SWI/SNF subunit BAF155 N-terminus structure informs the impact of cancer-associated mutations and reveals a potential drug binding site.

SWI/SNF (BAF) chromatin remodelling complexes are key regulators of gene expression programs, and attractive drug targets for cancer therapies. Here we show that the N-terminus of the BAF155/SMARCC1 subunit contains a putative DNA-binding MarR-like domain, a chromodomain and a BRCT domain that are interconnected to each other to form a distinct module. In this structure the chromodomain makes interdomain interactions and has lost its canonical function to bind to methylated lysines. The structure provides new insights into the missense mutations that target this module in cancer. This study also reveals two adjacent, highly-conserved pockets in a cleft between the domains that form a potential binding site, which can be targeted with small molecules, offering a new strategy to target SWI/SNF complexes.

Biophysical and Structural Methods to Study the bHLHZip Region of Human c-MYC.

The C-terminal region of the c-MYC transcription factor consists of approximately 100 amino acids that in its native state does not adopt a stable structure. When this region binds to the obligatory partner MAX via a coupled folding-and-binding mechanism, it forms a basic-helix-loop-helix-leucine zipper (bHLHZip) heterodimeric complex. The C-terminal region of MYC is the target for numerous drug discovery programs for direct MYC inhibition via blocking the dimerization event and/or binding to DNA, and a proper understanding of the partially folded, dynamic nature of the heterodimeric complex is essential to these efforts. The bHLHZip motif also drives protein-protein interactions with cofactors that are crucial for both transcriptional repression and activation of MYC target genes. Targeting these interactions could potentially provide a means of developing alternative approaches to halt MYC functions; however, the molecular mechanism of these regulatory interactions is poorly understood. Herein we provide methods to produce high-quality human c-MYC C-terminal by itself and in complex MAX, and how to study them using Nuclear Magnetic Resonance spectroscopy and X-ray crystallography. Our protein expression and purification protocols have already been used to study interactions with cofactors.

Structure of the BRK domain of the SWI/SNF chromatin remodeling complex subunit BRG1 reveals a potential role in protein-protein interactions.

BRG1/SMARCA4 and its paralog BRM/SMARCA2 are the ATPase subunits of human SWI/SNF chromatin remodeling complexes. These multisubunit assemblies can act as either tumor suppressors or drivers of cancer, and inhibiting both BRG1 and BRM, is emerging as an effective therapeutic strategy in diverse cancers. BRG1 and BRM contain a BRK domain. The function of this domain is unknown, but it is often found in proteins involved in transcription and developmental signaling in higher eukaryotes, in particular in proteins that remodel chromatin. We report the NMR structure of the BRG1 BRK domain. It shows similarity to the glycine-tyrosine-phenylalanine (GYF) domain, an established protein-protein interaction module. Computational peptide-binding-site analysis of the BRK domain identifies a binding site that coincides with a highly conserved groove on the surface of the protein. This sets the scene for experiments to elucidate the role of this domain, and evaluate the potential of targeting it for cancer therapy.

Crystal Structures and Nuclear Magnetic Resonance Studies of the Apo Form of the c-MYC:MAX bHLHZip Complex Reveal a Helical Basic Region in the Absence of DNA.

The c-MYC transcription factor is a master regulator of cell growth and proliferation and is an established target for cancer therapy. This basic helix-loop-helix Zip protein forms a heterodimer with its obligatory partner MAX, which binds to DNA via the basic region. Considerable research efforts are focused on targeting the heterodimerization interface and the interaction of the complex with DNA. The only available crystal structure is that of a c-MYC:MAX complex artificially tethered by an engineered disulfide linker and prebound to DNA. We have carried out a detailed structural analysis of the apo form of the c-MYC:MAX complex, with no artificial linker, both in solution using nuclear magnetic resonance (NMR) spectroscopy and by X-ray crystallography. We have obtained crystal structures in three different crystal forms, with resolutions between 1.35 and 2.2 Å, that show extensive helical structure in the basic region. Determination of the α-helical propensity using NMR chemical shift analysis shows that the basic region of c-MYC and, to a lesser extent, that of MAX populate helical conformations. We have also assigned the NMR spectra of the c-MYC basic helix-loop-helix Zip motif in the absence of MAX and showed that the basic region has an intrinsic helical propensity even in the absence of its dimerization partner. The presence of helical structure in the basic regions in the absence of DNA suggests that the molecular recognition occurs via a conformational selection rather than an induced fit. Our work provides both insight into the mechanism of DNA binding and structural information to aid in the development of MYC inhibitors.

