RAF inhibitor LY3009120 sensitizes RAS or BRAF mutant cancer to CDK4/6 inhibition by abemaciclib via superior inhibition of phospho-RB and suppression of cyclin D1.

KRAS, NRAS and BRAF mutations are among the most important oncogenic drivers in many major cancer types, such as melanoma, lung, colorectal and pancreatic cancer. There is currently no effective therapy for the treatment of RAS mutant cancers. LY3009120, a pan-RAF and RAF dimer inhibitor advanced to clinical study has been shown to inhibit both RAS and BRAF mutant cell proliferation in vitro and xenograft tumor growth in vivo. Abemaciclib, a CDK4/6-selective inhibitor, is currently in phase III studies for ER-positive breast cancer and KRAS mutant lung cancer. In this study, we found that combinatory treatment with LY3009120 and abemaciclib synergistically inhibited proliferation of tumor cells in vitro and led to tumor growth regression in xenograft models with a KRAS, NRAS or BRAF mutation at the doses of two drugs that were well tolerated in combination. Further in vitro screen in 328 tumor cell lines revealed that tumor cells with KRAS, NRAS or BRAF mutation, or cyclin D activation are more sensitive, whereas tumor cells with PTEN, PIK3CA, PIK3R1 or retinoblastoma (Rb) mutation are more resistant to this combination treatment. Molecular analysis revealed that abemaciclib alone inhibited Rb phosphorylation partially and caused an increase of cyclin D1. The combinatory treatment cooperatively demonstrated more complete inhibition of Rb phosphorylation, and LY3009120 suppressed the cyclin D1 upregulation mediated by abemaciclib. These results were further verified by CDK4/6 siRNA knockdown. Importantly, the more complete phospho-Rb inhibition and cyclin D1 suppression by LY3009120 and abemaciclib combination led to more significant cell cycle G0/G1 arrest of tumor cells. These preclinical findings suggest that combined inhibition of RAF and d-cyclin-dependent kinases might provide an effective approach to treat patients with tumors harboring mutations in RAS or RAF genes.

Structural analysis of a set of proteins resulting from a bacterial genomics project.

The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB.

The promise of structural genomics in the discovery of new antimicrobial agents.

Structural Genomics stands out among the emerging fields of proteomics since it influences the drug discovery process at so many points. Recent developments in protein expression technologies, x-ray crystallography and NMR spectroscopy provide the essential elements for high-throughput structure determination platforms. Bioinformatics methods to interrogate the resulting data will provide comprehensive, genome-wide databases of protein structure. Genomic sequencing and methods for high-throughput expression and protein purification are furthest advanced for microbial genes and so these have been the early targets for structural genomics initiatives. The information will be invaluable in understanding gene function, designing broad-spectrum small molecule inhibitors and in better understanding drug-host interactions.

A structural genomics approach to the study of quorum sensing: crystal structures of three LuxS orthologs.

BACKGROUND: Quorum sensing is the mechanism by which bacteria control gene expression in response to cell density. Two major quorum-sensing systems have been identified, system 1 and system 2, each with a characteristic signaling molecule (autoinducer-1, or AI-1, in the case of system 1, and AI-2 in system 2). The luxS gene is required for the AI-2 system of quorum sensing. LuxS and AI-2 have been described in both Gram-negative and Gram-positive bacterial species and have been shown to be involved in the expression of virulence genes in several pathogens. RESULTS: The structure of the LuxS protein from three different bacterial species with resolutions ranging from 1.8 A to 2.4 A has been solved using an X-ray crystallographic structural genomics approach. The structure of LuxS reported here is seen to have a new alpha-beta fold. In all structures, an equivalent homodimer is observed. A metal ion identified as zinc was seen bound to a Cys-His-His triad. Methionine was found bound to the protein near the metal and at the dimer interface. CONCLUSIONS: These structures provide support for a hypothesis that explains the in vivo action of LuxS. Specifically, acting as a homodimer, the protein binds a methionine analog, S-ribosylhomocysteine (SRH). The zinc atom is in position to cleave the ribose ring in a step along the synthesis pathway of AI-2.

Promoter sequence and expression of the leucine-rich repeat gene LRR47: evidence for cytoplasmic and nuclear localization in Drosophila embryos and cells.

In Drosophila, proteins containing leucine-rich repeats (LRR) play diverse roles during embryonic development. In particular, they function in cell adhesion and cellular signalling and have in common the ability to mediate reversible protein-protein interactions. The sequence and chromosomal location of Drosophila LRR47, which encodes a protein with eight LRR repeats, were reported previously. In this paper the 5' flanking region of the LRR47 gene is described and the initiation point for the maternal transcription unit is defined. LRR47 belongs to a subfamily of LRR proteins that have in common the ability to interact with ras GTPase. Whole-mount in situ hybridization studies show that the LRR47 transcript is uniformly distributed in early cleavage embryos but becomes depleted at the termini by the blastoderm stage. There is a specific requirement for ras function in the embryonic termini at this developmental stage. The distribution of LRR47 protein in embryos and tissue culture cells was also studied. The protein is present in both the cytoplasm and nuclei of embryos until gastrulation and is seen to persist in the nuclei of the amnioserosa until later stages of development. The protein is also constitutively present in growing SL2 culture cells and again is present in both cytoplasm and nuclei. These results suggest that LRR47 function may be modulated in the cell or nuclear division cycle.

