ABSTRACT
Src homology 2 (SH2) domains are 100 amino acid modular units, which recognize and bind to tyrosyl-phosphorylated peptide sequences on their target proteins, and thereby mediate intracellular protein-protein interactions. This review summarizes the progress towards the development of synthetic agents that disrupt the function of the SH2 domains in different proteins as well as the clinical relevance of targeting a specific SH2 domain. Since 1986, SH2 domains have been identified in over 110 human proteins, including kinases, transcription factors, and adaptor proteins. A number of these proteins are over-activated in many diseases, including cancer, and their function is highly dependent on their SH2 domain. Thus, inhibition of a protein's function through disrupting that of its SH2 domain has emerged as a promising approach towards the development of novel therapeutic modalities. Although targeting the SH2 domain is a challenging task in molecular recognition, the progress reported here demonstrates the feasibility of such an approach.
Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Protein-Tyrosine Kinases/chemistry , Transcription Factors/chemistry , Adaptor Proteins, Signal Transducing/metabolism , GRB2 Adaptor Protein/antagonists & inhibitors , GRB2 Adaptor Protein/metabolism , GRB7 Adaptor Protein/antagonists & inhibitors , GRB7 Adaptor Protein/metabolism , Humans , Protein-Tyrosine Kinases/metabolism , STAT3 Transcription Factor/antagonists & inhibitors , STAT3 Transcription Factor/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/metabolism , Transcription Factors/metabolism , src Homology DomainsABSTRACT
The concise total synthesis of dermostatin A is described. Highlights include a two-directional application of the asymmetric acetate aldol method developed in our lab, a novel diastereotopic-group-selective acetal isomerization for terminus differentiation, and a selective cross-metathesis reaction between a terminal olefin and a trienal. A study of the scope and viability of similar cross-metathesis reactions is also described. The synthesis is convergent and utilizes fragments of roughly equal complexity.
Subject(s)
Chemistry Techniques, Synthetic/methods , Polyenes/chemical synthesis , Polyenes/chemistry , Polymers/chemistry , Stereoisomerism , Substrate SpecificityABSTRACT
We have recently characterized sliding motility in Bacillus subtilis strains that lack functional flagella, and here describe the discovery of inhibitors of colony spreading in these strains as well as the aflagellate pathogen, Bacillus anthracis. Aflagellate B. subtilis strains were used to screen for new types of antibacterials that might inhibit colony spreading on semi-solid media. From a diverse set of organic structures, p-nitrophenylglycerol (NPG), an agent used primarily in clinical laboratories to control Proteus swarming, was found to inhibit colony spreading. The four stereoisomers of NPG were synthesized and tested, and only the 1R,2S-(1R-anti) and 1R,2R-(1R-syn) NPG isomers had significant activity in a quantitative colony-spreading assay. Twenty-six NPG analogs and related structures were synthesized and tested to identify more active inhibitors. p-Methylsulfonylphenylglycerol (p-SPG), but not its ortho or meta analogs, was found to be the most effective of these compounds, and synthesis and testing of all four p-SPG stereoisomers showed that the 1R-anti-isomer was the most active with an average IC(50) of 16 µM (3-5 µg mL(-1)). For B. anthracis, the colony-spreading IC(50) values for 1R-anti-SPG and 1R-anti-NPG are 12 µM (2-4 µg mL(-1)) and >150 µM, respectively. For both Bacillus species tested, 1R-anti-SPG inhibits colony spreading of surface cultures on agar plates, but is not bacteriostatic or bacteriocidal in liquid cultures. Work is in progress to find the cellular target(s) of the NPG/SPG class of compounds, since this could lead to an understanding of the mechanism(s) of colony spreading as well as design and development of more potent inhibitors for the control of B. anthracis surface cultures.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacillus anthracis/drug effects , Bacillus subtilis/drug effects , Glycerol/analogs & derivatives , Nitrobenzenes/pharmacology , Sulfones/pharmacology , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Bacillus anthracis/cytology , Bacillus anthracis/growth & development , Bacillus subtilis/cytology , Bacillus subtilis/growth & development , Dose-Response Relationship, Drug , Drug Discovery , Glycerol/chemical synthesis , Glycerol/chemistry , Glycerol/pharmacology , Microbial Sensitivity Tests , Molecular Structure , Nitrobenzenes/chemical synthesis , Nitrobenzenes/chemistry , Stereoisomerism , Structure-Activity Relationship , Sulfones/chemical synthesis , Sulfones/chemistryABSTRACT
The majority of chronic myeloid leukemia (CML) cases are caused by a chromosomal translocation linking the breakpoint cluster region (BCR) gene to the Abelson murine leukemia viral oncogene-1 (ABL1), creating the mutant fusion protein BCR-ABL1. Downstream of BCR-ABL1 is growth factor receptor-bound protein-2 (GRB2), an intracellular adapter protein that binds to BCR-ABL1 via its src-homology-2 (SH2) domain. This binding constitutively activates growth pathways, downregulates apoptosis, and leads to an over proliferation of immature and dysfunctional myeloid cells. Utilizing novel synthetic methods, we developed four furo-quinoxaline compounds as GRB2 SH2 domain antagonists with the goal of disrupting this leukemogenic signaling. One of the four antagonists, NHD2-15, showed a significant reduction in proliferation of K562 cells, a human BCR-ABL1+ leukemic cell line. To elucidate the mode of action of these compounds, various biophysical, in vitro, and in vivo assays were performed. Surface plasmon resonance (SPR) assays indicated that NHD2-15 antagonized GRB2, binding with a KD value of 119 ± 2 µM. Cellulose nitrate (CN) assays indicated that the compound selectively bound the SH2 domain of GRB2. Western blot assays suggested the antagonist downregulated proteins involved in leukemic transformation. Finally, NHD2-15 was nontoxic to primary cells and adult zebrafish, indicating that it may be an effective clinical treatment for CML.
