Joe Lewis

Inhibition of aurora kinases for tailored risk-adapted treatment of multiple myeloma

Genetic instability and cellular proliferation have been associated with aurora kinase expression in several cancer entities, including multiple myeloma. Therefore, the expression of aurora-A, -B, and -C was determined by Affymetrix DNA microarrays in 784 samples including 2 independent sets of 233 and 345 CD138-purified myeloma cells from previously untreated patients. Chromosomal aberrations were assessed by comprehensive interphase fluorescence in situ hybridization and proliferation of primary myeloma cells by propidium iodine staining. We found aurora-A and -B to be expressed at varying frequencies in primary myeloma cells of different patient cohorts, but aurora-C in testis cell samples only. Myeloma cell samples with detectable versus absent aurora-A expression show a significantly higher proliferation rate, but neither a higher absolute number of chromosomal aberrations (aneuploidy), nor of subclonal aberrations (chromosomal instability). The clinical aurora kinase inhibitor VX680 induced apoptosis in 20 of 20 myeloma cell lines and 5 of 5 primary myeloma cell samples. Presence of aurora-A expression delineates significantly inferior event-free and overall survival in 2 independent cohorts of patients undergoing high-dose chemotherapy, independent from conventional prognostic factors. Using gene expression profiling, aurora kinase inhibitors as a promising therapeutic option in myeloma can be tailoredly given to patients expressing aurora-A, who in turn have an adverse prognosis.

Residues in the HIV-1 Capsid Assembly Inhibitor Binding Site Are Essential for Maintaining the Assembly-competent Quaternary Structure of the Capsid Protein.

Morphogenesis of infectious HIV-1 involves budding of immature virions followed by proteolytic disassembly of the Gag protein shell and subsequent assembly of processed capsid proteins (CA) into the mature HIV-1 core. The dimeric interface between C-terminal domains of CA (C-CA) has been shown to be important for both immature and mature assemblies. We previously reported a CA-binding peptide (CAI) that blocks both assembly steps in vitro. The three-dimensional structure of the C-CA/CAI complex revealed an allosteric effect of CAI that alters the C-CA dimer interface. Based on this structure, we now investigated the phenotypes of mutations in the binding pocket. CA variants carrying mutations Y169A, L211A, or L211S had a reduced affinity for CAI and were unable to form mature-like particles in vitro. These mutations also blocked morphological conversion to mature virions in tissue culture and abolished infectivity. X-ray crystallographic analyses of the variant C-CA domains revealed that these alterations induced the same allosteric change at the dimer interface observed in the C-CA/CAI complex. These results point to a role of key interactions between conserved amino acids in the CAI binding pocket of C-CA in maintaining the correct conformation necessary for mature core assembly.

Improved synthesis of N-Benzylaminoferrocene-based prodrugs and evaluation of their toxicity and antileukemic activity

We report on an improved method of synthesis of N-benzylaminoferrocene-based prodrugs and demonstrate its applicability by preparing nine new aminoferrocenes. Their effect on the viability of selected cancer cells having different p53 status was studied. The obtained data are in agreement with the hypothesis that the toxicity of aminoferrocenes is not dependent upon p53 status. Subsequently the toxicity of a selected prodrug (4) was investigated ex vivo using rat precision cut liver slices and in vivo on hybrid male mice BDF1. In both experiments no toxicity was observed: ex vivo, up to 10 μM; in vivo, up to 6 mg/kg. Finally, prodrug 4 was shown to extend the survival of BDF1 mice carrying L1210 leukemia from 13.7 ± 0.6 days to 17.5 ± 0.7 days when injected daily 6 times at a dose of 26 μg/kg starting from the second day after injection of L1210 cells.

A Direct Role for ATP1A1 in Unconventional Secretion of Fibroblast Growth Factor 2

