Cropsolution utilizes two primary technologies, Targeted Biology™
and Evolutionary Chemistry™.

Targeted Biology™ - Overview
Targeted Biology™ is a proprietary technology platform practiced by Cropsolution comprised of know how and patented components.  Targeted Biology™ utilizes state-of-the-art target-based approaches coupled to high-throughput 96 well in vivo screening to rapidly identify and optimize lead compounds in all three indication areas – herbicides, fungicides and insecticides. Specifics regarding the team’s capabilities are detailed in the sections below.

Implementation of Targeted Biology™ for Agrochemical Discovery
Cropsolution’s process for discovering new agrochemicals starts with defining market opportunities and validated targets that can address these opportunities. Critical information for the prioritization process includes:

• biochemistry of the validated target
• the current product pipelines of the multinational agrochemical companies
• the type and cost of current products
• toxicological and ecological profiles of current products
• motivation of potential partners to license new products in a particular sub-market

Cropsolution has defined the first several accessible markets and prioritized sufficient public-sector targets to fill our product pipeline for several years.

Following target prioritization, chemical libraries that specifically suit the validated targets are designed at Cropsolution.  For an early stage target-specific R&D partnership with a major agrochemical company, chemists from the partner company would also likely be involved in library design.

Critical issues in designing libraries are:

• structure of substrate for the target
• structure(s) of known inhibitor(s) or agonist(s) of the target
• ease of production of the library inputs
• likelihood of lead compounds to be produced cost-effectively at a commercial scale
• reactivity and  stability of library inputs and lead compounds
• likely toxicological properties and environmental persistence of lead compounds
• likelihood of the lead compounds to be taken up and translocated within a crop plant
• likelihood of lead compounds to be protectable by patent claims

Library design for our first target is currently underway.

Concomitant with input chemical production, validated target expression occurs at Cropsolution using multiple protein expression systems to yield purified active validated target protein.  Systems are available to express signaling, enzyme and membrane-associated proteins.  An assay for the validated biochemical target is produced, keeping in mind, cost, speed, sensitivity, reproducibility and quality/reliability of data.

Following synthesis of the simple inputs, library construction and isolation of lead compounds is carried out.  The lead compounds are screened by in the format most suitable to both the target and the compounds.  The result is a small number (1-10) of lead compounds that bind to the validated target, and have properties favorable for agrochemicals.

The small molecule families are then produced in microgram quantities for micro-scale biological testing on plants (for all indications), insects (for insecticide leads) and fungi (fungicide leads).  At this stage, relative activity to a known chemical standard and spectrum of activity can be crudely estimated.  Chemical production for such testing will be performed at Cropsolution.

Once biological activity is confirmed with a test compound, the synthesis of related analogs is initiated to optimize compound performance. During this iterative process of synthesis and biological evaluation, several lead structures are likely to serve as starting points for analog synthesis. During optimization on the target of interest, attention is also given to the physicochemical properties of the analogs such as molecular weight, pKa, and logP that will affect the uptake and transport of the compounds in plants. By designing for optimal performance, the chances of commercial success are significantly increased. Cropsolution chemists have a wealth of agrochemical experience and a proven record of success in hit/lead optimization.

Once a molecule with an appropriate level and spectrum of activity has been found, greenhouse evaluation and the first of several patent filings occurs.  The greenhouse testing is conducted at contractors under carefully regulated growth conditions of important target crops.  The crops and pests selected match the market opportunity for which the target was chosen as well as a standard set of large opportunity crops/pests that are used by most in the industry.  Examples include wheat and Erysiphe, corn and Setaria, cotton and Heliothus.  Chemical production for this and subsequent testing is performed at Cropsolution.  The cost of production of suitable quantities for greenhouse and initial field characterization is $5,000 to 50,000 depending on the chemistry, number of steps, input cost and yields.

Following greenhouse characterization, formulation and field evaluation occurs.  Formulation occurs at Cropsolution while the field testing is conducted by select contract research organizations using methods and protocols identical to those employed by the multinational agrochemical companies.

Acetyl-CoA Carboxylase

Cropsolution has identified a validated biochemical target applicable for selection of a broad-spectrum fungicide with activity against the three major classes of disease causing fungi - ascomycetes, basidomycetes and oomycetes.  We estimate that a chemical with good inhibition of this target, low use rates and other appropriate agronomic characteristics has a market potential of at least $1 billion per year (25% of the global cereal and fruit/vegetable fungicide markets).

