducing the levels of the target protein in a specific and catalytic manner. Initially, PROTAC design was based upon known protein-inhibitor combinations with the aim of reducing the amount of the target protein catalytically, rather than inhibiting its function, as with conventional small-molecule therapies. Because the targeting moiety of a PROTAC does not need to inhibit the function of the target protein this greatly expands the scope of PROTACs and opens up ‘undruggable’ targets, such as non-enzymatic proteins with no obvious binding or catalytic site. To this end, the aim of this research is to exploit the selectivity, size, and stability of single-domain antibodies (sdAb) to target intra-cellular proteins via proteasomal degradation. Single-domain antibodies or Nanobodies® (Ablynx NV) are significantly smaller than mammalian antibodies and are typically between 12-15 kD compared to 150 kD, yet they retain the specific antigen binding domain and are reportedly more stable, with regard to temperature and pH[5], and more soluble than the equivalent mammalian Ab consisting of 2 heavy chains and 2 light chains. Camelids (camels, llamas and alpacas) and cartilaginous fish (sharks and rays) naturally produce homodimeric heavy chain antibodies and sdAbs can be produced from immunised animals by extracting lymphocyte mRNA, followed by PCR and phage display to identify and isolate antigen-binding sdAbs[6].
Research plan
The aim of this research project is to construct sdAb PROTAC conjugates for the intra-cellular proteosomal degradation of target proteins. Proof of concept will be achieved using commercially available anti-GFP sdAbs[7], which will be conjugated to a chemical fluorophore, such as Cy5 via a cell-penetrating peptide (CPP), such as Pep-1[8], for transport through the cell membrane. Thiol-maleimide chemistry will be used for sdAb conjugation and solid-supported peptide synthesis for the CPP. The co-localisation of the GFP-fusion protein and chemical fluorophore using confocal microscopy will provide evidence that the sdAb constructs are able to pass into cells and bind to the protein target of interest.
Single-domain Ab-fluorophore conjugates have been used for super-resolution microscopy[9] and are superior to Ab-based approaches due to their much smaller size, affording them increased permeability and higher spatial accuracy. Cell-membrane permeability and selective intra-cellular localisation of sdAbs in living cells has been demonstrated by Herce et al. [10] using CPP-sdAb conjugates highlighting the utility of this emerging technology. Once methods for sdAb conjugates have been established, the chemical fluorophore will be replaced with an E3 ligase ligand, such as thalidomide or VHL ligand, and the sbAb-PROTAC-dependant proteosomal degradation will be characterised, by loss of GFP by microscopy and target protein by Western blot. We have previously developed synthetic methodology to shown that a resveratrol-thalidomide PROTAC can significantly reduce the intracellular levels of ribosyldihydronicotinamide dehydrogenase [quinone] (NQO2) in a colorectal cancer cell-line, and also used conjugation chemistry to measure the Kd of resveratrol with NQO2 using ligand-immobilised biolayer interferometry. Once proof of concept has been established, a tool-kit comprising E3 ligase ligands, CPPs and flexible linkers will be used to produce sdAb-PROTACs against protein targets of interest as part of well-established collaborations within the School of Chemistry (Dr. R. Doveston, Dr. J. Hodgkinson, Dr. Suntharalingam), Leicester Institute of Structural and Chemical Biology (Prof. M. Carr) and the Leicester Cancer Research Centre (Prof. K. Brown, Dr. A. Rufini, Prof. D. Fennell).
Entry requirements:
• Those who have a 1st or a 2.1 undergraduate degree in a relevant field are eligible.
• Evidence of quantitative training is required. For example, AS or A level Maths, IB Standard or Higher Maths, or university level maths/statistics course.
• Those who have a 2.2 and an additional Masters degree in a relevant field may be eligible.
• Those who have a 2.2 and at least three years post-graduate experience in a relevant field may be eligible.
• Those with degrees abroad (perhaps as well as postgraduate experience) may be eligible if their qualifications are deemed equivalent to any of the above
• University English language requirements apply. https://le.ac.uk/study/research-degrees/entry-reqs/eng-lang-reqs/ielts-65
For further information please contact [Email Address Removed]
Application advice:
To apply please refer the application instructions at https://le.ac.uk/study/research-degrees/funded-opportunities/bbsrc-mibtp
You will need to apply for the PhD place and also submit your online application notification to MIBTP. Links for both are on the above web page.
Project / Funding Enquiries: For further information please contact [Email Address Removed]
Application enquiries to [Email Address Removed]
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