The structure of INI1/hSNF5 RPT1 and its interactions with the c-MYC:MAX heterodimer provide insights into the interplay between MYC and the SWI/SNF chromatin remodeling complex.

c-MYC and the SWI/SNF chromatin remodeling complex act as master regulators of transcription, and play a key role in human cancer. Although they are known to interact, the molecular details of their interaction are lacking. We have determined the structure of the RPT1 region of the INI1/hSNF5/BAF47/SMARCB1 subunit of the SWI/SNF complex that acts as a c-MYC-binding domain, and have localized the interaction regions on both INI1 and on the c-MYC:MAX heterodimer. c-MYC interacts with a highly conserved groove on INI1, while INI1 binds to the c-MYC helix-loop-helix region. The binding site overlaps with the c-MYC DNA-binding region, and we show that binding of INI1 and E-box DNA to c-MYC:MAX are mutually exclusive.

A selective high affinity MYC-binding compound inhibits MYC:MAX interaction and MYC-dependent tumor cell proliferation.

MYC is a key player in tumor development, but unfortunately no specific MYC-targeting drugs are clinically available. MYC is strictly dependent on heterodimerization with MAX for transcription activation. Aiming at targeting this interaction, we identified MYCMI-6 in a cell-based protein interaction screen for small inhibitory molecules. MYCMI-6 exhibits strong selective inhibition of MYC:MAX interaction in cells and in vitro at single-digit micromolar concentrations, as validated by split Gaussia luciferase, in situ proximity ligation, microscale thermophoresis and surface plasmon resonance (SPR) assays. Further, MYCMI-6 blocks MYC-driven transcription and binds selectively to the MYC bHLHZip domain with a KD of 1.6 ± 0.5 µM as demonstrated by SPR. MYCMI-6 inhibits tumor cell growth in a MYC-dependent manner with IC50 concentrations as low as 0.5 µM, while sparing normal cells. The response to MYCMI-6 correlates with MYC expression based on data from 60 human tumor cell lines and is abrogated by MYC depletion. Further, it inhibits MYC:MAX interaction, reduces proliferation and induces massive apoptosis in tumor tissue from a MYC-driven xenograft tumor model without severe side effects. Since MYCMI-6 does not affect MYC expression, it is a unique molecular tool to specifically target MYC:MAX pharmacologically and it has good potential for drug development.

A New Way Forward in Cancer Drug Discovery: Inhibiting the SWI/SNF Chromatin Remodelling Complex.

Mutations in subunits of the SWI/SNF chromatin remodelling complex are found in 20 % of human cancers. At face value, this would appear to indicate that this multiprotein complex is a potent tumour suppressor. However, it has recently emerged that some mutations in the SWI/SNF complex can have a gain-of-function effect and that in other tumours, such as pancreatic cancer, leukaemia, and breast cancer, the wild-type complex is used to drive cancer. Thus, paradoxically, this "tumour suppressor" has become an attractive target for developing anticancer agents. The SWI/SNF complex makes several protein-protein interactions both within the complex and with a wide range of transcription factors, and targeting these protein-protein interactions is emerging as the best approach to modulating the activity of the complex selectively.

Targeting protein-protein interactions (PPIs) of transcription factors: Challenges of intrinsically disordered proteins (IDPs) and regions (IDRs).

In this review we discuss recent progress in targeting the protein-protein interactions made by oncogenic transcription factors. We particularly focus on the challenges posed by the prevalence of intrinsically disordered regions in this class of protein and the strategies being used to overcome them.

The SWI/SNF Subunit INI1 Contains an N-Terminal Winged Helix DNA Binding Domain that Is a Target for Mutations in Schwannomatosis.