Background: Unconventional secretion of FGF2 occurs by direct translocation across plasma membranes. Results: The cytoplasmic domain of ATP1A1 directly interacts with FGF2 and is required for FGF2 secretion. Conclusion: ATP1A1 supports unconventional secretion by recruiting FGF2 to the inner leaflet of plasma membranes. Significance: A new machinery component required for unconventional secretion of FGF2 was identified and validated. Previous studies proposed a role for the Na/K-ATPase in unconventional secretion of fibroblast growth factor 2 (FGF2). This conclusion was based upon pharmacological inhibition of FGF2 secretion in the presence of ouabain. However, neither independent experimental evidence nor a potential mechanism was provided. Based upon an unbiased RNAi screen, we now report the identification of ATP1A1, the α1-chain of the Na/K-ATPase, as a factor required for efficient secretion of FGF2. As opposed to ATP1A1, down-regulation of the β1- and β3-chains (ATP1B1 and ATP1B3) of the Na/K-ATPase did not affect FGF2 secretion, suggesting that they are dispensable for this process. These findings indicate that it is not the membrane potential-generating function of the Na/K-ATPase complex but rather a so far unidentified role of potentially unassembled α1-chains that is critical for unconventional secretion of FGF2. Consistently, in the absence of β-chains, we found a direct interaction between the cytoplasmic domain of ATP1A1 and FGF2 with submicromolar affinity. Based upon these observations, we propose that ATP1A1 is a recruitment factor for FGF2 at the inner leaflet of plasma membranes that may control phosphatidylinositol 4,5-bisphosphate-dependent membrane translocation as part of the unconventional secretory pathway of FGF2.

5-azacytidine inhibits nonsense-mediated decay in a MYC-dependent fashion

Nonsense‐mediated RNA decay (NMD) is an RNA‐based quality control mechanism that eliminates transcripts bearing premature translation termination codons (PTC). Approximately, one‐third of all inherited disorders and some forms of cancer are caused by nonsense or frame shift mutations that introduce PTCs, and NMD can modulate the clinical phenotype of these diseases. 5‐azacytidine is an analogue of the naturally occurring pyrimidine nucleoside cytidine, which is approved for the treatment of myelodysplastic syndrome and myeloid leukemia. Here, we reveal that 5‐azacytidine inhibits NMD in a dose‐dependent fashion specifically upregulating the expression of both PTC‐containing mutant and cellular NMD targets. Moreover, this activity of 5‐azacytidine depends on the induction of MYC expression, thus providing a link between the effect of this drug and one of the key cellular pathways that are known to affect NMD activity. Furthermore, the effective concentration of 5‐azacytidine in cells corresponds to drug levels used in patients, qualifying 5‐azacytidine as a candidate drug that could potentially be repurposed for the treatment of Mendelian and acquired genetic diseases that are caused by PTC mutations.

High throughput screening against the peroxidase cascade of African trypanosomes identifies antiparasitic compounds that inactivate tryparedoxin.

Background: Hydroperoxide detoxification in African trypanosomes relies on a cascade composed of trypanothione, trypanothione reductase, tryparedoxin, and tryparedoxin peroxidases. Results: A library screening against the peroxidase system unraveled trypanocidal compounds that inactivate tryparedoxin in vitro and in the intact parasite. Conclusion: Tryparedoxin is a druggable target. Significance: Detection of novel target molecules is a crucial step to overcome the unsatisfactory chemotherapy of sleeping sickness. In African trypanosomes, the detoxification of broad spectrum hydroperoxides relies on a unique cascade composed of trypanothione (T(SH)2), trypanothione reductase, tryparedoxin (Tpx), and nonselenium glutathione peroxidase-type enzymes. All three proteins are essential for Trypanosoma brucei. Here, we subjected the complete system to a high throughput screening approach with nearly 80,000 chemicals. Twelve compounds inhibited the peroxidase system. All but one carried chloroalkyl substituents. The detailed kinetic analysis showed that two compounds weakly inhibited trypanothione reductase, but none of them specifically interacted with the peroxidase. They proved to be time-dependent inhibitors of Tpx-modifying Cys-40, the first cysteine of its active site WCPPC motif. Importantly, gel shift assays verified Tpx as a target in the intact parasites. T(SH)2, present in the in vitro assays and in the cells in high molar excess, did not interfere with Tpx inactivation. The compounds inhibited the proliferation of bloodstream T. brucei with EC50 values down to <1 μm and exerted up to 83-fold lower toxicity toward HeLa cells. Irreversible inhibitors are traditionally regarded as unfavorable. However, a large number of antimicrobials and anticancer therapeutics acts covalently with their target protein. The compounds identified here also interacted with recombinant human thioredoxin, a distant relative of Tpx. This finding might even be exploited for thioredoxin-based anticancer drug development approaches reported recently. The fact that the T(SH)2/Tpx couple occupies a central position within the trypanosomal thiol metabolism and delivers electrons also for the synthesis of DNA precursors renders the parasite-specific oxidoreductase an attractive drug target molecule.

ELR510444 inhibits tumor growth and angiogenesis by abrogating HIF activity and disrupting microtubules in renal cell carcinoma.

