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[Image: see text] BH3 peptide analogues are generally believed to exhibit great potency as cancer therapeutics via targeting antiapoptotic Bcl-2 proteins. Here, we describe the synthesis and identification of a new class of palmitoylated peptide BH3 analogues derived from the core region (h1–h4) of BH3 domains of proapoptotic Bcl-2 proteins and as alternative PTP1B inhibitors with antidiabetic potency in vitro and in vivo. PTP1B inhibitors are attractive for treatment of type 2 diabetes. We design the analogues using a simple lipidation approach and discovered novel lead analogues with promising antidiabetic potency in vitro and in vivo. The results presented here expanded the alternative target and function for the BH3 peptide analogues from one member Bim to other members of the proapoptotic Bcl-2 proteins and emphasize their therapeutic potential in T2DM. Furthermore, our findings may provide new proof of the regulatory function of Bcl-2 family proteins in mitochondrial nutrient and energy metabolism.

Modulating disease-relevant protein-protein interactions (PPIs) using pharmacological tools is a critical step toward the design of novel therapeutic strategies. Over the years, however, targeting PPIs has proven a very challenging task owing to the large interfacial areas. Our recent efforts identified possible novel routes for the design of potent and selective inhibitors of PPIs using a structure-based design of covalent inhibitors targeting Lys residues. In this present study, we report on the design, synthesis, and characterizations of the first Lys-covalent BH3 peptide that has a remarkable affinity and selectivity for hMcl-1 over the closely related hBfl-1 protein. Our structural studies, aided by X-ray crystallography, provide atomic-level details of the inhibitor interactions that can be used to further translate these discoveries into novel generation, Lys-covalent pro-apoptotic agents.

Hydrocarbon-stapled peptides are a class of bioactive α-helical ligands developed to target protein-protein interactions. Peptide stapling has benefited from the development of several chemical reactions to modulate their membrane permeability and binding affinity. However, in most current programs, choosing the best stapling positions is usually a trial-and-error process. Here, we develop a protocol to obtain optimal stapling positions computationally. Our method is based on molecular dynamics simulations and free energy calculations with nonequilibrium approaches; here, we predict the binding poses, hot-spot residues, and binding affinity differences of a set of perfluoroarene stapled α-helical peptides of the BIM BH3 peptide to the BCLXL receptor. The prediction of the hot-spot residues within the target peptide through computational alanine scanning anticipates not only the key residues for the receptor-peptide complex formation but also which positions should be avoided when applying the stapling groups. The staple moieties introduce local conformational changes not only in the replaced positions but also on their neighbor residues of the template peptide further affecting their binding behavior. Our approach is successful at rank-ordering the binding affinities of these stapled peptides with respect to the BIM BH3 peptide.

BCL-2 family proteins are central regulators of apoptosis and represent prime therapeutic targets for overcoming cell death resistance in malignancies. However, plasticity of anti-apoptotic members, such as MCL-1, often allows for a switch in cell death dependency patterns that lie outside the binding profile of targeted BH3-mimetics. Therefore discovery of therapeutics that effectively inactivate all anti-apoptotic members is a high priority. To address this we tested the potency of a hydrocarbon stapled BIM BH3 peptide (BIM SAHB A ) to overcome both BCL-2 and MCL-1 apoptotic resistance given BIM's naturally wide ranging affinity for all BCL-2 family multidomain members. BIM SAHB A effectively killed diffuse large B-cell lymphoma (DLBCL) cell lines regardless of their anti-apoptotic dependence. Despite BIM BH3's ability to bind all BCL-2 anti-apoptotic proteins, BIM SAHB A 's dominant intracellular target was MCL-1 and this specificity was exploited in sequenced combination BH3-mimetic treatments targeting BCL-2, BCL-XL, and BCL-W. Extending this MCL-1 functional dependence, mouse embryonic fibroblasts (MEFs) deficient in MCL-1 were resistant to mitochondrial changes induced by BIM SAHB A . This study demonstrates the importance of understanding BH3 mimetic functional intracellular affinities for optimized use and highlights the diagnostic and therapeutic promise of a BIM BH3 peptide mimetic as a potential MCL-1 inhibitor.

