7.2.1. Core A. Chemistry of probes and therapeuticsCore Leader: Paul WenderInvestigators: Lawrence Marnett, David OstrovAim 1: Review probe designs for the major projects and task-specific projects including those based onpeptides and cox-2 inhibitors, and optimize conjugation chemistries for fluorescent and Raman tags (Fig25).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 withendoscopy procedures, but this approach can miss difficult to observed tissue and flat neoplasias. Inaddition for some situations the difference between normal and precancerous tissue is only detectable atthe 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 conjugationand evaluation in cells and in animals. This expertise will be employed in the design and synthesis of novelprobe conjugates, as well as the development and even invention of synthetic methods as necessary. Avariety of linkage strategies to make both non-releasable and releasable compounds as well asselectively releasable conjugates has been developed. The laboratories offer considerable experience inthe design, synthesis, and evaluation of molecular probes that are indispensable tools for molecularimaging strategies.A significant challenge in the development of probes for optical imaging is in the attachment of labels fordetection. The selection of fluorophores .and how they are conjugated to specific probes can drasticallyalter the specificity and usefulness of selected peptides or small molecules.
We aim to address thischallenge by using structure-based molecular dynamic simulations to assess the preferred orientation ofthe probe bound to the target protein. This process is adapted from the molecular docking method weutilize in the high-throughput mode to select small molecules specific for target proteins (by parallelprocessing 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), solventexposed atoms in the probe are selected as points of attachment for the label (Fig. 25). Real-timeAutomated Combinatorial Heuristic Enhancement of Lead compounds, RACHEL (Tripos, Inc.) is a drugoptimization 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 leadcompounds 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 maximizethe likelihood of identifying optimized labeled probes that retain specificity.
Aim 2 : Design next generation therapeutic agents and develop conjugates with directed delivery andcontrolled release.The Wender lab has considerable experience with the design, synthesis and evaluation of probeconjugates that enable or enhance cell and tissue entry. Procedures and systems have been developedthat allow for quantification of uptake in real time in cells and in transgenic animals. These studies havebeen translated into the development of conjugates for drug delivery, including studies that haveprogressed to phase II human clinical trials. Much of this work has been applied the delivery of a plethoraof therapeutics via a molecular transporter, most specifically but not limited to octaarginine. This moleculartransporter 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 ofrelease mechanisms have been developed. The transporter technology and bioreleasable linker strategiesdeveloped in our laboratories have numerous applications which transcend the focus of this particulareffort 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 methodspreviously developed in our lab related to drug delivery and expand on this methodology to realize the goalof selective delivery to gastrointestinal cancers, as well as other diseases. All facets of this applicationthat rely on molecular detection and visualization can be facilitated and enhanced through access todesign and synthetic expertise available in the core laboratory.
Aim 3 : Work with investigators and the FDA to first develop and then get approval for newreagents 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 ofgastrointestinal cancers is intended for eventual application in a clinical setting. The Wender lab hasexperience in this area with the development of octaarginine-cyclosporine A agents for the topicaltreatment of psoriasis, with conjugates advancing into Phase II human clinical trials. Studies have alsobeen advanced based on IP, ocular, buccal, and lung drug administration, procedures that not unlike theproposed colon studies involve local administration. This expertise is available to enable and facilitate thedesign, synthesis, and evaluation of new probes and drug conjugates as needed to achieve the aims ofthe program.
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