Structural Basis of Molecular Mechanisms in Cell Guidance and Adhesion.

The cells of a multicellular organism, such as a human, are subject to an exquisite choreography during development. Each cell must achieve the particular balance between adhesion and motility appropriate to its role at a specific time and place in the developing organism. Much of the information to direct each cell must be garnered through interaction with its immediate environment including neighbouring cells. These interactions are mediated by various types of receptor molecules embedded in the cell surface. We wish to understand how one family of molecules, the semaphorins, work together with their receptors, the plexins, to control the ability of a cell to stick (adhere) or to move in a specific direction. Using the techniques of structural biology we aim to uncover, in atomic detail, the mechanisms by which the semaphorins and plexins control cell adhesion and guidance, for example in directing the wiring of the brain. These mechanisms must integrate receptor interactions occurring between cells and on the same cell surface, as well as spanning from the extracellular to the intracellular environment. To generate insight into such systems we will need to use state of the art techniques to produce suitable samples of the semaphorins, the plexins and their complexes, and to combine in vitro structural studies on the isolated molecules with in situ analyses in the functionally relevant context of the cell surface. This is basic research into the mechanisms by which biology works to build the nervous system and the blood vessels, to maintain bones and to activate the immune system. By understanding these mechanisms we will also be better equipped to explore molecular factors which may contribute to the failure of severed nerves to regenerate following spinal cord injury, to bone-related disorders, for example osteoporosis, to neurodevelopmental disorders such as autism disorder spectrum, and to cancer. Ultimately this knowledge can be used by the biotechnology and pharmaceutical industries, to inform and guide the design of novel therapeutics.

The semaphorins were first characterized as guidance cues exerting repulsive effects on axon growth cones during neural development. The number of tissues and biological processes for which we now know that the semaphorins and their type 1 single membrane spanning receptors, the plexins, play key roles has hugely expanded. Examples in the nervous system include neuronal migration, axon and dendrite guidance, branching, and synaptogenesis. Semaphorin function or dysfunction is also implicated in neurogenetic disorders, nerve regeneration, bone maintenance, immune cell differentiation and function, heart development, and cancer. Our knowledge of the molecular level mechanisms which deliver these myriad biological processes is still sparse. In this research programme we will apply an integrated structural and cellular biology approach to investigate the molecular mechanisms responsible for the biological activities of the class 5 and 6 semaphorins and their cognate plexin receptors. We will address three cross-cutting project areas:

1. Extracellular determinants of function: activation and signalling mode

2. Forward vs reverse signalling: inhibition and ligand as receptor

3. Full length signalling assemblies: extracellular, transmembrane and cytoplasmic systems

We will generate atomic level detail using protein crystallography and electron cryo microscopy. We will evaluate the functional hypotheses we generate in these molecular studies by using cellular imaging methods including electron cryo tomography as well as advanced fluorescence microscopy techniques such as localization microscopy (dSTORM) and FRET-FLIM. Our studies will also link through directly to in vivo and in vitro studies of cellular morphology and neuronal connectivity in collaborators' laboratories. Ultimately our results will inform and guide investigations into the semaphorin-plexin system as a point for therapeutic intervention (see Impact Summary).

As discussed in the Academic Beneficiaries section this research will be of interest and benefit to the structural biology community because it will generate new methods, protocols and reagents. Likewise it will provide information on molecular mechanisms which will underpin the design and interpretation of studies on biological function and clinical pathologies in a broad range of tissues including the nervous system, bone, the vasculature and the immune system.

The proposed programme is discovery research of potential value to the commercial private sector, specifically the biotechnology and pharmaceutical industries, to inform and guide the design of novel therapeutics. Because the molecular mechanisms of semaphorin-plexin function targeted in this application have been found to play fundamental roles in the morphogenesis and homeostasis of many human tissues these molecules have emerged as potential points for therapeutic intervention. Several biotech companies have active development programmes in this arena, for example SemaThera Inc. has a lead compound (ST-102) in development to target VEGF and SEMA3A in degenerative retinopathies such as diabetic macular edema. Most studies to date are at the pre-clinical stage, however, Vaccinex Inc is currently conducting Phase II clinical trials for an anti-semaphorin (SEMA4D) monoclonal antibody (VX15/2503) in Huntington's patients with late prodromal (before clinical diagnosis) and early manifest disease (ClinicalTrials.gov Identifier: NCT02481674), and (in combination with an anti-PD-L1 monoclonal antibody) in patients with advanced non-small cell lung cancer (ClinicalTrials.gov Identifier: NCT03268057).

The third area of impact for this research programme will be in the training and career development of a cohort of young research scientists. I have been very fortunate in the high level of talent and enthusiasm shown by the young scientists who have worked with me and I believe my laboratory has provided them with an excellent training environment. Of the graduate students I have supervised, or co-supervised, nearly all are pursuing science associated careers (11 in academic research, 9 in biotech or big pharma, plus one science teacher, a patent agent and a science writer). Past pre- and post-doctoral members of my laboratory include group leaders at universities or research institutes in the UK (MRC Laboratory of Molecular Biology, Cambridge, Imperial College London, Newcastle, Oxford), Australia, Japan, Spain, The Netherlands and the USA, as well as the occupants of leadership roles in a range of biotech and big pharma companies (Adaptimmune, Evotec, GlaxoSmithKline, Immunocore, Novartis and Roche).