SWI/SNF complexes use the energy of ATP hydrolysis to remodel chromatin. In mammals they play a central role in regulating gene expression during differentiation and proliferation. Mutations in SWI/SNF subunits are among the most frequent gene alterations in cancer. The INI1/hSNF5/SMARCB1 subunit is mutated in both malignant rhabdoid tumor, a highly aggressive childhood cancer, and schwannomatosis, a tumor-predisposing syndrome characterized by mostly benign tumors of the CNS. Here, we show that mutations in INI1 that cause schwannomatosis target a hitherto unidentified N-terminal winged helix DNA binding domain that is also present in the BAF45a/PHF10 subunit of the SWI/SNF complex. The domain is structurally related to the SKI/SNO/DAC domain, which is found in a number of metazoan chromatin-associated proteins.

Investigation of the protein alkylation sites of the STAT3:STAT3 inhibitor Stattic by mass spectrometry.

STAT3 (Signal Transducer and Activator of Transcription factor 3) is constitutively active in a wide range of human tumours. Stattic is one of the first non-peptidic small molecules reported to inhibit formation of the STAT3:STAT3 protein dimer complex. A mass spectrometry method has been developed to investigate the binding of Stattic to the un-phosphorylated STAT3ßtc (U-STAT3) protein. Alkylation of four cysteine residues has been observed with possible reaction at a fifth which could account for the mechanism of action.

Observation of unphosphorylated STAT3 core protein binding to target dsDNA by PEMSA and X-ray crystallography.

The STAT3 transcription factor plays a central role in a wide range of cancer types where it is over-expressed. Previously, phosphorylation of this protein was thought to be a prerequisite for direct binding to DNA. However, we have now shown complete binding of a purified unphosphorylated STAT3 (uSTAT3) core directly to M67 DNA, the high affinity STAT3 target DNA sequence, by a protein electrophoretic mobility shift assay (PEMSA). Binding to M67 DNA was inhibited by addition of increasing concentrations of a phosphotyrosyl peptide. X-ray crystallography demonstrates one mode of binding that is similar to that known for the STAT3 core phosphorylated at Y705.

Small-molecule inhibition of c-MYC:MAX leucine zipper formation is revealed by ion mobility mass spectrometry.

The leucine zipper interaction between MAX and c-MYC has been studied using mass spectrometry and drift time ion mobility mass spectrometry (DT IM-MS) in addition to circular dichroism spectroscopy. Peptides comprising the leucine zipper sequence with (c-MYC-Zip residues 402-434) and without a postulated small-molecule binding region (c-MYC-ZipΔDT residues 406-434) have been synthesized, along with the corresponding MAX leucine zipper (MAX-Zip residues 74-102). c-MYC-Zip:MAX-Zip complexes are observed both in the absence and in the presence of the reported small-molecule inhibitor 10058-F4 for both forms of c-MYC-Zip. DT IM-MS, in combination with molecular dynamics (MD), shows that the c-MYC-Zip:MAX-Zip complex [M+5H](5+) exists in two conformations, one extended with a collision cross section (CCS) of 1164 ± 9.3 Å(2) and one compact with a CCS of 982 ± 6.6 Å(2); similar values are observed for the two forms of c-MYC-ZipΔDT:MAX-Zip. Candidate geometries for the complexes have been evaluated with MD simulations. The helical leucine zipper structure previously determined from NMR measurements (Lavigne, P.; et al. J. Mol. Biol. 1998, 281, 165), altered to include the DT region and subjected to a gas-phase minimization, yields a CCS of 1247 Å(2), which agrees with the extended conformation we observe experimentally. More extensive MD simulations provide compact complexes which are found to be highly disordered, with CCSs that correspond to the compact form from experiment. In the presence of the ligand, the leucine zipper conformation is completely inhibited and only the more disordered species is observed, providing a novel method to study the effect of interactions of disordered systems and subsequent inhibition of the formation of an ordered helical complex.

Molecular dynamics studies of the STAT3 homodimer:DNA complex: relationships between STAT3 mutations and protein-DNA recognition.