X-ray diffraction and far-UV CD studies of filaments formed by a leucine-rich repeat peptide: structural similarity to the amyloid fibrils of prions and Alzheimer's disease beta-protein.

The development of neuro-degenerative diseases often involves amyloidosis, that is the formation of polymeric fibrillar structures from normal cellular proteins or peptides. For example, in Alzheimer's disease, a 42 amino acid peptide processed from the amyloid precursor protein forms filaments with a beta-sheet structure. Because of this, the structure and dynamics of polymeric peptide filaments is of considerable interest. We showed previously that a 23 amino acid peptide constituting a single leucine-rich repeat (LRRN) polymerises spontaneously in solution to form long filaments of a beta-sheet structure, a property similar to that of Alzheimer's beta-amyloid and prion peptides. Here we report that a variant of LRRN in which a highly conserved asparagine residue is replaced by aspartic acid does not form either filaments or beta structure. By contrast, a variant which replaces this asparagine residue with glutamine forms filaments ultrastructurally indistinguishable from those of LRRN. Electron micrographs of LRRN filaments show that many consist of two interleaved strands which appear to have a ribbon-like morphology. X-ray diffraction patterns from oriented LRRN fibres reveal that they are composed of long beta-sheet arrays, with the interstrand hydrogen bonding parallel to the filament axis. This 'cross-beta' structure is similar to that adopted by beta-amyloid and prion derived fibres. Taken together, these results indicate that the LRR filaments are stabilised by inter- or intra-strand hydrogen bonded interactions comparable to the asparagine ladders of beta-helix proteins or the 'glutamine zippers' of poly-glutamine peptides. We propose that similar stabilising interactions may underlie a number of characterised predispositions to neuro-degenerative diseases that are caused by mutations to amide residues. Our finding that amyloid-like filaments can form from a peptide motif not at present correlated with degenerative disease suggests that a propensity for beta-filament formation is a common feature of protein sub-domains.

Sequence and expression of LRR47, a novel embryonic leucine rich repeat protein of Drosophila.

Leucine-rich repeats (LRRs) are 22-28 amino acid long sequence motifs found in a variety of extracellular, membrane and cytoplasmic proteins. They are believed to mediate specific protein-protein interactions and to function in cellular adhesion. In Drosophila, four LRR proteins are known and each plays an important role in embryogenesis. In this paper we report the cloning of a cDNA that encodes a fifth Drosophila embryonic LRR protein, LRR47. The sequence includes a hydrophobic N-terminal which may constitute an ER signal sequence, eight LRR copies and a unique C-terminal. The transcript of the LRR47 gene is detected in adult females and in early embryogenesis. It is not found in adult males and is only present at low levels in embryos after 6 h of development. In Western blot experiments, a protein of approx. 47 kDa, which is expressed in a similar developmental profile and purifies in peripheral membrane protein extracts, is detected by an antibody specific for LRR47. The LRR47 gene maps to position 32A on the left arm of chromosome 2, an interval in which three genes with semi-lethal maternal effects (dal, hup and wdl) are located.

Drosophila ribosomal protein L18a: cDNA sequence, expression and chromosomal localization of the gene.

We describe the cDNA sequence of the Drosophila homologue of the rat ribosomal protein L18a. The protein sequence predicted has identical or conservatively substituted amino acids in 80% of positions. It is distinctly basic in character with an overall net positive charge of + 20. Analysis of L18a RNA with the Northern blot technique shows it to be expressed both during embryonic development and in the adult fly. In situ hybridisation to polytene chromosomes reveals that the L18a gene(s) is located at 54B on the second chromosome.

Analysis of physician and hospital differences in 'negative' coronary angiogram rates.

A direct relationship has been postulated between high "negative" coronary angiogram rates and physician payment. We conducted a prospective study of coronary angiography in a teaching and community hospital staffed, respectively, by cardiologists who were performing cardiac catheterization as salaried or fee-for-service physicians. The lower overall rate of negative angiograms at the teaching hospital correlated with the presence of a cardiac surgery unit and the increased referral of patients with documented coronary artery disease. The percentage of completely normal angiograms did not differ significantly between hospitals. The number of angiograms positive by a 70% occlusion criterion in patients not previously known to have coronary artery disease also did not differ greatly. Negative angiogram rates appeared to vary inversely with physician ability to set preangiogram probabilities of coronary artery disease. Our findings do not discount reimbursement as a strong incentive, but suggest other important determinants of coronary angiographic variation.