Jeffrey M. Holzbeierlein

Engineering an Antibiotic to Fight Cancer: Optimization of the Novobiocin Scaffold to Produce Anti-proliferative Agents

Development of the DNA gyrase inhibitor, novobiocin, into a selective Hsp90 inhibitor was accomplished through structural modifications to the amide side chain, coumarin ring, and sugar moiety. These species exhibit ∼700-fold improved anti-proliferative activity versus the natural product as evaluated by cellular efficacies against breast, colon, prostate, lung, and other cancer cell lines. Utilization of structure-activity relationships established for three novobiocin synthons produced optimized scaffolds, which manifest midnanomolar activity against a panel of cancer cell lines and serve as lead compounds that manifest their activities through Hsp90 inhibition.

The design, synthesis, and evaluation of coumarin ring derivatives of the novobiocin scaffold that exhibit antiproliferative activity

Novobiocin, a known DNA gyrase inhibitor, binds to a nucleotide-binding site located on the Hsp90 C-terminus and induces degradation of Hsp90-dependent client proteins at approximately 700 microM in breast cancer cells (SKBr3). Although many analogues of novobiocin have been synthesized, it was only recently demonstrated that monomeric species exhibit antiproliferative activity against various cancer cell lines. To further refine the essential elements of the coumarin core, a series of modified coumarin derivatives was synthesized and evaluated to elucidate structure-activity relationships for novobiocin as an anticancer agent. Results obtained from these studies have produced novobiocin analogues that manifest low micromolar activity against several cancer cell lines.

Development of Novobiocin Analogues That Manifest Anti-proliferative Activity against Several Cancer Cell Lines

Recent studies have shown that the DNA gyrase inhibitor, novobiocin, binds to a previously unrecognized ATP-binding site located at the C-terminus of Hsp90 and induces degradation of Hsp90-dependent client proteins at approximately 700 microM. As a result of these studies, several analogues of the coumarin family of antibiotics have been reported and shown to exhibit increased Hsp90 inhibitory activity; however, the monomeric species lacked the ability to manifest anti-proliferative activity against cancer cell lines at concentrations tested. In an effort to develop more efficacious compounds that produce growth inhibitory activity against cancer cell lines, structure-activity relationships were investigated surrounding the prenylated benzamide side chain of the natural product. Results obtained from these studies have produced the first novobiocin analogues that manifest anti-proliferative activity against several cancer cell lines.

Hsp90: a drug target?

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in the trafficking of proteins in the cell. Under stressful conditions, Hsp90 stabilizes its client proteins and provides protection to the cell against cellular stressors such as in cancer cells. Disruption of Hsp90 leads to client protein degradation and often cell death. As Hsp90 has been found to be either overexpressed or constitutively more active in cancer cells, inhibitors of Hsp90 may have cancer cell selectivity. The N-terminal inhibitors, geldanamycin and radiciol, were the first two described inhibitors of Hsp90, but were not clinically useful. Subsequent analogues—17 allylamino-17demethoxygeldanamycin and 17 dimethylaminoethylamino-17-demethoxygeldanamycin—were found to be more clinically appropriate and have been studied in a number of clinical trials since 1999. In addition, to the N-terminal site of Hsp90, the C-terminal site appears to be another target for inhibition of Hsp90. More recently, inhibitors of the C terminus of Hsp90 have been developed and studied in vitro with promising results.

Development and characterization of a novel C-terminal inhibitor of Hsp90 in androgen dependent and independent prostate cancer cells

BackgroundThe molecular chaperone, heat shock protein 90 (Hsp90) has been shown to be overexpressed in a number of cancers, including prostate cancer, making it an important target for drug discovery. Unfortunately, results with N-terminal inhibitors from initial clinical trials have been disappointing, as toxicity and resistance resulting from induction of the heat shock response (HSR) has led to both scheduling and administration concerns. Therefore, Hsp90 inhibitors that do not induce the heat shock response represent a promising new direction for the treatment of prostate cancer. Herein, the development of a C-terminal Hsp90 inhibitor, KU174, is described, which demonstrates anti-cancer activity in prostate cancer cells in the absence of a HSR and describe a novel approach to characterize Hsp90 inhibition in cancer cells.MethodsPC3-MM2 and LNCaP-LN3 cells were used in both direct and indirect in vitro Hsp90 inhibition assays (DARTS, Surface Plasmon Resonance, co-immunoprecipitation, luciferase, Western blot, anti-proliferative, cytotoxicity and size exclusion chromatography) to characterize the effects of KU174 in prostate cancer cells. Pilot in vivo efficacy studies were also conducted with KU174 in PC3-MM2 xenograft studies.ResultsKU174 exhibits robust anti-proliferative and cytotoxic activity along with client protein degradation and disruption of Hsp90 native complexes without induction of a HSR. Furthermore, KU174 demonstrates direct binding to the Hsp90 protein and Hsp90 complexes in cancer cells. In addition, in pilot in-vivo proof-of-concept studies KU174 demonstrates efficacy at 75 mg/kg in a PC3-MM2 rat tumor model.ConclusionsOverall, these findings suggest C-terminal Hsp90 inhibitors have potential as therapeutic agents for the treatment of prostate cancer.