Anti-apoptotic Bcl-2 family proteins are vital for cancer cells to escape apoptosis, which make them attractive targets for cancer therapy. Recently, a lead compound 1 was found to modestly inhibit the binding of BH3 peptide to Bcl-2 protein with a Ki value of 5.2 µM. Based on this, a series of substituted tyrosine derivatives were developed and tested for their binding affinities to Bcl-2 protein. Results indicated that these compounds exhibited potent binding affinities to Bcl-2 and Mcl-1 protein but not to Bcl-XL protein. Promisingly, compound 6i inhibited the binding of BH3 peptide to Bcl-2 and Mcl-1 protein with a Ki value of 450 and 190 nM respectively, and showed obvious anti-proliferative activities against tested cancer cells.

This chapter presents a new method that allows one to qualitatively determine the effect of point mutations in peptides on the stability of the formed complex with full-length proteins. On the basis of the developed approach, a qualitative correlation of the obtained results with the dissociation constant was revealed using the example of the formation of the BH3 peptide biological complex of Bmf, Puma, Bad, Hrk, Bax, Bik, Noxa, Bid, Bim, and Bak proteins with the Bcl-xl protein and the BH3 peptides protein Bax with the Bcl-2 protein, taking into account the replacement of amino acid residues.

To investigate the kinetics of Cas9-mediated double strand break generation and repair in vivo, we developed two new tools. The first, chemically inducible Cas9 (ciCas9), is a rapidly-activated, single-component Cas9 variant engineered using a novel domain replacement strategy. ciCas9 can be activated in a matter of minutes, and the level of ciCas9 specificity and activity can be tuned. The second tool, DSB-ddPCR, is a droplet digital PCR-based assay for double strand breaks. DSB-ddPCR is the first assay to demonstrate time-resolved, highly quantitative and targeted measurement of DSBs. Combining these tools facilitated an unprecedented exploration of the kinetics of Cas9-mediated DNA cleavage and repair. We find that sgRNAs targeting different sites generally produce cleavage within minutes and repair within an hour or two. However, we observe distinct kinetic profiles, even for proximal sites, suggesting that target sequence and chromatin state modulate cleavage and repair kinetics.

A direct binding screen of 100 000 sp(3)-rich molecules identified a single diastereomer of a macrolactam core that binds specifically to myeloid cell leukemia 1 (MCL1). A comprehensive toolbox of biophysical methods was applied to validate the original hit and subsequent analogues and also established a binding mode competitive with NOXA BH3 peptide. X-ray crystallography of ligand bound to MCL1 reveals a remarkable ligand/protein shape complementarity that diverges from previously disclosed MCL1 inhibitor costructures.

We report the synthesis of a series of distance-matching aryl and vinylaryl cross-linkers for constructing stapled peptides containing cysteines at i,i+7 positions. Langevin dynamics simulation studies helped to classify these cross-linkers into two categories: the rigid cross-linkers with narrower S-S distance distribution and the flexible cross-linkers with wider S-S distance distribution. The stapled Noxa BH3 peptides with the flexible distance-matching cross-linkers gave the highest degree of helicity as well as the most potent inhibitory activity against Mcl-1. However, the stapled peptides with the highest hydrophobicity showed the most efficient cellular uptake. Together, this work illustrates the divergent nature of binding affinity and cellular uptake, and the vital importance of choosing appropriate cross-linkers in constructing stapled peptides with the drug-like properties.

Bcl2 Homology (BH) proteins can either trigger or prevent programmed cell death or apoptosis. Deregulation of the BH protein family network leads to evasion of apoptosis, uncontrolled proliferation and is a hallmark of cancer. Inhibition of pro-survival BH proteins is a promising chemotherapeutic strategy for certain cancers. We have examined whether helix-constrained peptides based on the BAD BH3 domain (residues 103–127) can be downsized to much smaller more drug-like peptides. We report the preparation, structural characterisation, in vitro Bcl-xL inhibition and leukemic T-cell killing ability of 45 linear, mono-, bi- and tricyclic helical peptidomimetics between 8- and 19-residues in length. We show that the BAD BH3 can be downsized to 8–14 residues and still maintain appreciable affinity for Bcl-xL. In addition, the binding efficiency indices (BEI) of the downsized mimetics are significantly higher than the BAD BH3 and similar stapled BH3 mimetics, approaching drug-like molecules. This suggests that bicyclic and monocyclic mimetics based on BH3 domains are much more efficient binding ligands than the longer peptides which they mimic.

