Muriel Bonnet

Targeting GLUT1 and the Warburg effect in renal cell carcinoma by chemical synthetic lethality

A screen identifies a drug that specifically kills glycolysis-dependent cancer cells by inhibiting glucose uptake. Cancer’s Achilles’ Heel A quick tug on a fuel line can stop a car dead in its tracks. Similarly, depriving a cancer cell of its energy source can bring proliferation to a standstill. Chan et al. devised a drug discovery assay that took advantage of the fact that some kidney cancer cells depend on glucose for survival. By screening 64,000 small molecules, the authors found a class of drug that inhibits the glucose transporter and selectively impairs growth of these cancer cells in cultures and in animals. Certain kidney and other types of cancer cells lack the von Hippel–Lindau (VHL) tumor suppressor protein. This deficiency reorients carbohydrate metabolism so that the cancer cells depend on aerobic glycolysis—the conversion of glucose to lactate—rather than the more typical oxidative phosphorylation for a supply of energy. The drug identified by the authors, STF-31, was toxic to the VHL-deficient kidney tumor cells but, unlike many other cancer drugs, did not induce autophagy, apoptosis, or DNA damage. Rather, STF-31 exploited the fact that inactivation of VHL increases the activity of hypoxia-inducible factor transcription factor, which in turn stimulates the transcription of genes involved in glucose metabolism, including the glucose transporter–encoding gene GLUT1. By binding directly to the transporter, STF-31 blocked glucose uptake in VHL-deficient cancer cells but not in those with intact VHL; with their sugar delivery system stymied, the tumor suppressor–deprived cancer cells ceased glycolysis and thus adenosine 5′-triphosphate production and succumbed to necrosis. An extra benefit of the new agent is that its activity can be easily visualized, even deep inside an animal. Glucose uptake in a tumor can be monitored by fluorodeoxyglucose positron emission tomography. The reduction in glucose metabolism forced on tumors by STF-31 was detected in mice with this method—an approach that can be readily applied to humans to test the drug’s efficacy. If it can thwart the fuel supply line in human cancers, this promising drug likely will bring tumor thriving to a halt. Identifying new targeted therapies that kill tumor cells while sparing normal tissue is a major challenge of cancer research. Using a high-throughput chemical synthetic lethal screen, we sought to identify compounds that exploit the loss of the von Hippel–Lindau (VHL) tumor suppressor gene, which occurs in about 80% of renal cell carcinomas (RCCs). RCCs, like many other cancers, are dependent on aerobic glycolysis for ATP production, a phenomenon known as the Warburg effect. The dependence of RCCs on glycolysis is in part a result of induction of glucose transporter 1 (GLUT1). Here, we report the identification of a class of compounds, the 3-series, exemplified by STF-31, which selectively kills RCCs by specifically targeting glucose uptake through GLUT1 and exploiting the unique dependence of these cells on GLUT1 for survival. Treatment with these agents inhibits the growth of RCCs by binding GLUT1 directly and impeding glucose uptake in vivo without toxicity to normal tissue. Activity of STF-31 in these experimental renal tumors can be monitored by [18F]fluorodeoxyglucose uptake by micro–positron emission tomography imaging, and therefore, these agents may be readily tested clinically in human tumors. Our results show that the Warburg effect confers distinct characteristics on tumor cells that can be selectively targeted for therapy.

4-Pyridylanilinothiazoles That Selectively Target von Hippel−Lindau Deficient Renal Cell Carcinoma Cells by Inducing Autophagic Cell Death

Renal cell carcinomas (RCC) are refractory to standard therapy with advanced RCC having a poor prognosis; consequently treatment of advanced RCC represents an unmet clinical need. The von Hippel-Lindau (VHL) tumor suppressor gene is mutated or inactivated in a majority of RCCs. We recently identified a 4-pyridyl-2-anilinothiazole (PAT) with selective cytotoxicity against VHL-deficient renal cells mediated by induction of autophagy and increased acidification of autolysosomes. We report exploration of structure-activity relationships (SAR) around this PAT lead. Analogues with substituents on each of the three rings, and various linkers between rings, were synthesized and tested in vitro using paired RCC4 cell lines. A contour map describing the relative spatial contributions of different chemical features to potency illustrates a region, adjacent to the pyridyl ring, with potential for further development. Examples probing this domain validated this approach and may provide the opportunity to develop this novel chemotype as a targeted approach to the treatment of RCC.

SAR Studies of 4-Pyridyl Heterocyclic Anilines that Selectively Induce Autophagic Cell Death in von Hippel-Lindau-Deficient Renal Cell Carcinoma Cells

We recently identified a class of pyridyl aniline thiazoles (PAT) that displayed selective cytotoxicity for von Hippel-Lindau (VHL) deficient renal cell carcinoma (RCC) cells in vitro and in vivo. Structure-activity relationship (SAR) studies were used to develop a comparative molecular field analysis (CoMFA) model that related VHL-selective potency to the three-dimensional arrangement of chemical features of the chemotype. We now report the further molecular alignment-guided exploration of the chemotype to discover potent and selective PAT analogues. The contribution of the central thiazole ring was explored using a series of five- and six-membered ring heterocyclic replacements to vary the electronic and steric interactions in the central unit. We also explored a positive steric CoMFA contour adjacent to the pyridyl ring using Pd-catalysed cross-coupling Suzuki-Miyaura, Sonogashira and nucleophilic displacement reactions to prepare of a series of aryl-, alkynyl-, alkoxy- and alkylamino-substituted pyridines, respectively. In vitro potency and selectivity were determined using paired RCC cell lines: the VHL-null cell line RCC4 and the VHL-positive cell line RCC4-VHL. Active analogues selectively induced autophagy in RCC4 cells. We have used the new SAR data to further develop the CoMFA model, and compared this to a 2D-QSAR method. Our progress towards realising the therapeutic potential of this chemotype as a targeted cytotoxic therapy for the treatment of RCC by exploiting the absence of the VHL tumour suppressor gene is reported.

