Pierre Beauparlant

Small molecule obatoclax (GX15-070) antagonizes MCL-1 and overcomes MCL-1-mediated resistance to apoptosis

Elevated expression of members of the BCL-2 pro-survival family of proteins can confer resistance to apoptosis in cancer cells. Small molecule obatoclax (GX15-070), which is predicted to occupy a hydrophobic pocket within the BH3 binding groove of BCL-2, antagonizes these members and induces apoptosis, dependent on BAX and BAK. Reconstitution in yeast confirmed that obatoclax acts on the pathway and overcomes BCL-2-, BCL-XL-, BCL-w-, and MCL-1-mediated resistance to BAX or BAK. The compound potently interfered with the direct interaction between MCL-1 and BAK in intact mitochondrial outer membrane and inhibited the association between MCL-1 and BAK in intact cells. MCL-1 has been shown to confer resistance to the BCL-2/BCL-XL/BCL-w-selective antagonist ABT-737 and to the proteasome inhibitor bortezomib. In both cases, this resistance was overcome by obatoclax. These findings support a rational clinical development opportunity for the compound in cancer indications or treatments where MCL-1 contributes to resistance to cell killing.

The Small Molecule GMX1778 Is a Potent Inhibitor of NAD+ Biosynthesis: Strategy for Enhanced Therapy in Nicotinic Acid Phosphoribosyltransferase 1-Deficient Tumors

ABSTRACT GMX1777 is a prodrug of the small molecule GMX1778, currently in phase I clinical trials for the treatment of cancer. We describe findings indicating that GMX1778 is a potent and specific inhibitor of the NAD+ biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT). Cancer cells have a very high rate of NAD+ turnover, which makes NAD+ modulation an attractive target for anticancer therapy. Selective inhibition by GMX1778 of NAMPT blocks the production of NAD+ and results in tumor cell death. Furthermore, GMX1778 is phosphoribosylated by NAMPT, which increases its cellular retention. The cytotoxicity of GMX1778 can be bypassed with exogenous nicotinic acid (NA), which permits NAD+ repletion via NA phosphoribosyltransferase 1 (NAPRT1). The cytotoxicity of GMX1778 in cells with NAPRT1 deficiency, however, cannot be rescued by NA. Analyses of NAPRT1 mRNA and protein levels in cell lines and primary tumor tissue indicate that high frequencies of glioblastomas, neuroblastomas, and sarcomas are deficient in NAPRT1 and not susceptible to rescue with NA. As a result, the therapeutic index of GMX1777 can be widended in the treatment animals bearing NAPRT1-deficient tumors by coadministration with NA. This provides the rationale for a novel therapeutic approach for the use of GMX1777 in the treatment of human cancers.

Biological and biochemical inhibitors of the NF-κB/Rel proteins and cytokine synthesis

Abstract The NF-κB/Rel family of transcription factors participates in the activation of a diverse range of genes involved in inflammation, immune response, lymphoid differentiation, growth control and development. The present review provides a brief overview of NF-κB/Rel activation and a detailed analysis of important biological and biochemical inhibitors of the NF-κB/Rel pathway. Given the pleiotropic role of NF-κB in controlling cytokines and other immunoregulatory genes, the inhibition of NF-κB activation by steroid hormones, antioxidants, protease inhibitors and other compounds may provide a pharmacological basis for interfering with pathological inflammatory conditions, cancer and AIDS.

Preclinical development of the nicotinamide phosphoribosyl transferase inhibitor prodrug Gmx1777

GMX1778 was recently shown to function as a potent inhibitor of nicotinamide phosphoribosyl transferase. To translate the discovery of GMX1778 mechanism of action into optimal clinical use of its intravenously administered prodrug, GMX1777, the efficacy of GMX1777 was evaluated in xenograft models and the pharmacokinetic profile of GMX1778 and its effect on nicotinamide adenine dinucleotide cellular levels was measured by liquid chromatography/mass spectrometry. Consistent with the requirement for a prolonged exposure for cytotoxicity in vitro, a dose of 75 mg/kg of GMX1777 administered as two bolus intravenous injections in 1 day were not effective at reducing the growth of multiple myeloma (IM-9) tumors, whereas the same dose of GMX1777 administered over a 24 h intravenous infusion caused tumor regression in the IM-9 model, a small-cell lung cancer (SHP-77) model, and a colon carcinoma (HCT-116) model. A 72 h continuous intravenous infusion of GMX1777 was also effective in the IM-9 model, but was associated with a smaller therapeutic index. GMX1777 at a dose of 75 mg/kg administered over a 24 h intravenous infusion produced GMX1778 steady-state plasma levels of approximately 1 μg/ml and caused nicotinamide adenine dinucleotide levels to decrease significantly in tumors. Consistent with the GMX1778 mechanism of action, nicotinic acid protected mice treated with a lethal dose of GMX1777. These data support the design of an open-label, dose-escalation trial, in which patients with refractory solid tumors and lymphomas receive 24 h infusions of GMX1777 as a single agent in 3-week cycles. Furthermore, these results indicate that nicotinic acid is a potent antidote to treat GMX1777 overdose.