Subject(s)
Cell Proliferation/drug effects , GRB2 Adaptor Protein/antagonists & inhibitors , Quinoxalines/pharmacology , Animals , Fusion Proteins, bcr-abl/antagonists & inhibitors , Fusion Proteins, bcr-abl/metabolism , GRB2 Adaptor Protein/chemistry , GRB2 Adaptor Protein/metabolism , Humans , K562 Cells , Kidney/cytology , Kinetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Protein Binding , Quinoxalines/chemistry , Quinoxalines/metabolism , Stromal Cells/cytology , Stromal Cells/drug effects , Stromal Cells/metabolism , Surface Plasmon Resonance , Zebrafish , src Homology DomainsABSTRACT
Brain metastases (BM) represent the most common tumor to affect the adult central nervous system. Despite the increasing incidence of BM, likely due to consistently improving treatment of primary cancers, BM remain severely understudied. In this study, we utilized patient-derived stem cell lines from lung-to-brain metastases to examine the regulatory role of STAT3 in brain metastasis initiating cells (BMICs). Annotation of our previously described BMIC regulatory genes with protein-protein interaction network mapping identified STAT3 as a novel protein interactor. STAT3 knockdown showed a reduction in BMIC self-renewal and migration, and decreased tumor size in vivo. Screening of BMIC lines with a library of STAT3 inhibitors identified one inhibitor to significantly reduce tumor formation. Meta-analysis identified the oncomir microRNA-21 (miR-21) as a target of STAT3 activity. Inhibition of miR-21 displayed similar reductions in BMIC self-renewal and migration as STAT3 knockdown. Knockdown of STAT3 also reduced expression of known downstream targets of miR-21. Our studies have thus identified STAT3 and miR-21 as cooperative regulators of stemness, migration and tumor initiation in lung-derived BM. Therefore, STAT3 represents a potential therapeutic target in the treatment of lung-to-brain metastases.
Subject(s)
Brain Neoplasms/secondary , Gene Expression Regulation, Neoplastic , Lung Neoplasms/pathology , MicroRNAs/metabolism , STAT3 Transcription Factor/metabolism , Animals , Cell Line, Tumor , Cell Movement , Genes, Regulator , Humans , Inhibitory Concentration 50 , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Metastasis , Neoplasm Transplantation , Protein Interaction Mapping , Proteomics , RNA, Small Interfering/metabolism , Stem Cells/cytologyABSTRACT
Mutations in the BCR-ABL1 kinase domain are an established mechanism of tyrosine kinase inhibitor (TKI) resistance in Philadelphia chromosome-positive leukemia, but fail to explain many cases of clinical TKI failure. In contrast, it is largely unknown why some patients fail TKI therapy despite continued suppression of BCR-ABL1 kinase activity, a situation termed BCR-ABL1 kinase-independent TKI resistance. Here, we identified activation of signal transducer and activator of transcription 3 (STAT3) by extrinsic or intrinsic mechanisms as an essential feature of BCR-ABL1 kinase-independent TKI resistance. By combining synthetic chemistry, in vitro reporter assays, and molecular dynamics-guided rational inhibitor design and high-throughput screening, we discovered BP-5-087, a potent and selective STAT3 SH2 domain inhibitor that reduces STAT3 phosphorylation and nuclear transactivation. Computational simulations, fluorescence polarization assays and hydrogen-deuterium exchange assays establish direct engagement of STAT3 by BP-5-087 and provide a high-resolution view of the STAT3 SH2 domain/BP-5-087 interface. In primary cells from chronic myeloid leukemia (CML) patients with BCR-ABL1 kinase-independent TKI resistance, BP-5-087 (1.0 µM) restored TKI sensitivity to therapy-resistant CML progenitor cells, including leukemic stem cells. Our findings implicate STAT3 as a critical signaling node in BCR-ABL1 kinase-independent TKI resistance, and suggest that BP-5-087 has clinical utility for treating malignancies characterized by STAT3 activation.