Novel nitroimidazole alkylsulfonamides as hypoxic cell radiosensitisers

A novel class of nitroimidazole alkylsulfonamides have been prepared and evaluated as hypoxia-selective cytotoxins and radiosensitisers. The sulfonamide side chain markedly influences the physicochemical properties of the analogues: lowering aqueous solubility and raising the electron affinity of the nitroimidazole group. The addition of hydroxyl or basic amine groups increased aqueous solubility, with charged amine groups contributing to increased electron affinity. The analogues covered the range of electron affinity for effective radiosensitisation with one-electron reduction potentials ranging from -503 to -342mV. Cytotoxicity under normoxia or anoxia against a panel of human tumour cell lines was determined using a proliferation assay. 2-Nitroimidazole sulfonamides displayed significant hypoxia-selective cytotoxicity (6 to 64-fold), while 4- and 5-nitroimidazole analogues did not display hypoxia-selective cytotoxicity. All analogues sensitised anoxic HCT-116 human colorectal cells to radiation at non-toxic concentrations. 2-Nitroimidazole analogues provided modest sensitisation due to the relatively low concentrations used while several 5-nitroimidazole analogues provided equivalent sensitisation to misonidazole and etanidazole at similar molar concentrations.

Identifying novel targets in renal cell carcinoma: design and synthesis of affinity chromatography reagents.

Two novel scaffolds, 4-pyridylanilinothiazoles (PAT) and 3-pyridylphenylsulfonyl benzamides (PPB), previously identified as selective cytotoxins for von Hippel-Lindau-deficient Renal Carcinoma cells, were used as templates to prepare affinity chromatography reagents to aid the identification of the molecular targets of these two classes. Structure-activity data and computational models were used to predict possible points of attachment for linker chains. In the PAT class, Click coupling of long chain azides with 2- and 3-pyridylanilinothiazoleacetylenes gave triazole-linked pyridylanilinothiazoles which did not retain the VHL-dependent selectivity of parent analogues. For the PPB class, Sonagashira coupling of 4-iodo-(3-pyridylphenylsulfonyl)benzamide with a propargyl hexaethylene glycol carbamate gave an acetylene which was reduced to the corresponding alkyl 3-pyridylphenylsulfonylbenzamide. This reagent retained the VHL-dependent selectivity of the parent analogues and was successfully utilized as an affinity reagent.

Next-generation hypoxic cell radiosensitizers: nitroimidazole alkylsulfonamides

Innovations in the field of radiotherapy such as stereotactic body radiotherapy, along with the advent of radio-immuno-oncology, herald new opportunities for classical oxygen-mimetic radiosensitizers. The role of hypoxic tumor cells in resistance to radiotherapy and in suppression of immune response continues to endorse tumor hypoxia as a bona fide, yet largely untapped, drug target. Only nimorazole is used clinically as a radiosensitizer, and there is a dearth of new radiosensitizers in development. Here we present a survey of novel nitroimidazole alkylsulfonamides and document their cytotoxicity and ability to radiosensitize anoxic tumor cells in vitro. We use a phosphate prodrug approach to increase aqueous solubility and to improve tumor drug delivery. A 2-nitroimidazole and a 5-nitroimidazole analogue demonstrated marked tumor radiosensitization in either ex vivo assays of surviving clonogens or tumor regrowth delay.

Antitubercular Nitroimidazoles Revisited: Synthesis and Activity of the Authentic 3-Nitro Isomer of Pretomanid

A published study of structural features associated with the aerobic and anaerobic activities of 4- and 5-nitroimidazoles had found that the 3-nitro isomer of pretomanid, 8, displayed interesting potencies, including against nitroreductase mutant Mycobacterium tuberculosis. However, recent nuclear magnetic resonance analyses of two trace byproducts, isolated from early process optimization studies toward a large-scale synthesis of pretomanid, raised structural assignment queries, particularly for 8, stimulating further investigation. Following our discovery that the reported compound was a 6-nitroimidazooxazole derivative, we developed a de novo synthesis of authentic 8 via nitration of the chiral des-nitro imidazooxazine alcohol 26 in trifluoroacetic or acetic anhydride, and verified its identity through an X-ray crystal structure. Unfortunately, 8 displayed no antitubercular activity (MICs > 128 μM), whereas the second byproduct (3′-methyl pretomanid) was eight-fold more potent than pretomanid in the aerobic assay. These findings further clarify target specificities for bicyclic nitroimidazoles, which may become important in the event of any future clinical resistance.