ABSTRACT
Proteins in the B cell CLL/lymphoma 2 (BCL-2) family are key regulators of the apoptotic process. This family comprises proapoptotic and prosurvival proteins, and shifting the balance toward the latter is an established mechanism whereby cancer cells evade apoptosis. The therapeutic potential of directly inhibiting prosurvival proteins was unveiled with the development of navitoclax, a selective inhibitor of both BCL-2 and BCL-2-like 1 (BCL-X(L)), which has shown clinical efficacy in some BCL-2-dependent hematological cancers. However, concomitant on-target thrombocytopenia caused by BCL-X(L) inhibition limits the efficacy achievable with this agent. Here we report the re-engineering of navitoclax to create a highly potent, orally bioavailable and BCL-2-selective inhibitor, ABT-199. This compound inhibits the growth of BCL-2-dependent tumors in vivo and spares human platelets. A single dose of ABT-199 in three patients with refractory chronic lymphocytic leukemia resulted in tumor lysis within 24 h. These data indicate that selective pharmacological inhibition of BCL-2 shows promise for the treatment of BCL-2-dependent hematological cancers.
Subject(s)
Antineoplastic Agents/pharmacology , Blood Platelets/drug effects , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Hematologic Neoplasms/drug therapy , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Sulfonamides/pharmacology , Aniline Compounds/pharmacology , Animals , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Cell Survival/drug effects , Dogs , Female , HeLa Cells , Humans , Mice , Mice, SCID , Proto-Oncogene Proteins c-bcl-2/chemistry , Tumor Burden , Xenograft Model Antitumor Assays , bcl-X Protein/antagonists & inhibitorsABSTRACT
Overexpression of the antiapototic proteins Bcl-2 and Bcl-xL provides a common mechanism through which cancer cells gain a survival advantage and become resistant to conventional chemotherapy. Inhibition of these prosurvival proteins is an attractive strategy for cancer therapy. We recently described the discovery of a selective Bcl-xL antagonist that potentiates the antitumor activity of chemotherapy and radiation. Here we describe the use of structure-guided design to exploit a deep hydrophobic binding pocket on the surface of these proteins to develop the first dual, subnanomolar inhibitors of Bcl-xL and Bcl-2. This study culminated in the identification of 2, which exhibited EC50 values of 8 nM and 30 nM in Bcl-2 and Bcl-xL dependent cells, respectively. Compound 2 demonstrated single agent efficacy against human follicular lymphoma cell lines that overexpress Bcl-2, and efficacy in a murine xenograft model of lymphoma when given both as a single agent and in combination with etoposide.
Subject(s)
Antineoplastic Agents/chemical synthesis , Biphenyl Compounds/chemical synthesis , Nitrophenols/chemical synthesis , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Sulfonamides/chemical synthesis , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Biphenyl Compounds/chemistry , Biphenyl Compounds/pharmacology , Cell Line, Tumor , Drug Screening Assays, Antitumor , Humans , Lymphoma , Mice , Mice, SCID , Models, Molecular , Nitrophenols/chemistry , Nitrophenols/pharmacology , Piperazines/chemical synthesis , Piperazines/chemistry , Piperazines/pharmacology , Proto-Oncogene Proteins c-bcl-2/chemistry , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacology , Transplantation, Heterologous , bcl-X Protein/antagonists & inhibitors , bcl-X Protein/chemistryABSTRACT
One of the primary objectives in the design of protein inhibitors is to shape the three-dimensional structures of small molecules to be complementary to the binding site of a target protein. In the course of our efforts to discover potent inhibitors of Bcl-2 family proteins, we found a unique folded conformation adopted by tethered aromatic groups in the ligand that significantly enhanced binding affinity to Bcl-XL. This finding led us to design compounds that were biased by nonbonding interactions present in a urea tether to adopt this bioactive, folded motif. To characterize the key interactions that induce the desired conformational bias, a series of substituted N,N'-diarylureas were prepared and analyzed using X-ray crystallography and quantum mechanical calculations. Stabilizing pi-stacking interactions and destabilizing steric interactions were predicted to work in concert in two of the substitution patterns to promote the bioactive conformation as a global energy minimum and result in a high target binding affinity. Conversely, intramolecular hydrogen bonding present in the third substitution motif promotes a less active, extended conformer as the energetically favored geometry. These findings were corroborated when the inhibition constant of binding to Bcl-XL was determined for fully elaborated analogues bearing these structural motifs. Finally, we obtained the NMR solution structure of the disubstituted N,N'-diarylurea bound to Bcl-XL demonstrating the folded conformation of the urea motif engaged in extensive pi-interactions with the protein.
Subject(s)
Drug Design , bcl-X Protein/antagonists & inhibitors , Binding Sites , Computer Simulation , Crystallography, X-Ray , Ligands , Magnetic Resonance Spectroscopy , Models, Molecular , Protein Structure, Tertiary , bcl-X Protein/chemistry , bcl-X Protein/metabolismABSTRACT
Development of a rationally designed potentiator of cancer chemotherapy, via inhibition of Bcl-X(L) function, is described. Lead compounds generated by NMR screening and directed parallel synthesis displayed sub-microM binding but were strongly deactivated in the presence of serum. The dominant component of serum deactivation was identified as domain III of human serum albumin (HSA); NMR solution structures of inhibitors bound to both Bcl-X(L) and HSA domain III indicated two potential optimization sites for separation of affinities. Modifications at both sites resulted in compounds with improved Bcl-X(L) binding and greatly increased activity in the presence of human serum, culminating in 73R, which bound to Bcl-X(L) with a K(i) of 0.8 nM. In a cellular assay 73R reversed the protection afforded by Bcl-X(L) overexpression against cytokine deprivation in FL5.12 cells with an EC(50) of 0.47 microM. 73R showed little effect on the viability of the human non small cell lung cancer cell line A549. However, consistent with the proposed mechanism, 73R potentiated the activity of paclitaxel and UV irradiation in vitro and potentiated the antitumor efficacy of paclitaxel in a mouse xenograft model.
Subject(s)
Aniline Compounds/chemical synthesis , Antineoplastic Agents/chemical synthesis , Piperidines/chemical synthesis , Sulfonamides/chemical synthesis , bcl-X Protein/antagonists & inhibitors , Aniline Compounds/chemistry , Aniline Compounds/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Biological Availability , Cell Line, Tumor , Drug Screening Assays, Antitumor , Drug Synergism , Fluorescence Polarization , Humans , Magnetic Resonance Spectroscopy , Mice , Mice, SCID , Paclitaxel/pharmacology , Piperidines/chemistry , Piperidines/pharmacology , Protein Binding , Protein Structure, Tertiary , Serum , Serum Albumin/chemistry , Stereoisomerism , Sulfonamides/chemistry , Sulfonamides/pharmacology , Transplantation, Heterologous , Ultraviolet RaysABSTRACT
Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.