Cellular and viral protein interactions regulating I kappa B alpha activity during human retrovirus infection.

NF‐κB/Rel transcription factors participate in the activation of numerous genes involved in immune regulation/inflammation including cytokines, cell surface receptors, adhesion molecules, and acute phase proteins. NF‐κB activity is controlled by inhibitory proteins, IκBs, that maintain the DNA‐binding forms of NF‐κB in an inactive state in the cytoplasm. Many viruses, including the human retroviruses HIV‐1 and HTLV‐1, also utilize the NF‐κB/IκB pathway to their transcriptional advantage during viral infection. Our recent studies have focused on the IκBα inhibitor and have characterized several protein interactions that modulate the functional activity of IκBα during human retrovirus infection. In this article, we summarize recent studies demonstrating that (1) chronic HIV‐1 infection of human myelomonoblastic PLB‐985 cells leads to constitutive NF‐κB activity, activated in part due to enhanced IκBα turnover and increased NF‐κB/Rel production; (2) HTLV‐1 Tax protein physically associates with the IκBα protein in vivo and in vitro and also mediates a 20‐ to 40‐fold stimulation of NF‐κB DNA binding activity mediated via an enhancement of NF‐κB dimer formation; (3) casein kinase II phosphorylates IκBα at multiple sites in the C‐terminal PEST domain and regulates IκBα function; (4) transdominant forms of IκBα, mutated in critical Ser or Thr residues required for inducer‐mediated (S32A,S36A) and/or constitutive phosphorylation block HIV LTR trans‐activation and also effectively inhibit HIV‐1 multiplication in a single cycle infection model; and (5) the amino‐terminal 55aa of IκBα (NIK) interacts with the human homologue of dynein light chain 1, a small 9‐kDa human homologue of the dynein light chain protein involved in microtubule and cytoskeletal dynamics. Together, our results highlight a number of intriguing molecular interactions between IκBα and cellular or viral proteins that modulate transcription factor activity and nuclear‐cytoplasmic flow of host proteins. J. Leukoc. Biol. 62: 82–92; 1997.

Therapeutic Small Molecule Inhibitors of Bcl-2

Although several studies suggest that suppression of apoptosis by Bcl-2 proteins is a critical early event in oncogenesis, its role in the maintenance of established tumors has still not been fully characterized. Similarly, the role of apoptosis inhibition by Bcl-2 proteins in normal proliferating cells has been studied only in few cell types. Without this information, it is difficult to predict the selectivity of Bcl-2-modulating therapeutic approaches in cancer treatment. Thus far, the best indication that cancer cells differ from their normal counterparts by their dependence on apoptosis inhibition is the significant up-regulation of anti-apoptotic members in many cancers and the successful treatment of mice bearing tumor xenograft with Bcl-2 AO. Early results from clinical trials for Bcl-2 AO are also providing the first validation for this approach in humans, but will most likely not reveal the full potential for Bcl-2 inhibition given the inherent clinical limitations of AOs (Dvorchik et al., 2002). Therapeutic small molecule inhibitors of Bcl-2 proteins are the preferred mean as they have the potential to be more stable and orally available. Recent successes for these compounds in xenograft animal models promise their clinical testing in the near future. One of the challenges for Bcl-2 inhibitors may be that different tumors may depend on a distinct Bcl-2 family member for their survival. Therefore, agents that act against more than one anti-apoptotic Bcl-2 family protein are desired. This goal has already been achieved with the design of Bcl-2/Bcl-Xl bispecific AOs. To achieve the same Bcl-2 pan-inhibition using small molecules, investigators will need to design inhibitors that contact structures within the BH3 binding site that are conserved among the Bcl-2 family. Results with TDDD, which inhibits both Bcl-2 and Bcl-Xl, as well as results with AA which appear to turn both Bcl-2 and Bcl-Xl into pro-apoptotic proteins, suggest that this could be achieved.