NDC for the Optical Control of Biological Function
A major challenge for biomedicine is to develop new ways of determining how proteins operate in complexes in cells. This requires molecularly focused methods for dynamic interrogation and manipulation. An attractive approach is to use light as both input and output to probe molecular machines in cells. While there has been significant progress in optical detection of protein function, little advance has been made in control. The NDC for the Optical Control of Biological Function is developing methods for rapidly turning select proteins in cells on and off with light. The strategies are broadly applicable across protein classes.
Biological and disease targets: Three central signaling systems. We are focusing on ion channels, Gprotein coupled receptors and kinases and phosphatases. These represent some of the largest protein families, together comprising about 7% of the human genome. The proteins are implicated as causes and drug targets in major human diseases. By enabling rapid, reversible and local switching these tools should make it possible to overcome two major problems in standard drug delivery: a) side effects due to systemic delivery, or systemic leak of locally delivered drugs, and b) long delays between delivery and action, and difficulty to terminate action.
Toolkits of light-gated signaling proteins and of light-gated peptides that selectively modify protein function. We are using structure-based design to chemically synthesize two classes of nanoscale photoswitches: 1) ligand photoswitches that attach to native and engineered target proteins, and remote control their function (with both activating and blocking ligands); and 2) light-gated peptides, which bind to target proteins (at interaction domains or substrates binding sites) in a light-dependent manner, interfering with function only in the bound state.
Addressing specific medical problems. A critical challenge is to transfer our methods from basic research in cell biology to therapeutics. We will focus on animal models of human disease, including using light-gated channels to restore vision to retinae that have lost photoreceptor cells, treatment of retinal disease due to angiogenesis, and prevention of cardiac damage due to ischemia.
In Yr 1 we made progress in several areas: a) the fundamental properties of new target proteins, b) new classes of photoswiches, and c) design of optical instrumentation for delivering light in vivo and in vitro.
Significance and Uniqueness
An important advance for improved pharmacology has been the development of tethered ligands, in which the targeting of a small molecule to a protein of interest depends not only on the usual single condition of the quality of fit into the binding site, but also on a second condition, namely proximity of the binding site to a covalent attachment site. This two-key approach makes it possible to obtain high specificity even when binding is weak. By using a short linker between the ligand and the protein-attachment site, the effective concentration of the ligand can be made very high, permitting even low affinity ligands to have high occupancy. On its own this approach produces chronic liganding and thus begs for a modification that would enable controlled switching. An attractive solution is the use of photoisomerizable moieties. Photochemically induced changes in the shape or electronic character of functionally important amino acids have been used to control the function of proteins in response to light or to alter the backbone structure of peptides, thereby controlling their interaction with other biological macromolecules. In an alternative strategy, which we propose here, photoisomerization of a tethered ligand or cross-linker can be used to gate protein activity.
The applications that we propose are generalizable to many classes of proteins, providing astonishingly rapid, reversibly, and spatially constrained remote control of protein function. Our plan is to produce a unique and powerful toolset of chemical photoswitches, and switched peptides and proteins, which can be used for basic research in cell and tissue biology. These tools will revolutionize the in vivo analysis of cell function, including in such challenging arenas as the analysis of neural circuits and the relation between the molecular vents in specific cells and the behavior of an entire organism. The methods also open a new avenue for reengineering of cells, or the production of synthetic cells, for targeted “smart-cell” therapy and for high throughput drug screening. Most enticing of all is the long-term goal of using light switches that control the function of specific proteins in specific cells in new strategies of therapy for human disease that circumvent problems of drug targeting by ensuring that the reagent acts only in a desired location that is pinpointed by light.
Pathway to Medicine
Blinding diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD) result from progressive degeneration of rods and cones. Downstream retinal neurons that receive and process signals from the photoreceptors are preserved for years after the onset of blindness in these diseases , giving hope that visual sensitivity might be restored by allowing the artificial input of information to these surviving cells. Previous retinal prosthetic devices used electrode arrays driven by a CCD video camera to control the firing of surviving retinal interneurons. While electrical stimulation of the retina produced light perception in patients receiving these devices, spatial and temporal resolution was poor, probably because of the relatively large size and imprecise positioning of electrodes in the arrays with relation to individual RGCs. The materials used in building these devices also present serious challenges of long-term biocompatibility. We propose using light-activated ion channels as an alternative therapeutic approach to avoid these problems.
By inserting minimally invasive micro-optical probes directly into deep tissues, cellular structures and dynamics never before visualized in vivo have recently become accessible to imaging with micron-scale resolution. This has begun to allow longitudinal studies in which cells in individual animals are imaged repeatedly over time, reducing the number of animals sacrificed for population statistics and allowing the progression of disease, biological processes, and experimental treatments to be monitored chronically in vivo. However, imaging is a powerful but purely passive technological approach in that it only collects information about the biological state. To alter biological states in active manner, imaging should ideally be combined with analogous capabilities for optical manipulation over protracted time periods, e.g. over the time course of disease progression. In the future, combined optical imaging and control will be performed in an intelligent manner, with the imaging data informing the spatiotemporal patterns of optical stimulation used for cellular control. This joint capability will impact biomedical research and clinical practice, which we illustrate with examples from three application areas:
Development of new drugs – It is common to test promising pharmaceutical compounds in rodent models of disease. The ability to perform minimally invasive imaging allows extended studies of the cellular effects of such compounds. A new generation of promising pharmaceuticals that will emerge from our NDC will be ‘smart’, optically activated drugs. Insertion of minimally invasive micro-optics into the body will allow both optical activation of these drugs in deep tissues and regular assessment of their performance at the cellular scale.
Creation of novel therapeutic strategies – Many therapeutic strategies of the future will involve aspects of optical control. This includes light-driven prosthetics for the visual system, light-activated gene therapies, and optically guided surgery for tissue ablation with cellular precision. The capability for minimally invasive delivery of light for imaging and control will be important for all these emerging therapeutic strategies, and microendoscopy is presently the only minimally invasive imaging modality with the ~1 micron resolution needed to resolve all cell types.
Cell biological studies of diseases processes in live animals – Combined capabilities for photonic imaging and control will allow increasingly sophisticated studies of disease mechanisms in live animal models. The ability to acquire imaging data, manipulate cells with micron-scale precision based on this data, and then to image the subsequent effects of manipulation will allow online, active intervention to become part of the disease researcher’s toolkit and to move beyond passive imaging. For example, laser-based precision control of cellular properties in a high-speed, three-dimensional manner may eventually allow us to simulate (and to repair) improper cellular dynamics.
This page last reviewed on July 24, 2013