Inhibition of the transcriptional kinase CDK7 overcomes therapeutic resistance in HER2-positive breast cancers.

Resistance of breast cancer to human epidermal growth factor receptor 2 (HER2) inhibitors involves reprogramming of the kinome through HER2/HER3 signaling via the activation of multiple tyrosine kinases and transcriptional upregulation. The heterogeneity of induced kinases prevents kinase targeting by a single kinase inhibitor and presents a major challenge to the treatment of therapeutically recalcitrant HER2-positive breast cancers (HER2+ BCs). As a result, there is a critical need for effective treatment that attacks the aberrant kinome activation associated with resistance to HER2-targeted therapy. Here, we describe a novel treatment strategy that targets cyclin-dependent kinase 7 (CDK7) in HER2 inhibitor-resistant (HER2iR) breast cancer. We show that both HER2 inhibitor-sensitive (HER2iS) and HER2iR breast cancer cell lines exhibit high sensitivity to THZ1, a newly identified covalent inhibitor of the transcription regulatory kinase CDK7. CDK7 promotes cell cycle progression through inhibition of transcription, rather than via direct phosphorylation of classical CDK targets. The transcriptional kinase activity of CDK7 is regulated by HER2, and by the receptor tyrosine kinases activated in response to HER2 inhibition, as well as by the downstream SHP2 and PI3K/AKT pathways. A low dose of THZ1 displayed potent synergy with the HER2 inhibitor lapatinib in HER2iR BC cells in vitro. Dual HER2 and CDK7 inhibition induced tumor regression in two HER2iR BC xenograft models in vivo. Our data support the utilization of CDK7 inhibition as an additional therapeutic avenue that blocks the activation of genes engaged by multiple HER2iR kinases.

Development of inhibitors of heterotrimeric Gαi subunits.

Heterotrimeric G-proteins are the immediate downstream effectors of G-protein coupled receptors (GPCRs). Endogenous protein guanine nucleotide dissociation inhibitors (GDIs) like AGS3/4 and RGS12/14 function through GPR/Goloco GDI domains. Extensive characterization of GPR domain peptides indicate they function as selective GDIs for Gαi by competing for the GPCR and Gßγ and preventing GDP release. We modified a GPR consensus peptide by testing FGF and TAT leader sequences to make the peptide cell permeable. FGF modification inhibited GDI activity while TAT preserved GDI activity. TAT-GPR suppresses G-protein coupling to the receptor and completely blocked α2-adrenoceptor (α2AR) mediated decreases in cAMP in HEK293 cells at 100nM. We then sought to discover selective small molecule inhibitors for Gαi. Molecular docking was used to identify potential molecules that bind to and stabilize the Gαi-GDP complex by directly interacting with both Gαi and GDP. Gαi-GTP and Gαq-GDP were used as a computational counter screen and Gαq-GDP was used as a biological counter screen. Thirty-seven molecules were tested using nucleotide exchange. STD NMR assays with compound 0990, a quinazoline derivative, showed direct interaction with Gαi. Several compounds showed Gαi specific inhibition and were able to block α2AR mediated regulation of cAMP. In addition to being a pharmacologic tool, GDI inhibition of Gα subunits has the advantage of circumventing the upstream component of GPCR-related signaling in cases of overstimulation by agonists, mutations, polymorphisms, and expression-related defects often seen in disease.

Sterolins ABCG5 and ABCG8: regulators of whole body dietary sterols.

ABCG5 and ABCG8 are two ATP-binding cassette half-transporters that belong to the G family members. They were identified as proteins that are mutated in a rare human disorder, sitosterolemia, and their identification led to the completion of the physiological pathways by which dietary cholesterol, as well as noncholesterol sterols, traffics in the mammalian body. These proteins are likely to function as heterodimers, and current evidence suggests that these proteins are responsible for the majority of sterol secretions into bile, thus may define the long sought-after biliary sterol transporters. This review will focus on some of the backgrounds of this physiology, the genetics and regulation of these genes, as well as our current understanding of their functions. This review will also highlight the current limitations in our knowledge gap.

Defining the CD59-C9 binding interaction.

CD59 is a membrane glycoprotein that regulates formation of the cytolytic membrane attack complex (MAC or C5b-9) on host cell membranes. It functions by binding to C8 (alpha chain) and C9 after their structural rearrangement during MAC assembly. Previous studies indicated that the CD59 binding site in C9 was located within a 25-residue disulfide-bonded loop, and in C8alpha was located within a 51-residue sequence that overlaps the CD59 binding region of C9. By peptide screens and the use of peptides in binding assays, functional assays, and computer modeling and docking studies, we have identified a 6-residue sequence of human C9, spanning residues 365-371, as the primary CD59 recognition domain involved in CD59-mediated regulation of MAC formation. The data also indicate that both C8alpha and C9 bind to a similar or overlapping site on CD59. Furthermore, data from CD59-peptide docking models are consistent with the C9 binding site on CD59 located at a hydrophobic pocket, putatively identified previously by CD59 mutational and modeling studies.