Macrophage Inhibitory Cytokine-1: Possible Bridge Molecule of Inflammation and Prostate Cancer

There is emerging evidence that inflammation may lead to prostate cancer development. Although inflammation is an essential response to injury or infection, chronic inflammation is harmful and causes tissue damage. Increasing evidence suggests that inflammation leads to the development of epithelial cancers; however, studies on inflammation-targeted genes that might contribute to the development of cancer are at the beginning stage. Here, we describe macrophage inhibitory cytokine-1, which provides a potential link between inflammation and prostate cancer. Understanding the regulation of macrophage inhibitory cytokine-1 in response to inflammation may have potential for novel therapeutic strategies.

Treatment of Non-Metastatic Muscle-Invasive Bladder Cancer: AUA/ASCO/ASTRO/SUO Guideline

Purpose: This multidisciplinary, evidence‐based guideline for clinically non‐metastatic muscle‐invasive bladder cancer focuses on the evaluation, treatment and surveillance of muscle‐invasive bladder cancer guided toward curative intent. Materials and Methods: A systematic review utilizing research from the Agency for Healthcare Research and Quality as well as additional supplementation by the authors and consultant methodologists was used to develop the guideline. Evidence‐based statements were based on body of evidence strengths Grade A, B or C and were designated as Strong, Moderate and Conditional Recommendations with additional statements presented in the form of Clinical Principles or Expert Opinions. Results: For the first time for any type of malignancy, the American Urological Association, American Society of Clinical Oncology, American Society for Radiation Oncology and Society of Urologic Oncology have formulated an evidence‐based guideline based on a risk‐stratified clinical framework for the management of muscle‐invasive urothelial bladder cancer. This document is designed to be used in conjunction with the associated treatment algorithm. Conclusions: The intensity and scope of care for muscle‐invasive bladder cancer should focus on the patient, disease and treatment response characteristics. This guideline attempts to improve a clinicians ability to evaluate and treat each patient, but higher quality evidence in future trials will be essential to improve level of care for these patients.

The perioperative management of an inferior vena caval tumor thrombus in patients with renal cell carcinoma

OBJECTIVES Inferior vena caval tumor thrombus (IVC-TT) occurs in 10% of patients diagnosed with renal cell carcinoma (RCC). The perioperative management of these patients remains challenging. Despite multiple publications outlining surgical approaches and outcomes there have been few studies detailing the best peri-operative management of patients with IVC-TT. Our goal was to define the optimal management of patients with RCC and IVC-TT. MATERIALS AND METHODS A review of all published literature regarding the management of RCC with IVC-TT was performed utilizing Pub Med and the Cochrane Database. Reviews were also made of all relevant literature regarding the need for cardiopulmonary bypass and recommendations regarding thrombus in any location in patients with malignancy. Specific items critically examined included: need for preoperative heart catheterization, need for anticoagulation and type of anticoagulation, need for additional studies such as lower extremity duplex or venogram, and indications for vena caval filter placement. The results were then presented to a multidisciplinary group made up of experts in the fields of Urology, Hematology, Oncology, Cardiothoracic Surgery, Interventional Radiology, and Pulmonary/Critical Care. Based on the available literature a best practice guidelines regarding the management of RCC with IVC-TT was established at our institution. RESULTS Our institutional recommendations include (1) preoperative cardiac catheterization in all patients believed to require cardiopulmonary bypass for removal of the thrombus but only cardiac clearance for those who bypass is unlikely, (2) preoperative anticoagulation using a low molecular weight heparin such as enoxaparin unless contraindicated due to bleeding from the tumor or other contraindication, (3) avoidance of vena caval filters whenever possible is recommended due the potential for caval thrombosis and the difficulties they present during surgical resection. CONCLUSION This study identified the available literature on the management of IVC-TT in association with RCC and was carefully reviewed by a multidisciplinary team. As a result, we have established a set of practice guidelines at our institution to help optimally manage patients with renal cell carcinoma and an inferior venal caval thrombus.

Targeting the Heat Shock Protein 90 Dimer with Dimeric Inhibitors

The design, synthesis, and biological evaluation of conformationally constrained coumermycin A1 analogues are reported. Compounds were evaluated against both breast cancer (SKBr3 and MCF7) and prostate cancer (PC3 mm2, A549, and HT29) cell lines. Non-noviosylated coumermycin A1 analogues that manifest potent antiproliferative activity resulting from Hsp90 inhibition are provided, wherein replacement of the stereochemically complex noviose sugar with readily available piperidine rings resulted in ∼100 fold increase in antiproliferative activities as compared to coumermycin A1, producing small molecule Hsp90 inhibitors that exhibit nanomolar activities.

Characterization of a novel novobiocin analogue as a putative C‐terminal inhibitor of heat shock protein 90 in prostate cancer cells

Hsp90 is important in the folding, maturation and stabilization of pro‐tumorigenic client proteins and represents a viable drug target for the design of chemotherapies. Previously, we reported the development of novobiocin analogues designed to inhibit the C‐terminal portion of Hsp90, which demonstrated the ability to decrease client protein expression. We now report the characterization of the novel novobiocin analogue, F‐4, which demonstrates improved cytotoxicity in prostate cancer cell lines compared to the N‐terminal inhibitor, 17‐AAG.