Validating “undruggable” or orphan cancer targets by combining fragment based screening peptidomimetic chemistry, phage display, structural biology and cell biology.

In comparison to prominent and successful target classes like GPCRs, the success rate in targeting protein – protein interactions (PPIs) is very low. The identification of chemical starting points in the initial phases of the hit and lead discovery process is often very limited. PPIs comprise highly diverse interactions which are often characterized by structural flexibility, high affinity, and large surface areas. The pharmaceutical industry is therefore cautious and hesitant to perform high throughput screens costing upwards of 1 million dollars on a PPI unless it is a fully validated drug target. This translates to around 50% of all human cancer-causing genes being missed. Target validation involves 2 aspects. (a) Biological validation, (b) Chemical validation, which involves the identification of a proven binding and inhibitory reagent, stopping function in biochemical, cellular and potentially model organism assays.

The suggested project aims at the establishment and exemplification of a generic process for chemical validation of proteins without known function which could represent important new targets in cancer drug discovery. Tentatively, these include i) Survivin (a key component of the Chromosomal Passenger Complex essential for error-free cell division), ii) the Chromosomal Passenger Complex (consists of Borealin, Survivin, Aurora B and INCENP), iii) the Ska complex (consists of Ska1, Ska2 and Ska3) essential for the mitotic spindle driven chromosome segregation and iv) CENP-32, an RNA methyltransferase implicated in maintaining the intact mitotic spindle assembly.

Step 1: Development of dimer, trimer and tetramer peptidomimetic libraries which contain one fluorinated aromatic amino acid by split & mix solid phase chemistry (chemistry candidate) and/or development of monobody and cyclotide based phage display libraries of high diversity (biology candidate).

Step 2: identification of a molecule which binds to the target.

Chemical and/or biological miniprotein libraries will be screened by on-bead confocal scanning and panning techniques on microbeads for target binding. Established screening platforms of the Auer lab will be applied, including LFAP, OBOC-CONA and Phage-CONA see website).

Step 3: Optimizing hit affinity by iterative synthesis, affinity determination and structural biology and/or by iterative sequence and affinity maturation of phage display derived monobody and cyclotide miniproteins.

Our biophysical methods allow identifying binders in the mM to pM KD range. Applying miniaturized OBOC chemistry intein based protein expression [1], a variety of label free and fluorescence single molecule assay techniques [2], and structural methods, micromolar to nanomolar binders will be developed.

Step 4: Functional evaluation of target modulation in cells

Affinity optimized peptidomimetic and biosimilar miniprotein hits will be tested for functional activity in cellular assays within the JP lab in unlabelled and fluorescently labelled forms. Single molecule and super resolution imaging will be applied using excellent microscopy equipment in the Auer lab and the WTCCB facility.

A chemist or a biologist with broad interest in biophysics, screening, structural biology and imaging and with a dedication to progress cancer translational biology will be required for this strongly interdisciplinary project. The completion of this PhD project will provide the candidate with excellent chances to progress a career in translational science.

Auer Lab website: https://sites.google.com/view/the-auer-lab-uoe/home
JP Lab website: http://jeyaprakash.bio.ed.ac.uk