Controlling cell death and proliferation with encodable visible light responsive proteins

Interactions between biomacromolecules play a crucial role in all cellular processes. They are usually weak, i.e. non-covalent and temporal and hence inherently difficult to address chemically. In many cases such as cell cycle control, it would be extremely important to find ways to target these interactions, which could open the way to control cellular processes such as cell death and cell proliferation. We have recently shown that we are able to induce cell death in cancer cells treated with biophotonic nanoswitches, short peptides that interact specifically with protein surfaces. In detail, the interactions between the cell cycle regulators p53/hdm-2, Bcl-xL/bak and Bcl-xL/bid depend on alpha-helices from one partner that bind into groves on the surface of the other. Peptides were synthesised with azobenzene-linkers that enable the light-controlled generation of a stable alpha-helical structure, which then interacts with the binding partner. Unfortunately, UV light is required for the conformational change and for the generation of the alpha-helical, active structure of these peptides and UV light can have damaging effects on cells. Furthermore, UV light cannot penetrate deeply into tissue. It would be advantageous to switch to longer, visible wavelengths of light, which are not damaging and penetrate deeper. An extremely promising approach is to combine such peptides with photo-sensitive domains from blue light receptors. These receptors are used by plants, fungi and bacteria to regulate physiological processes upon a stimulus with blue light. The light-sensitive parts in these proteins are LOV domains, which bind a molecule of a coenzyme called flavine mononucleotide, which in turn acts as the light-harvesting chromophore. Upon illumination a covalent photoadduct between the cofactor and the apoprotein is formed which induces a conformational change in the LOV domain: a C-terminal alpha-helix that is bound to a beta-sheet of the core of the protein becomes flexible and therefore accessible for binding to other partners. We will develop genetically encoded photo-activatable proteins, in which residues of p53 and bid/bak recognition alpha-helices are introduced into the C-terminal helix of LOV domains. In the dark these helices will be tightly bound to the core of the LOV protein and cannot be accessed for binding to other proteins. A blue light pulse will then set the alpha-helix free and allow interaction/binding to hdm-2 and p53 to influence the targeted pathways in live cells. The absorption characteristics of LOV domains allow for wavelengths up to 500 nm, the use of FMN analogues will extend this range up to 550 nm. Light of this range has no detrimental effects on cells and is currently used in dentistry to harden polymer fillings. Thus we will be able to regulate cell cycles processes in a time-dependant manner in exactly defined spatial areas with minimal cell damage. The life-time of the active state can be regulated by the period of illumination, the choice of the LOV domain used and the type of light-sensitive pigment present. The photo-switchable proteins developed will be delivered to cells using our established approach, in which the protein will be tagged with a peptide that enables cellular uptake. Additionally, we will use transient expression of our constructs for short term investigations of the cell cycle and using viral vectors we will engineer cellular systems that allow for stable expression of our proteins for long term cell cycle tracking. The approach described here to intervene in biological process in a targeted fashion is generic in that photoactivatable proteins will be generally applicable to all biomacromolecular interactions based on alpha-helices. It will establish a novel research approach for the reversible modulation of the way in which proteins interact in real time and within live cells with enormous potential for the study of biological processes and for therapy.

Nature uses proteins to target other proteins to gain subtle control of cellular pathways such as those involved in the response to stress or the control of the cell cycle. The key to this method of control is a complex cassette of reversible exchanges between interacting protein surfaces. Our development of new peptide-based reagents, exploiting extensive preliminary studies, has shown the feasibility of mimicking nature's strategy by engineering the span, form and selectivity of synthetic alpha-helical structures. These novel agents use photo-activatable peptides, the activity of which can be controlled in live cells. Delivery to mammalian cells of octa-Arg mediated photo-crosslinked peptides derived from the pro-apoptotic peptides bid and bak revealed induction of cell death in a light dependent fashion. It is desirable however, to design molecules that can be stably integrated in cells and activated with visible light of low toxicity and significant penetration. Our proposal is to engineer LOV-domains, light-sensing domains present in many photo-reactive proteins, to target hdm-2 and Bcl-xL and influence cell cycle arrest and cell death in a biomimetic manner. The objectives are to develop genetically-encodable, photo-activatable proteins, which carry recognition sequences for hdm-2 and Bcl-xL in the Jalpha-helix of LOV domains to control their activity with respect to binding to hdm-2 and p53. Options include tuning to higher wavelengths and of the lifetime of the photo-excited state. We have established model cellular systems and assays for testing the activity of each of the constructs. Our high-resolution cell-tracking methods will be exploited to monitor the consequences on mitochondrial membrane potential and downstream apoptotic indicators for the LOV2-Bid complex and how modulation of p53/hdm2 dynamics influences the cell cycle under stress. Our work will deliver novel generic investigational tools and insights into the biology of cellular pathways.

The project area impact will be improvements in healthcare through cell-based discovery and evaluation systems for new medicines. We also see a primary impact on the development of professional and strategic skills of staff working on the project being an exposure of chemists to a cross-disciplinary environment that faces an unmet need in medicine. Early stage beneficiaries of the research will be academic research and commercial drug developers, as our focus is to seek an impact of healthcare improvement via translational research. All involved in this project have established links with industry which could facilitate future translational development of the IP. Cardiff RACD has established procedures for evaluating, protecting, developing and management of intellectual ideas. Exploitation may range from licensing of the technology in the commercial life sciences sector to the development of a spin-out company. We would judge a successful outcome to have a wide and sustained impact on the life sciences community. Beneficiaries include: 1. Basic researchers - interested in finding new tools or methods to understand mechanisms through hypothesis-driven research. 2. Optoelectronic engineers exploiting the availability of light controlled probes within new designs for HTS platforms. 3. Computational biologists interested in encoding the complex outputs of fundamental cellular pathways. Or systems engineers studing feedback, black boxes and derived concepts such as communication and control in living organisms. 4. Drug delivery developers exploring the use of light to activate novel medicines and refining target selection in drug screens. How will they benefit from this research? The impact of the proposed research is firmly in the translational path from tools developed in basic research through to the highly active commercial exploitation of those tools for the development of new medicines. The investigators understand this process and have a successful track record of commercial exploitation in this field. We seek to provide the field with sensor technologies that can be readily incorporated into a target cell screening systems - making screening protocols more informative and multi-scalar. Future commercial beneficiaries - The beneficiaries in the commercial life sciences will be groups involved in knowledge-led target discovery, molecular modelling and high content/throughput screening: (i) reagent developers; (ii) medium-sized pharmaceutical drug development companies that seek advantages for screening and agent validation; (iii) major pharmaceutical drug-screening operations that seek generic platforms for drug efficacy or toxicity testing. Beneficiaries within policy-makers and regulatory bodies - Regulatory demands on the safety of medicines and judgment of efficacy versus cost increasingly depends on the specificity of molecular and cellular targeting. The negative impact of the inability to capture unwanted cellular responses (toxicity, genotoxic or resistance-generating outcomes) is a cause of failure of medicines at the costly and ethically unacceptable later stages (eg at phase I-III trial). Beneficiaries within the wider public an impact on the nation's health & wealth - Simply stated, our commitment is to support and enhance that long translational path for the delivery of safe and effective medicines based on improved screening methods. We are aware of global changes in the funding and commercial re-location of R&D in healthcare. The need to deliver personalised drug-based therapies demands highly selective agent and target evaluation. Agents developed with regard to those issues are less likely to fail, more likely to achieve regulatory approval and return economic value to the originators. Our research will support the generation of licensed technologies that will provide economic competitiveness for the threatened United Kingdom pharmaceutical sector.