Bim BH3 peptide features an α-helix with hotspot residues on multiple faces. Compound 5 (6-bromo-2,3-dihydroxyanthracene-9,10-dione), which adopts a rigid-plan amphipathic conformation, was designed and evaluated as a scaffold to mimic two faces of Bim α-helix. It reproduced the functionalities of both D67 and I65 on two opposing helical sides. Moreover, it maintained the two-faced binding mode during further evolution. A putative BH3 α-helix mimic and nanomolar Bcl-2/Mcl-1 dual inhibitor, 6, was obtained based on the structure of 5.

Forster (fluorescence) resonance energy transfer (FRET) and fluores- cence polarization (FP) are widely used technologies for monitoring bimolecular interactions and have been extensively used in high- throughput screening (HTS) for probe and drug discovery. Despite their popularity in HTS, it has been recognized that different assay tech- nologies may generate different hit lists for the same biochemical interaction. Due to the high cost of large-scale HTS campaigns, one has to make a critical choice to employee one assay platform for a particular HTS. Here we report the design and development of a dual- readout HTS assay that combines two assay technologies into one system using the Mcl-1 and Noxa BH3 peptide interaction as a model system. In this system, both FP and FRET signals were simultaneously monitored from one reaction, which is termed ''Dual-Readout F 2 as- say'' with F 2 for FP and FRET. This dual-readout technology has been optimized in a 1,536-well ultra-HTS format for the discovery of Mcl-1 protein inhibitors and achieved a robust performance. This F 2 assay was further validated by screening a library of 102,255 compounds. As two assay platforms are utilized for the same target simultaneously, hit information is enriched without increasing the screening cost. This strategy can be generally extended to other FP-based assays and is expected to enrich primary HTS information and enhance the hit quality of HTS campaigns.

Hydrocarbon stapling has been applied to restore and stabilize the α-helical structure of bioactive peptides for biochemical, structural, cellular, and in vivo studies. The peptide sequence, in addition to the composition and location of the installed staple, can dramatically influence the properties of stapled peptides. As a result, constructs that appear similar can have distinct functions and utilities. Here, we perform a side-by-side comparison of stapled peptides modeled after the pro-apoptotic BIM BH3 helix to highlight these principles. We confirm that replacing a salt-bridge with an i, i + 4 hydrocarbon staple does not impair target binding affinity and instead can yield a biologically and pharmacologically enhanced α-helical peptide ligand. Importantly, we demonstrate by electron microscopy that the pro-apoptotic activity of a stapled BIM BH3 helix correlates with its capacity to achieve cellular uptake without membrane disruption and accumulate at the organellar site of mechanistic activity.

The ratio of the levels of pro-survival and pro-apoptotic members of the Bcl-2 protein family is thought to be an important regulatory factor for determining the sensitivity of the mammalian cells to apoptotic stimuli. High levels of expression of pro-survival members such as Bcl(XL) in human cancers were frequently found to be a good prognostic indicator predicting poor response to chemotherapy. The pro-survival members of the Bcl-2 family mediate their effects through heterodimerization with the BH3 region of the pro-apoptotic members. Structural analyses of the binding complex of the BH3 peptide and Bcl(XL) showed that a hydrophobic groove termed the BH3 binding cleft is the docking site for the BH3 region. Chemical mimetics of the BH3 region such as BH3I-1 that target the BH3 binding cleft indeed exhibit pro-apoptotic activities. Chelerythrine (CHE) and sanguinarine (SAN) are natural benzophenanthridine alkaloids that are structurally homologous to each other. CHE was previously identified as an inhibitor of Bcl(XL) function from a high-throughput screen of natural products, but its mode of interaction with Bcl(XL) is not known. By determining the effect of site-directed mutagenesis on ligand binding and using saturation transfer difference (STD) NMR experiments, we have verified locations of these docked ligands. Surprisingly, CHE and SAN bind separately at the BH groove and BH1 region of Bcl(XL) respectively, different from the BH3 binding cleft where other known inhibitors of Bcl(XL) target. Interestingly, certain residues on the flexible loop between helices alpha1 and alpha2 of Bcl(XL) are also perturbed upon CHE, but not SAN or BH3I-1 binding. Although CHE and SAN are similarly effective as BH3I-1 in displacing bound BH3 peptide, they are much more effective in inducing apoptosis, raising the possibility that CHE and SAN might be able to antagonize other pro-survival mechanisms in addition to the one that involves BH3 region binding.