Background Hypoxia-inducible factor (HIF) is an attractive therapeutic target for renal cell carcinoma (RCC) as its high expression due to the loss of von Hippel-Lindau (VHL) promotes RCC progression. Considering this, we hypothesized that ELR510444, a novel orally available small molecule inhibitor of HIF activity, would reduce angiogenesis and possess significant activity in RCC. The mechanism of action and therapeutic efficacy of ELR510444 were investigated in in vitro and in vivo models of RCC. Principal Findings ELR510444 decreased HIF-1α and HIF-2α levels, reduced RCC cell viability and clonogenic survival, and induced apoptosis. VHL-deficient RCC cells were more sensitive to ELR510444-mediated apoptosis and restoration of VHL promoted drug resistance. Higher concentrations of ELR51044 promoted apoptosis independently of VHL status, possibly due to the microtubule destabilizing properties of this agent. ELR510444 significantly reduced tumor burden in the 786-O and A498 RCC xenograft models. These effects were associated with increased necrosis and apoptosis and inhibition of angiogenesis. Conclusions ELR510444 is a promising new HIF inhibitor that reduced RCC cell viability, induced apoptosis, and diminished tumor burden in RCC xenograft models. ELR510444 also destabilized microtubules suggesting that it possesses vascular disrupting and anti-angiogenic properties. Further investigation of ELR510444 for the therapy of RCC is warranted.

Toluidinesulfonamide Hypoxia-Induced Factor 1 Inhibitors: Alleviating Drug–Drug Interactions through Use of PubChem Data and Comparative Molecular Field Analysis Guided Synthesis

Inhibitors of hypoxia-inducible factor 1 (HIF-1) represent promising anticancer therapeutics. We have identified a series of potent toluidinesulfonamide HIF-1 inhibitors. However, the series was threatened by a potential liability to inhibit CYP2C9 which could cause dangerous drug-drug interactions when being coadministered with other drugs. We used structure-activity data from the PubChem database to develop a topomer CoMFA model that guided the design of novel sulfonamides with high selectivity for HIF-1 over CYP2C9 inhibition.

Evaluation of Different Glutathione S-Transferase–Tagged Protein Captures for Screening E6/E6AP Interaction Inhibitors Using AlphaScreen®:

Human papillomavirus (HPV) infection is responsible for the development of cervical cancer and its premalignant lesions in women. The virus-encoded oncogene E6 is a promising target for an anti-HPV drug therapy. The authors describe the development of a homogenous screening assay for inhibitors of the E6 interaction with its cellular target, the E6-associated protein (E6AP), based on AlphaScreen® technology. The E6 protein was expressed and purified as glutathione S-transferase (GST) fusion protein, and the binding to a biotinylated E6AP peptide was monitored using GST-detecting Acceptor beads coated either with anti-GST antibody or glutathione. After optimization of the assay conditions, a commercial library of 3000 compounds was screened for inhibitors. Active compounds were retested and counterscreened for E6/E6AP specificity using biotinylated GST as a control protein. The results obtained with both types of GST-detecting reagents correlated very well and demonstrated the great potential of the newly developed glutathione-coated Acceptor beads as a detection reagent for GST fusion proteins. (Journal of Biomolecular Screening 2007:560-567)

Tripolin A, a novel small-molecule inhibitor of aurora A kinase, reveals new regulation of HURP's distribution on microtubules.

Mitotic regulators exhibiting gain of function in tumor cells are considered useful cancer therapeutic targets for the development of small-molecule inhibitors. The human Aurora kinases are a family of such targets. In this study, from a panel of 105 potential small-molecule inhibitors, two compounds Tripolin A and Tripolin B, inhibited Aurora A kinase activity in vitro. In human cells however, only Tripolin A acted as an Aurora A inhibitor. We combined in vitro, in vivo single cell and in silico studies to demonstrate the biological action of Tripolin A, a non-ATP competitive inhibitor. Tripolin A reduced the localization of pAurora A on spindle microtubules (MTs), affected centrosome integrity, spindle formation and length, as well as MT dynamics in interphase, consistent with Aurora A inhibition by RNAi or other specific inhibitors, such as MLN8054 or MLN8237. Interestingly, Tripolin A affected the gradient distribution towards the chromosomes, but not the MT binding of HURP (Hepatoma Up-Regulated Protein), a MT-associated protein (MAP) and substrate of the Aurora A kinase. Therefore Tripolin A reveals a new way of regulating mitotic MT stabilizers through Aurora A phosphorylation. Tripolin A is predicted to bind Aurora A similarly but not identical to MLN8054, therefore it could be used to dissect pathways orchestrated by Aurora kinases as well as a scaffold for further inhibitor development.