7.2.1. Core A. Chemistry of probes and therapeutics Core Leader: Paul Wender Investigators: Lawrence Marnett, David Ostrov Aim 1: Review probe designs for the major projects and task-specific projects including those based on peptides and cox-2 inhibitors, and optimize conjugation chemistries for fluorescent and Raman tags (Fig 25). It is generally accepted that early detection leads to improved survival rates in the treatment of cancers. Detection of cancers and precancerous polyps in the gastrointestinal tract has been improved with endoscopy procedures, but this approach can miss difficult to observed tissue and flat neoplasias. In addition for some situations the difference between normal and precancerous tissue is only detectable at the molecular level. This necessitates the use of molecular probes for visualization of tissue differences. The Wender lab has considerable expertise in the innovation and use of drug/probe delivery systems, which includes the development of peptide and probe chemistry, as well as methods for their conjugation and evaluation in cells and in animals. This expertise will be employed in the design and synthesis of novel probe conjugates, as well as the development and even invention of synthetic methods as necessary. A variety of linkage strategies to make both non-releasable and releasable compounds as well as selectively releasable conjugates has been developed. The laboratories offer considerable experience in the design, synthesis, and evaluation of molecular probes that are indispensable tools for molecular imaging strategies. A significant challenge in the development of probes for optical imaging is in the attachment of labels for detection. The selection of fluorophores .and how they are conjugated to specific probes can drastically alter the specificity and usefulness of selected peptides or small molecules.
We aim to address this challenge by using structure-based molecular dynamic simulations to assess the preferred orientation of the probe bound to the target protein. This process is adapted from the molecular docking method we utilize in the high-throughput mode to select small molecules specific for target proteins (by parallel processing using DOCK6 at the University of Florida High Performance Computing Center). After determining the most likely orientation of the probe bound to the target protein (Fig. 25), solvent exposed atoms in the probe are selected as points of attachment for the label (Fig. 25). Real-time Automated Combinatorial Heuristic Enhancement of Lead compounds, RACHEL (Tripos, Inc.) is a drug optimization package designed to optimize weak binding lead compounds in an automated, combinatorial fashion. We routinely use RACHEL to add chemical groups, such as fluorophores, to lead compounds that bind with Kd values in the micromolar range in order to increase their specific activities (Fig. 25). This process can be applied to 5ROX and a large number of potential fluorophores to maximize the likelihood of identifying optimized labeled probes that retain specificity.
Aim 2 : Design next generation therapeutic agents and develop conjugates with directed delivery and controlled release. The Wender lab has considerable experience with the design, synthesis and evaluation of probe conjugates that enable or enhance cell and tissue entry. Procedures and systems have been developed that allow for quantification of uptake in real time in cells and in transgenic animals. These studies have been translated into the development of conjugates for drug delivery, including studies that have progressed to phase II human clinical trials. Much of this work has been applied the delivery of a plethora of therapeutics via a molecular transporter, most specifically but not limited to octaarginine. This molecular transporter has been used to deliver therapeutic agents such as taxol, cyclosporine A, SN-38, rapamycin, peptides, proteins, siRNA, plasmid DMA, metals, imaging agents and even magnetic particles. A variety of release mechanisms have been developed. The transporter technology and bioreleasable linker strategies developed in our laboratories have numerous applications which transcend the focus of this particular effort and could have applications for the entire field of drug delivery as well as conjugation chemistry. With the growing importance of selectivity in the delivery of therapeutic agents, we can apply methods previously developed in our lab related to drug delivery and expand on this methodology to realize the goal of selective delivery to gastrointestinal cancers, as well as other diseases. All facets of this application that rely on molecular detection and visualization can be facilitated and enhanced through access to design and synthetic expertise available in the core laboratory.
Aim 3 : Work with investigators and the FDA to first develop and then get approval for new reagents that can be used in the Gl tract for imaging and therapy. Development of new selective probe and therapeutic agents for the detection and treatment of gastrointestinal cancers is intended for eventual application in a clinical setting. The Wender lab has experience in this area with the development of octaarginine-cyclosporine A agents for the topical treatment of psoriasis, with conjugates advancing into Phase II human clinical trials. Studies have also been advanced based on IP, ocular, buccal, and lung drug administration, procedures that not unlike the proposed colon studies involve local administration. This expertise is available to enable and facilitate the design, synthesis, and evaluation of new probes and drug conjugates as needed to achieve the aims of the program.
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