To investigate the possibility of intracellular delivery of Bak BH3 peptide using sonoporation effect by microbubble-enhanced ultrasound.HeLa and BJAB cells were exposed to 1.696-Mhz focused ultrasound with 2% microbubble contrast agents (OPTISON). Cell-impermeable calcein was used as an indicator for successful sonoporation, and propidium iodide staining was used for cell viability assessment. Peptides were also exposed to ultrasound with OPTISON and analyzed with mass spectrometry for evaluation of stability under ultrasound exposure. The effect of transduced Bak BH3 peptide was evaluated by the cell viability of successfully sonoporated cells.Bak BH3 peptides did not undergo mechanical degradation with microbubble-enhanced ultrasound exposure. With the increase of acoustic energy exposure, the sonoporation efficiency saturated both in BJAB and HeLa cells, while direct cell death rate by ultrasound exposure tended to increase. When BJAB cells were treated with 100 microM Bak BH3 peptides, and ultrasound exposure with ultrasound contrast agents (OPTISON), an increased 35% cell death was confirmed. On the other hand, although HeLa cells had a similar trend, they failed to exhibit statistical significance.Our results suggest that microbubble-enhanced focused ultrasound peptide transduction is possible. Further optimization of ultrasound exposure conditions may be necessary.

The design of a cross-acridine scaffold mimicking the i, i+3, i+5, and i+7 residues distributed over a two-face, two-turn α-helix is described. Docking studies and 2D (1)H, (15)N HSQC NMR spectroscopy provide compelling evidence that compound 3 d accurately reproduces the arrangement of four hotspots in the Bim BH3 peptide to permit binding to the Mcl-1 and Bcl-2 proteins (Ki 0.079 and 0.056 μM, respectively). Furthermore, the hotspot mutation could also be mimicked by individual or multiple deletions of side chains on the scaffold.

Proteins of the Bcl-2 family regulate apoptosis through the formation of heterodimers between antiapoptotic or pro-survival proteins and proapoptotic or pro-death proteins. Overexpression of antiapoptotic proteins not only contributes to the progression of many cancers, but also confers resistance to the chemo- and radiotherapeutic treatments. It has been demonstrated that peptides containing the BH3 domain of proapoptotic Bcl-2 family members are able to bind and inhibit antiapoptotic proteins. For this reason, the design of small molecules mimicking the BH3 domain of proapoptotic proteins has emerged as a promising therapeutic strategy for cancer treatment during the last years. However, BH3 domains exhibit different affinities for binding to antiapoptotic proteins; whereas Bim(BH3) and Puma(BH3) are able to bind all antiapoptotic proteins, others like Bad(BH3) and Bmf(BH3) show preference for some proteins over others. Consequently, the ability of a BH3-mimetic to kill tumor cells will depend on the BH3 peptide used as template and thus will have a selective or pan-inhibition effect. Recently, it has been suggested that this last approach could be interesting. Therefore, the present work is aimed to elucidate how the nonselective peptide Bim(BH3) is able to bind to all of the Bcl-2 family antiapoptotic proteins. To unravel the molecular determinants of this pan-inhibition, we used the MM-PB/GBSA approaches to calculate the binding free energy of the different complexes studied and to determine which residues of the peptide have the largest contribution to complex formation. Results obtained in the present work show that the binding of Bim(BH3) to pro-survival proteins is mainly hydrophobic and that specific interactions are fully distributed along the peptide sequence.

Cancer cells subvert the natural balance between cellular life and death, achieving immortality through pathologic enforcement of survival pathways and blockade of cell death mechanisms. Pro-apoptotic BCL-2 family proteins are frequently disarmed in relapsed and refractory cancer through genetic deletion or interaction-based neutralization by overexpressed antiapoptotic proteins, resulting in resistance to chemotherapy and radiation treatments. New pharmacologic strategies are urgently needed to overcome these formidable apoptotic blockades. We harnessed the natural killing activity of BCL-2–interacting mediator of cell death (BIM), which contains one of the most potent BH3 death domains of the BCL-2 protein family, to restore BH3-dependent cell death in resistant hematologic cancers. A hydrocarbon-stapled peptide modeled after the BIM BH3 helix broadly targeted BCL-2 family proteins with high affinity, blocked inhibitory antiapoptotic interactions, directly triggered proapoptotic activity, and induced dose-responsive and BH3 sequence–specific cell death of hematologic cancer cells. The therapeutic potential of stapled BIM BH3 was highlighted by the selective activation of cell death in the aberrant lymphoid infiltrates of mice reconstituted with BIM-deficient bone marrow and in a human AML xenograft model. Thus, we found that broad and multimodal targeting of the BCL-2 family pathway can overcome pathologic barriers to cell death.

