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Prostate cancer (PCa) is the most commonly diagnosed cancer in men in the United States and the second leading cause of cancer death. Death is not caused by the primary tumor but rather by the formation of distinct metastatic tumors. Therefore, prevention of metastasis is of utmost importance. The natural product genistein, found in high amounts in soy products, has been implicated in preventing PCa formation and metastasis in men who consume high amounts of soy. In vitro studies and in vivo rodent models that used human PCa cells, as well as prospective human clinical trials, provide a mechanistic explanation directly supporting genistein as an antimetastatic agent. Specifically, our group showed that genistein inhibits cell detachment, protease production, cell invasion, and human PCa metastasis at concentrations achieved in humans with dietary intake. Finally, phase I and phase II clinical trials conducted by us and others showed that concentrations of genistein associated with antimetastatic efficacy in preclinical models are achievable in humans, and treatment with genistein inhibits pathways that regulate metastatic transformation in human prostate tissue.

Targeting prostate cancer metastasis has very high therapeutic potential. Prostate cancer is the second most common cause of cancer death among men in the USA, and death results from the development of metastatic disease. In order to metastasize, cancer cells must complete a series of steps that together constitute the metastatic cascade. Each step therefore offers the opportunity for therapeutic targeting. However, practical limitations have served as limiting roadblocks to successfully targeting the metastatic cascade. They include our still-emerging understanding of the underlying biology, as well as the fact that many of the dysregulated processes have critical functionality in otherwise normal cells. We provide a discussion of the underlying biology, as it relates to therapeutic targeting. Therapeutic inroads are rapidly being made, and we present a series of case studies to highlight key points. Finally, future perspectives related to drug discovery for antimetastatic agents are discussed.

A component of the shikimate biosynthetic pathway, dehydroquinate dehydratase (DHQD) catalyzes the dehydration of 3-dehydroquniate (DHQ) to 3-dehydroshikimate. In the type I DHQD reaction mechanism a lysine forms a Schiff base intermediate with DHQ. The Schiff base acts as an electron sink to facilitate the catalytic dehydration. To address the mechanism of Schiff base formation, we determined structures of the Salmonella enterica wild-type DHQD in complex with the substrate analogue quinate and the product analogue shikimate. In addition, we determined the structure of the K170M mutant (Lys170 being the Schiff base forming residue) in complex with quinate. Combined with nuclear magnetic resonance and isothermal titration calorimetry data that revealed altered binding of the analogue to the K170M mutant, these structures suggest a model of Schiff base formation characterized by the dynamic interplay of opposing forces acting on either side of the substrate. On the side distant from the substrate 3-carbonyl group, closure of the enzyme's β8-α8 loop is proposed to guide DHQ into the proximity of the Schiff base-forming Lys170. On the 3-carbonyl side of the substrate, Lys170 sterically alters the position of DHQ's reactive ketone, aligning it at an angle conducive for nucleophilic attack. This study of a type I DHQD reveals the interplay between the enzyme and substrate required for the correct orientation of a functional group constrained within a cyclic substrate.

Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. Here we identify a Bifidobacterium longum protein with high sequence homology to type II DHQDs but no detectable DHQD activity under standard assay conditions. A crystal structure reveals that the B. longum protein adopts a DHQD-like tertiary structure but a distinct quaternary state. Apparently forming a dimer, the B. longum protein lacks the active site aspartic acid contributed from a neighboring protomer in the type II DHQD dodecamer. Relating to the absence of protein–protein interactions established in the type II DHQD dodecameric assembly, substantial conformational changes distinguish the would-be active site of the B. longum protein. As B. longum possess no other genes with homology to known DHQDs, these findings imply a unique DHQD activity within B. longum.

Enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes a critical step in the biosynthesis of a number of aromatic metabolites. An essential prokaryotic enzyme and the molecular target of the herbicide glyphosate, EPSPSs are the subject of both pharmaceutical and commercial interest. Two EPSPS classes that exhibit low sequence homology, differing substrate/glyphosate affinities, and distinct cation activation properties have previously been described. Here, we report structural studies of the monovalent cation-binding class II Coxiella burnetii EPSPS (cbEPSPS). Three cbEPSPS crystal structures reveal that the enzyme undergoes substantial conformational changes that alter the electrostatic potential of the active site. A complex with shikimate-3-phosphate, inorganic phosphate (Pi), and K(+) reveals that ligand induced domain closure produces an unusual cation-binding site bordered on three sides by the N-terminal domain, C-terminal domain, and the product Pi. A crystal structure of the class I Vibrio cholerae EPSPS (vcEPSPS) clarifies the basis of differential class I and class II cation responsiveness, showing that in class I EPSPSs a lysine side chain occupies the would-be cation-binding site. Finally, we identify distinct patterns of sequence conservation at the domain-domain interface and propose that the two EPSPS classes have evolved to differently optimize domain opening-closing dynamics.