A detailed Hapmap of the Sitosterolemia locus spanning 69 kb; differences between Caucasians and African-Americans.

BACKGROUND: Sitosterolemia is an autosomal recessive disorder that maps to the sitosterolemia locus, STSL, on human chromosome 2p21. Two genes, ABCG5 and ABCG8, comprise the STSL and mutations in either cause sitosterolemia. ABCG5 and ABCG8 are thought to have evolved by gene duplication event and are arranged in a head-to-head configuration. We report here a detailed characterization of the STSL in Caucasian and African-American cohorts. METHODS: Caucasian and African-American DNA samples were genotypes for polymorphisms at the STSL locus and haplotype structures determined for this locus RESULTS: In the Caucasian population, 13 variant single nucleotide polymorphisms (SNPs) were identified and resulting in 24 different haplotypes, compared to 11 SNPs in African-Americans resulting in 40 haplotypes. Three polymorphisms in ABCG8 were unique to the Caucasian population (E238L, INT10-50 and G575R), whereas one variant (A259V) was unique to the African-American population. Allele frequencies of SNPs varied also between these populations. CONCLUSION: We confirmed that despite their close proximity to each other, significantly more variations are present in ABCG8 compared to ABCG5. Pairwise D' values showed wide ranges of variation, indicating some of the SNPs were in strong linkage disequilibrium (LD) and some were not. LD was more prevalent in Caucasians than in African-Americans, as would be expected. These data will be useful in analyzing the proposed role of STSL in processes ranging from responsiveness to cholesterol-lowering drugs to selective sterol absorption.

Tannic acid mimicking dendrimers as small intestine submucosa stabilizing nanomordants.

Chemical stabilization resulting in increased resistance to proteolytic degradation is one of the approaches in prevention of post-implantational aneurysm development in decellularized natural vascular scaffolds. Recently, tannic acid (TA) and tannic acid mimicking dendrimers (TAMD) have been suggested as potential stabilization agents for collagen and elastin. The aim of this work was to determine the stabilizing effects of TAMD on decellularized natural scaffolds. Vascular scaffolds fabricated from small intestine submucosa (SIS) and SIS plane sheets (Cook Biotech Inc.) were used. The biomechanical properties of the SIS vascular graft segments treated with TA and TAMD were tested. The effect of TAMD treatment on resistance to proteolytic degradation was evaluated by measuring biomechanical properties of TAMD stabilized and non-stabilized SIS specimens after incubation in collagenase solution. It was shown that treatment with TA as well as with TAMD increased the strength of tubular SIS as well as their resistance to proteolytic biodegradation manifested by preservation of biomechanical properties after collagenase treatment. Transmission electron microscopy demonstrated that treatment with TAMD increased the periodical pattern typical of collagen fiber ultrastructure as a result of the "mordant" effect. The possible collagen cross-linking effect of TAMD on SIS was investigated by differential scanning calorimetry (DSC). The treatment with TAMD induced a small, but detectable cross-linking effect, suggesting that TAMD do not establish extensive covalent cross links within the extracellular matrix but rather interact with collagen, thus rendering SIS scaffolds more resistant to proteolytic degradation.

Identification of structural features in the G-protein regulatory motif required for regulation of heterotrimeric G-proteins.

The G-protein regulatory (GPR) motif, a conserved 25-30 amino acid domain found in multiple mammalian proteins, stabilizes the GDP-bound conformation of Galpha(i), inhibits guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding to Galpha(i) and competes for Gbetagamma binding to Galpha. To define the core GPR motif and key amino acid residues within a GPR peptide (TMGEEDFFDLLAKSQSKRMDDQRVDLAG), we determined the effect of truncation, insertion, and alanine substitutions on peptide-mediated inhibition of GTPgammaS binding to purified Galpha(i1). The bioactive core GPR peptide consists of 17 amino acids ((7)F-R(23)). Within this core motif, two hydrophobic sectors ((7)FF(8) and (10)LL(11)) and Q(22) are required for bioactivity, whereas M19A and R23A increased IC(50) values by 70-fold. Disruption of spatial relationships between the required sectors in the amino and carboxyl regions of the peptide also resulted in a loss of biological activity. Mutation of three charged sectors ((4)EED(6), R(18), (20)DD(21)) within the 28-amino acid GPR decreased peptide affinity by approximately 10-fold. Alanine substitutions of selected residues within the core GPR peptide differently influenced peptide inhibition of GTPgammaS binding to Galpha(i) versus Galpha(o). These data provide a platform for the development of novel, G-protein-selective therapeutics that inhibit Galpha(i)- mediated signaling, selectively activate Gbetagamma-sensitive effectors, and/or disrupt specific regulatory input to G-proteins mediated by GPR-containing proteins.