SummaryDefining protein interactions forms the basis for discovery of biological pathways, disease mechanisms, and opportunities for therapeutic intervention. To harness the robust binding affinity and selectivity of structured peptides for interactome discovery, we engineered photoreactive stapled BH3 peptide helices that covalently capture their physiologic BCL-2 family targets. The crosslinking α helices covalently trap both static and dynamic protein interactors, and enable rapid identification of interaction sites, providing a critical link between interactome discovery and targeted drug design.

2190-Pos B160, The proteins of the Bcl-2 family are key regulators of apoptosis, but their molecular mechanisms remains controversial. Two important aspects that center the debate involve the interaction network between the pro- and antiapoptotic family members and the role of their translocation to the mitochondrial outer membrane (MOM) during apoptosis. We have used FCCS to examine quantitatively the dynamic interactions of Bid and tBid with Bcl-xLΔCt in solution and in lipid membranes. We found that only the active form tBid binds to Bcl-xLΔCt and that the membrane strongly promotes binding between them. Importantly for drug design, a BH3 peptide from Bid disrupts the tBid/Bcl-xL complex in solution but not in lipid bilayers. Our findings convincingly suggest that the most relevant interaction between tBid and Bcl-xL happens in the membrane and reveal its significance as an additional regulatory stage for MOM permeabilization.

Correction for ‘Downsizing the BAD BH3 peptide to small constrained α-helices with improved ligand efficiency’ by Nicholas E. Shepherd et al., Org. Biomol. Chem., 2016, DOI: 10.1039/c6ob02185a.

International audience; The mechanism by which some BH3-only proteins of the Bcl-2 family directly activate the “multidomain” proapoptotic member Bax is poorly characterized. We report that the first alpha helix (Halpha1) of Bax specifically interacts with the BH3 domains of Bid and PUMA but not with that of Bad. Inhibition of this interaction, by a peptide comprising Halpha1 or by a mutation in this helix, prevents ligand-induced activation of Bax by Bid, PUMA, or their BH3 peptides. Halpha1-mutated Bax, which can mediate death induced by Bad or its BH3 peptide, does not mediate that induced by Bid, PUMA, or their BH3 peptides. The response of Halpha1-mutated Bax to Bid can be restored by a compensating mutation in Bid BH3. Thus, a specific interaction between Bax Halphaj1 and their BH3 domains allows Bid and PUMA to function as “death agonists” of Bax, whereas Bad recruits Bax activity through a distinct pathway.

The interactions between B-cell lymphoma 2 (BCL-2) family members are known to be mediated through the binding of the BH3 domain of a proapoptotic member to the BH3-binding groove of an antiapoptotic member. We determined the crystal structure of antiapoptotic CED-9, which reveals a unique C-terminal helix altering the common BH3-binding region. A coexpression system to produce CED-9 in complex with proapoptotic EGL-1 enabled us to show that the binding of EGL-1 to CED-9 is extremely stable, raising the melting temperature (T(M)) of CED-9 by 25 degrees C, and that the binding surface of CED-9 extends beyond the BH3-binding region and reaches the BH4 domain. Consistently, the T(M) and a 1H-15N correlation NMR spectrum of CED-9 in complex with EGL-1 are drastically different from those of CED-9 in complex with the EGL-1 BH3 peptide. The data suggest that the recognition between other BCL-2 family members may also involve much wider protein surfaces than is previously thought.

Commentary to:Bcl2/bcl-xl Inhibitor Engenders Apoptosis and Increases Chemo-sensitivity In MesotheliomaXiaobo Cao, Charles Rodarts, Lidong Zhang, Clinton D. Morgan, James Littlejohn and W. Roy SmytheCancer Biology & Therapy Volume: 6 | Issue: 2 | Pages: 246 - 252