Signal Transducers and Activators of Transcription (STAT) proteins are a group of latent cytoplasmic transcription factors involved in cytokine signaling. STAT3 is a member of the STAT family and is expressed at elevated levels in a large number of diverse human cancers and is now a validated target for anticancer drug discovery.. Understanding the dynamics of the STAT3 dimer interface, accounting for both protein-DNA and protein-protein interactions, with respect to the dynamics of the latent unphosphorylated STAT3 monomer, is important for designing potential small-molecule inhibitors of the activated dimer. Molecular dynamics (MD) simulations have been used to study the activated STAT3 homodimer:DNA complex and the latent unphosphorylated STAT3 monomer in an explicit water environment. Analysis of the data obtained from MD simulations over a 50 ns time frame has suggested how the transcription factor interacts with DNA, the nature of the conformational changes, and ways in which function may be affected. Examination of the dimer interface, focusing on the protein-DNA interactions, including involvement of water molecules, has revealed the key residues contributing to the recognition events involved in STAT3 protein-DNA interactions. This has shown that the majority of mutations in the DNA-binding domain are found at the protein-DNA interface. These mutations have been mapped in detail and related to specific protein-DNA contacts. Their structural stability is described, together with an analysis of the model as a starting-point for the discovery of novel small-molecule STAT3 inhibitors.

A novel small-molecule inhibitor of IL-6 signalling.

A small library of pyrrolidinesulphonylaryl molecules has been synthesized via an efficient 4-step route, and members evaluated for their ability to inhibit IL-6 signalling. One molecule (6a) was found to have promising activity against IL-6/STAT3 signalling at the low micromolar level, and to selectively inhibit phosphorylation of STAT3 (but not STAT1) in IL-6 stimulated MDA-MB-231 breast cancer and HeLa cell lines. It was also selectively cytostatic in MDA-MB-231 (STAT3-dependent) versus A4 (STAT3-null) cells suggesting STAT3-specific inhibitory properties.

Facile nucleophilic substitution at the C3a tertiary carbon of the 3a-bromohexahydropyrrolo[2,3-b]indole scaffold.

The synthesis of 3a-substituted hexahydropyrrolo[2,3-b]indole derivatives via nucleophilic substitution at the C3a position is reported. Nitrogen-, oxygen-, sulfur-, fluoro- and carbon-based nucleophiles have been employed, using both conventional organic solvents and ionic liquids. The C3a-substituted derivatives were obtained in good to excellent yields.

Facile oxidation of electron-poor benzo[b]thiophenes to the corresponding sulfones with an aqueous solution of H2O2 and P2O5.

A facile oxidation for the clean conversion of benzo[b]thiophenes to their corresponding sulfones is described employing an aqueous solution of H(2)O(2) and P(2)O(5); the solution can be prepared and stored on a multi-gram scale with a shelf-life of up to two weeks.

Targeting protein-protein interactions for therapeutic intervention: a challenge for the future.

BACKGROUND: Over the last two decades, an increasing research effort in academia and industry has focused on the modulation (both inhibition and stabilization) of protein-protein interactions (PPIs) in order to develop novel therapeutic approaches and target-selective agents in drug discovery. DISCUSSION: The diversity and complexity of highly dynamic systems such as PPIs present many challenges for the identification of drug-like molecules with the ability to modulate the PPI with the necessary selectivity and potency. In this review, a number of these strategies will be presented along with a critical overview of the challenges and potential solutions relating to the exploitation of PPIs as molecular targets. CONCLUSIONS: Both traditional drug discovery approaches and some more recently developed innovative strategies have already provided valuable tools for the discovery of PPI modulators, and a number of successful examples have highlighted the potential of targeting PPIs for therapeutic intervention, especially in the oncology area.

Natural-product-like spiroketals and fused bicyclic acetals as potential therapeutic agents for B-cell chronic lymphocytic leukaemia.

B-cell chronic lymphocytic leukaemia (CLL) is the most common form of leukaemia in the Western world for which no curative treatments are currently available. Purine nucleotide analogues and alkylating agents feature frequently in combination regimens to treat the malignant state, but their use has not led to any significant improvement in patient survival. Consequently, there still remains a need for alternative small-molecule chemotherapeutics. Natural products are an unparalleled source of drug leads, and an unending inspiration for the design of small-molecule libraries for drug discovery. The screening of focused libraries of natural-product-like spiroketal and fused bicyclic acetal small molecules against primary CLL cells has led to the identification of a small series of novel and potent cytotoxic agents towards primary CLL cells. The validation of the activity of these molecules is delineated through a series of synthesis and screening iterations, whereas preliminary mode of action studies positively indicate their ability to induce cell death via an apoptotic pathway with no evidence of necrosis to further support their potential as novel chemotherapeutic agents.