Cropsolution has a proprietary assay for the soraphen-like inhibitors of ACCase.  Please contact the company for licensing information.

Background. Acetyl-CoA carboxylase (ACC) catalyzes the conversion of acetyl-CoA to malonyl-CoA, the two-carbon donor in fatty acid biosynthesis. ACC is a multifunctional enzyme in that this conversion proceeds via two half reactions: the ATP-dependent carboxylation of enzyme-bound biotin followed by the transfer of the carboxyl group to acetyl-CoA (Figure 1). The biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT) domains are contained on a single, large (~250 kD), multidomain polypeptide in the ACC’s found in animals, fungi, and plant cytosols (eukaryotic ACC’s). In contrast, these functions reside on separate protein subunits in the ACC’s found in bacteria and the plastids of most plants (prokaryotic ACC’s).

Due to its role in primary metabolism, ACC has been exploited as a commercial agrochemical target. Two classes of commercial herbicides, the aryloxyphenoxypropionates and the cyclohexanediones, inhibit the CT domain of the eukaryotic ACC’s found in the plastids of grasses. Additionally, two new insecticides, spirodiclofen and spiromesifen, are reported to target ACC. Finally, ACC is a chemically validated fungicide target (discussed below).

Soraphen. The most potent small molecule inhibitor of eukaryotic ACCs identified to date is the natural product soraphen (Figure 2). Soraphen was first identified by virtue of its antifungal activity and later shown to target the BC domain of ACC. While soraphen exhibits commercial efficacy as a broad-spectrum fungicide under field conditions, it is not suitable as a commercial product due to high production costs and toxic side-effects (1). To our knowledge, ACC is the only chemically validated fungicide target for which no commercial product currently exists. As such, it presents an attractive opportunity for a target-based discovery program.

Fungicide Discovery.  The macrocyclic structure of soraphen does not lend itself to analoging in order to identify related but safer compounds with similar efficacy. Clearly, however, novel small molecules that target the soraphen binding site have great potential as fungicide leads. Understanding soraphen-BC binding interactions and developing sensitive assays to monitor this binding site are invaluable tools in the identification of such compounds. To this end, we have developed a high level expression system for full-length fungal ACC. The recombinant enzyme exhibits similar enzymatic properties as the native enzyme, and can be used to assay compounds for activity inhibition. In addition, we have isolated small and stable BC domains from several organisms that retain high-affinity soraphen binding.  The isolated BC domains proved optimal for structural studies and allowed us to obtain a high resolution crystal structure of the yeast BC domain in complex with soraphen (Figure 3). This structure has given us detailed information about the soraphen binding site, and has led us to propose a novel mechanism for ACC inhibition whereby soraphen binds to the BC dimer interface and disrupts subunit interactions that are required for enzyme activity.

Access to this crystal structure is allowing us to incorporate computational methods into our small molecule discovery efforts. Furthermore, knowledge obtained from this structure was instrumental in designing and synthesizing a fluorescent soraphen derivative that retains native soraphen binding properties. Using this derivative, we have developed a robust, sensitive, high-throughput fluorescence polarization competition binding assay that allows us to screen for novel small molecules that interact with the soraphen binding site. To complement our in vitro screening capabilities, we also have developed high-throughput, plate-based in vivo screens that include representative oomycetes, basidiomycetes, deuteromycetes, and ascomycetes. Finally, our chemistry team has a wealth of experience in assessing structure activity relationships and using them to direct lead optimization that ultimately results in commercial success.

Drug Discovery. In animals, the malonyl-CoA produced by ACC inhibits fatty acid oxidation in addition to serving as the two-carbon donor for fatty acid synthesis. Because of this central role as a regulator of whether fat gets stored or burned, ACC is being pursued as a pharma target for the development of therapeutics to treat obesity and related diseases. Knockout studies in mice (2, 3) and animal studies with ACC inhibitors (4, 5) have validated this approach. Furthermore, soraphen has been demonstrated to have pharmalogical properties consistent with the potential to treat metabolic disorders like obesity (6). Therefore, we are leveraging our in vitro screening capabilities and computational assets by using them as a dual platform for both drug discovery and fungicide discovery. For drug discovery efforts, we are developing in vitro assays to monitor human ACC activity, and cell-based assays to monitor fatty acid synthesis and fatty acid oxidation. Additionally, we are developing contractual arrangements to carry out pre-clinical animal studies on leads that we optimize. We have received an NIH SBIR grant to fund these studies.