The scientific community has been saddened and diminished by the loss of Stanley J. KorsmeyerStanley J. Korsmeyer, M.D., who died of lung cancer last month at the age of 54. Over the last 25 years, Korsmeyer had been an international leader in the fields of cancer biology and programmed cell death (apoptosis). Through his studies of apoptosis, Korsmeyer helped revolutionize our concepts of carcinogenesis. Korsmeyer, a medical oncologist by training, also helped translate this knowledge into the development of novel cancer therapeutics that are just now entering into human clinical trials.Stan didn’t start out to be a research scientist. He grew up the oldest of four on a livestock farm in southern Illinois. It was his involvement in the 4-H club that spawned his interest in science. From the beginning, his scientific experiments met with success. The pair of Hampshire Hogs he raised at age 14 were named Grand Champion at the Illinois State Fair, and Stan went off to the University of Illinois determined to become a veterinarian. However, on the advice of one of his veterinary mentors, he switched to pre-med. Following receipt of an M.D. degree from the University of Illinois and an internship and residency at UCSF, he arrived at the NIH as a Research Fellow at a time when molecular biology was just beginning to revolutionize the study of human disease. There, under the tutelage of scientists like Tom Waldmann and Phil Leder, Stan soon joined the vanguard of research scientists characterizing the molecular features of the chromosomal translocations that had been observed in patients with leukemia/lymphoma. Over his career, Stan contributed to the characterization of a variety of chromosomal translocations and identified several widely studied oncogenes including MLL, AF-4, and Ttg-1 (rhombotin). Stanley J. KorsmeyerView Large Image | View Hi-Res Image | Download PowerPoint SlideAlthough he had many accomplishments, Stan will be best known for his contributions to the discovery and characterization of the role that programmed cell death plays in the pathogenesis of cancer. In 1985, the Korsmeyer laboratory was one of three laboratories to describe the molecular aspects of a chromosomal translocation between chromosomes 14 and 18 observed in the majority of patients with follicular lymphoma. This translocation placed the immunoglobulin gene enhancer from chromosome 14 in close proximity to a previously uncharacterized gene on chromosome 18 that was given the name Bcl-2. From the beginning, Bcl-2 was a problem. It didn’t score easily in any of the assays usually used to characterize oncogenes. In 1988, a group from The Walter and Eliza Hall Institute reported that Bcl-2 could promote the survival of hematopoietic cells in culture. Shortly thereafter, Korsmeyer, who had been pursuing an independent investigation to determine whether Bcl-2 overexpression could alter B cell development and/or transformation in vivo, confirmed and extended this observation. Korsmeyer demonstrated that a Bcl-2 transgene expressed under the control of an immunoglobulin enhancer led to the progressive accumulation of B cells in vivo. The transgenic B cells were characterized by an extended cell survival, and this extended survival led ultimately to an increased incidence of B cell lymphomas in the mice. These studies provided the first evidence that inhibiting cell death by apoptosis could contribute to development of cancer.Following his pioneering discoveries of the role of Bcl-2 in preventing programmed cell death and in promoting carcinogenesis, Stan remained at the forefront of apoptosis research. Not only did he demonstrate that overexpression of Bcl-2 could enhance lymphocyte survival in vivo, he also demonstrated that Bcl-2s’ endogenous role was to regulate cell survival. He showed that Bcl-2-deficient mice were unable to maintain the survival of mature lymphocytes. Bcl-2-deficient animals developed profound immunodeficiency soon after birth. Korsmeyer also led investigations to identify how Bcl-2 proteins work. His laboratory localized Bcl-2 to the mitochondria; discovered Bax, a Bcl-2 homolog whose function was to execute cells rather than keep cells alive; and cloned Bad and Bid, two BH3-only-containing Bcl-2 homologs that modulate the functions of other family members. In recent years, many others have contributed to the current understanding of apoptosis regulation, but Stan continued to be a leading figure. As the Bcl-2 family expanded to include a dizzying array of death inhibitors and death effectors and their intermolecular regulatory network became increasingly complex, it was Stan who came up with a simple model of Bcl-2 function that focused on the family’s central activity. Stan proposed that the combined activities of Bcl-2-related proteins acted like a rheostat to regulate a cell’s sensitivity to apoptotic stimuli. He hoped to take advantage of this by developing cancer therapeutics specifically designed to either inhibit the antiapoptotic members or activate the proapoptotic members.Stan’s ability to reduce problems to first principles kept the field’s attention focused on the big picture. Stan’s summation to a science writer of his interest in the Bcl-2 family said it best: “It turns out to be a family in which there are good guys and bad guys.” Last year, Stan’s laboratory collaborated with investigators in the Harvard Chemistry Department to develop peptide mimetics of the BH3 proteins that suppress the antiapoptotic function of Bcl-2. Using a chemical technique called hydrocarbon stapling, they replaced the natural amino acids within a BH3 peptide sequence to produce a stable helical structure with enhanced killing activity and provided preliminary evidence that treatment of cultured leukemia cells with these modified peptides led to selective inhibition of leukemic cell survival. Treatment of mice with leukemia with these new compounds slowed disease progression. In addition, Stan’s collaboration with a company he helped found, Idun Pharmaceuticals, and Abbott Laboratories has led to the development of small molecule inhibitors of antiapoptotic Bcl-2 proteins. These mimetics appear to shift the Bcl-2 rheostat in favor of the proapoptotic family members and induce programmed cell death in cancer cells with elevated levels of antiapoptotic Bcl-2-related proteins. Ironically, in a paper soon to be published, these new Bcl-2 inhibitors are shown to have single agent activity in treating lung cancer cells.As numerous and significant as Stan’s scientific achievements were, what those of us who worked with him will remember is the enthusiasm and perspective that he brought to his research. No matter how competitive the situation, he always reminded his own laboratory, collaborators, and competitors that the important goal was to focus on developing a deeper understanding of cancer and utilize that information to improve cancer treatment. Stan’s dedication to science was surpassed only by his dedication and love for his family. Despite being a nonsmoker, he developed lung cancer. Even his illness couldn’t change his approach to life. Right through the final stages of his treatment, he faced his illness with courage and optimism, actively participating in the choice of therapy he received. Stan was never one to stay on the sidelines.Stan’s contributions to cancer research were recognized widely in his lifetime. He was appointed a tenured Professor, first at Washington University and then at Harvard. Stan was appointed an Investigator within the Howard Hughes Medical Institute at both universities. He was elected to membership of the National Academy of Sciences, the Institute of Medicine, and the American Philosophical Society. His awards for cancer research include the Bristol Meyers Squibb Award for Distinguished Achievement in Cancer Research, the Charles S. Mott Prize of General Motor Cancer Research Foundation, the 2000 Louisa Gross Horwitz Prize, the Stratton Medal from the American Society of Hematology, the Leukemia and Lymphoma Society deVilliers International Achievement Award, and the International Award for Cancer Research from the Pezcoller Foundation and the American Association for Cancer Research. Nevertheless, those of us who knew him will remember Stan best for his qualities as a colleague, mentor, and friend. When Stan Korsmeyer died on March 31, 2005, we lost one of the good guys.