Prostate cancer (PCa) is the second highest cause of cancer death in United States males. If the metastatic movement of PCa cells could be inhibited, then mortality from PCa could be greatly reduced. Mitogen-activated protein kinase kinase 4 (MAP2K4) has previously been shown to activate pro-invasion signaling pathways in human PCa. Recognizing that MAP2K4 represents a novel and validated therapeutic target, we sought to develop and characterize an efficient process for the identification of small molecules that target MAP2K4. Using a fluorescence-based thermal shift assay (FTS) assay, we first evaluated an 80 compound library of known kinase inhibitors, thereby identifying 8 hits that thermally stabilized MAP2K4 in a concentration dependent manner. We then developed two in vitro MAP2K4 kinase assays to evaluate kinase inhibitory function, one a western-blot based assay employing the biologically relevant downstream substrates, JNK1 and p38 MAPK, and the other an Alphascreen based activity assay. In this manner, we validated the performance of our initial FTS screen. Finally, by coupling our structure-activity relationship data to MAP2K4's crystal structure, we constructed a model for inhibitor binding. It predicted binding of our identified inhibitors to MAP2K4's ATP pocket. We next applied this approach in a high-throughput fashion to over 2200 chemically diverse compounds and identified new inhibitors of MAP2K4. Herein we report the creation of a robust inhibitor-screening platform with the ability to inform the discovery and design of new and potent MAP2K4 inhibitors. Citation Format: Sankar Narayan Krishna, Chi-Hao Luan, Matthew R. Clutter, Rama K. Mishra, Karl A. Scheidt, Wayne F. Anderson, Raymond C. Bergan. A high-throughput screening platform for the identification of small molecule inhibitors of MAP2K4. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5394. doi:10.1158/1538-7445.AM2014-5394

Prostate cancer (PCa) is the second highest cause of cancer death in United States males. If the metastatic movement of PCa cells could be inhibited, then mortality from PCa could be greatly reduced. Mitogen-activated protein kinase kinase 4 (MAP2K4) has previously been shown to activate pro-invasion signaling pathways in human PCa. Recognizing that MAP2K4 represents a novel and validated therapeutic target, we sought to develop and characterize an efficient process for the identification of small molecules that target MAP2K4. Using a fluorescence-based thermal shift assay (FTS) assay, we first evaluated an 80 compound library of known kinase inhibitors, thereby identifying 8 hits that thermally stabilized MAP2K4 in a concentration dependent manner. We then developed an in vitro MAP2K4 kinase assay employing the biologically relevant downstream substrates, JNK1 and p38 MAPK, to evaluate kinase inhibitory function. In this manner, we validated the performance of our initial FTS screen. We next applied this approach to a 2000 compound chemically diverse library, identified 7 hits, and confirmed them in the in vitro kinase assay. Finally, by coupling our structure-activity relationship data to MAP2K4's crystal structure, we constructed a model for ligand binding. It predicts binding of our identified inhibitory compounds to the ATP binding pocket. Herein we report the creation of a robust inhibitor-screening platform with the ability to inform the discovery and design of new and potent MAP2K4 inhibitors.

Introduction: Metastasis of Prostate Cancer (PCa) represents the second highest cause of death due to cancer among men in the United States. If this unregulated movement of cells can be inhibited, mortality arising from PCa will be greatly reduced. Our group has shown that Mitogen-activated protein kinase kinase 4 (MAP2K4/MEK4), a 399 amino acid protein, activates pro-invasion signaling pathways in human PCa. Further, we have shown that 4,5,7-trihydroxyisoavone (genistein) inhibits MEK4 kinase activity, cell invasion and metastasis of human PCa cells in a murine model. While therapeutically effective, genistein represents a non-chemically optimized natural product with additional undesired effects such as estrogenic receptor stimulation. Recognizing that MEK4 represents a novel and important therapeutic target, we have sought to characterize its structure, biochemical function and have synthesized genistein analogs designed to target it. Summary of Results: Using X-ray crystallography we have obtained a 3.38Å resolution structure of the MEK4 catalytic domain. We are in the process of improving our resolution, and of deriving 3D structures with inhibitors bound to MEK4. We have evaluated biophysical interactions between MEK4 and ligands (i.e., genistein and its analogs). Ligand binding to a protein tends to increase its stability, thereby increasing the temperature of denaturation, or melting. Using a fluorescence thermal shift (FTS) assay, we demonstrated that genistein and 6 of 39 novel analogs thermally stabilized MEK4 in a concentration dependent manner, consistent with MEK4 binding. We identified a similar pattern in 5 compounds from a commercial library, one of which was staurosporine - a broad spectrum protein kinase inhibitor. We developed and characterized a novel in vitro MEK4 kinase assay, using kinase dead p38MAPK (K53A) as substrate. In human PCa cells, MEK4 activates the p38 MAPK pro-invasion pathway. To date, we have used this assay system to demonstrate that staurosporine inhibits MEK4 with nanomolar IC50 values. Conclusions and Future Directions: MEK4 is a novel target for therapeutic intervention in PCa. Our ultimate goal is to synergistically utilize structural and biochemical information to inform the synthesis of inhibitors that specifically and selectively act upon MEK4 to inhibit human PCa metastasis in man. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4751. doi:1538-7445.AM2012-4751