References

1. Pridzun, L., Sasse, F., and Reichenbach, H. (1995) in Antifungal Agents:  Discovery and Mode of Action (Dixon, G. K., Copping, L. G., and Hollomon, D., Eds.) pp 99-109, Bios Scientific, Oxford.
   
2. Abu-Elheiga, L., Matzuk, M. M., Abo-Hashema, K. A., and Wakil, S. J. (2001) Science291, 2613-6.
   
3. Abu-Elheiga, L., Oh, W., Kordari, P., and Wakil, S. J. (2003) Proc Natl Acad Sci U S A100, 10207-12.
   
4. Harwood, H. J., Jr. (2004) Curr Opin Investig Drugs5, 283-9.
   
5. Harwood, H. J., Jr., Petras, S. F., Shelly, L. D., Zaccaro, L. M., Perry, D. A., Makowski, M. R., Hargrove, D. M., Martin, K. A., Tracey, W. R., Chapman, J. G., Magee, W. P., Dalvie, D. K., Soliman, V. F., Martin, W. H., Mularski, C. J., and Eisenbeis, S. A. (2003) J Biol Chem278, 37099-111.
   
6. Gubler, M., and Mizrahi, J. (2003), Hoffman-La Roche AG, PCT.

Cropsolution Publications

• Weatherly, S.C., Volrath, S.L., and Elich, T.D. (2004). Expression and characterization of recombinant fungal acetyl-CoA carboxylase and isolation of a soraphen-binding domain. Biochem J 380, 105-110.
  
• Shen, Y., Volrath, S.L., Weatherly, S.C., Elich, T.D., and Tong, L. (2004). A mechanism for the potent inhibition of eukaryotic acetyl-coenzyme a carboxylase by soraphen a, a macrocyclic polyketide natural product. Mol Cell 16, 881-891.

Evolutionary Chemistry™

Evolutionary Chemistry™ starts with design of a chemical library by our chemistry team. Design takes advantage of all available information about a validated biochemical target, including structures of substrates, natural ligands, and known inhibitors. The resulting library scheme contains up to millions of compounds specific for the target.

Cropsolution has an exclusive worldwide license to use Evolutionary Chemistry™ for agrochemical discovery.

Evolutionary Chemistry™ projects at Cropsolution include a NSF-SBIR funded proof of concept experiment aimed at extending the capability of RNA catalysts to synthesize small molecules.  This proof of concept was successful, demonstrating the ability of the technology to evolve RNA catalysts for the synthesis of sulfonylureas, an important class of herbicides and pharmaceuticals.

An RNA catalyst for sulfonylurea synthesis was evolved after 10 rounds of selection.  Specifically, the selection converged on a single sequence.  This catalyst, Acy1, demonstrated a rate enhancement of 60-fold over background. 

A second ECTM experiment was then designed in which the DNA template for the Acy1 catalyst was mutated through mutagenic PCR and used as the template for the RNA library in the second ECTM selection.  This mutagenized selection allowed for the exploration of sequences related to Acy1 that resulted in the evolution of new sequences with improved catalytic ability.  A family of sequences that were variants of the original Acy1 catalyst evolved from this selection.  The best catalyst identified, Acy279, demonstrated a rate enhancement of 600-fold over background. 

This proof of concept experiment demonstrated the ECTM technology to be successful in the selection of RNA catalyst s from a large pool (10e14) of random sequences.  This RNA-catalyzed sulfonylurea formation broadens the scope of compounds demonstrated to be synthesized by RNA.

General reaction scheme for RNA catalyzed sulfonylurea synthesis - acylation of a RNA-tethered sulfonamide with an activated carbamate (L = linker).
Kinetic characterization of Acy1. Data fit to Michaelis-Menten rate	Kinetic characterization of Acy279. Data fit to Michaelis-Menten rate expression for single turnover reaction at a single saturable substrate-binding site. Kcat = 6.7x10-5 min-1, Km = 257 mM.
Proposed Acy1 structure.	Proposed Acy279 structure. Differences from the Acy1 structure are indicated by arrows. Increased thermal stability of the Acy279 structure may have contributed to the improved substrate binding that was selected for in this catalyst.

Cropsolution currently has a research partnership with a major agrochemical company utilizing Evolutionary Chemistry™ technology to identify novel inhibitors to a proprietary herbicide target. 

Future Evolutionary Chemistry™ projects will involve discovery of additional chemistries catalyzed by RNA that are relevant for the synthesis of novel, commercial compounds.