Alkenylglycine amino acids were assessed as potential candidates for hydrocarbon stapling and shown to be effective in stapling of the BID BH3 peptide.

The identification of selective inhibitors of the anti-apoptotic Bcl-2 family remains a principal target for cancer drug development. With the recent recognition of Mcl-1 as the primary Bcl-2 family member expressed in solid tumors and its ability to enable therapeutic resistance, identification of peptides that specifically bind to Mcl-1 has become critical for the development of improved therapeutics. To identify novel peptides that bind to the BH3 cleft of Mcl-1, we screened a randomized twelve residue phage display library using a pan-active Bcl-2 small-molecule inhibitor. This screen identified a unique non-canonical BH3 sequence, rBH3, which selectively binds to Mcl-1 over Bcl-xL. NMR analysis of the rBH3 interaction with Mcl-1 shows that it binds to the BH3 binding groove and an alanine scan of the sequence highlights the key residues responsible for its binding. A search for proteins with the rBH3 motif has identified a number of interesting targets which have previously been associated with apoptosis regulation. Thus we believe that this sequence represents a unique, Mcl-1-specific peptide sequence that may identify a previously unknown family of Mcl-1 interacting proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 8. doi:10.1158/1538-7445.AM2011-8

Programmed cell death regulating protein motifs play an essential role in the development of an organism, its immune response, and disease-related cellular mechanisms. Among those motifs the BH3 domain of the BCL-2 family is found to be of crucial importance. Recent experiments showed how the isolated, otherwise unstructured BH3 peptide can be modified by a hydrocarbon linkage to regain function. We parametrized a reduced, dynamic model for the stability effects of such covalent cross-linking and confirmed that the model reproduces the reinforcement of the structural stability of the BH3 motif by cross-linking. We show that an analytically solvable model for thermostability around the native state is not capable of reproducing the stabilization effect. This points to the crucial importance of the peptide dynamics and the fluctuations neglected in the analytic model for the cross-linking system to function properly. This conclusion is supported by a thorough analysis of a simulated Go model. The resulting model is suitable for rational design of generic cross-linking systems in silicio.