Discovery and biological evaluation of 1-{2,7-diazaspiro[3.5]nonan-2-yl}prop-2-en-1-one derivatives as covalent inhibitors of KRAS G12C with favorable metabolic stability and anti-tumor activity.

RAS protein plays a key role in cellular proliferation and differentiation. RAS gene mutation is a known driver of oncogenic alternation in human cancer. RAS inhibition is an effective therapeutic treatment for solid tumors, but RAS protein has been classified as an undruggable target. Recent reports have demonstrated that a covalent binder to KRAS protein at a mutated cysteine residue (G12C) is effective for the treatment of solid tumors. Here, we report a series of 1-{2,7-diazaspiro[3.5]nonan-2-yl}prop-2-en-1-one derivatives as potent covalent inhibitors against KRAS G12C identified throughout structural optimization of an acryloyl amine moiety to improve in vitro inhibitory activity. From an X-ray complex structural analysis, the 1-{2,7-diazaspiro[3.5]nonan-2-yl}prop-2-en-1-one moiety binds in the switch-II pocket of KRAS G12C. Further optimization of the lead compound (5c) led to the successful identification of 1-[7-[6-chloro-8-fluoro-7-(5-methyl-1H-indazol-4-yl)-2-[(1-methylpiperidin-4-yl)amino]quinazolin-4-yl]-2,7-diazaspiro[3.5]nonan-2-yl]prop-2-en-1-one (7b), a potent compound with high metabolic stabilities in human and mouse liver microsomes. Compound 7b showed a dose-dependent antitumor effect on subcutaneous administration in an NCI-H1373 xenograft mouse model.

Azoreductase from alkaliphilic Bacillus sp. AO1 catalyzes indigo reduction.

Indigo is an insoluble blue dye historically used for dyeing textiles. A traditional approach for indigo dyeing involves microbial reduction of polygonum indigo to solubilize it under alkaline conditions; however, the mechanism by which microorganisms reduce indigo remains poorly understood. Here, we aimed to identify an enzyme that catalyzes indigo reduction; for this purpose, from alkaline liquor that performed microbial reduction of polygonum indigo, we isolated indigo carmine-reducing microorganisms. All isolates were facultative anaerobic and alkali-tolerant Bacillus spp. An isolate termed AO1 was found to be an alkaliphile that preferentially grows at pH 9.0-11.0 and at 30-35 °C. We focused on flavin-dependent azoreductase as a possible enzyme for indigo carmine reduction and identified its gene (azoA) in Bacillus sp. AO1 using homology-based strategies. azoA was monocistronic but clustered with ABC transporter genes. Primary sequence identities were < 50% between the azoA product (AzoA) and previously characterized flavin-dependent azoreductases. AzoA was heterologously produced as a flavoprotein tolerant to alkaline and organic solvents. The enzyme efficiently reduced indigo carmine in an NADH-dependent manner and showed strict specificity for electron acceptors. Notably, AzoA oxidized NADH in the presence, but not the absence, of indigo. The reaction rate was enhanced by adding organic solvents to solubilize indigo. Absorption spectrum analysis showed that indigo absorption decreased during the reaction. These observations suggest that AzoA can reduce indigo in vitro and potentially in Bacillus sp. AO1. This is the first study that identified an indigo reductase, providing a new insight into a traditional approach for indigo dyeing.

Terminal bridging of siRNA duplex at the ribose 2' position controls strand bias and target sequence preference.

Small interfering RNA (siRNA) and short hairpin RNA (shRNA) are widely used as RNA interference (RNAi) reagents. Recently, truncated shRNAs that trigger RNAi in a Dicer-independent manner have been developed. We generated a novel class of RNAi reagent, designated enforced strand bias (ESB) RNA, in which an siRNA duplex was chemically bridged between the 3' terminal overhang region of the guide strand and the 5' terminal nucleotide of the passenger strand. ESB RNA, which is chemically bridged at the 2' positions of ribose (2'-2' ESB RNA), functions in a Dicer-independent manner and was highly effective at triggering RNAi without the passenger strand-derived off-target effect. In addition, the 2'-2' ESB RNA exhibited a unique target sequence preference that differs from siRNA and silenced target sequences that could not be effectively suppressed by siRNA. Our results indicate that ESB RNA has the potential to be an effective RNAi reagent even when the target sequence is not suitable for siRNA.

Immaturin-Nuclease as a Model System for a Gene-Programmed Sexual Development and Rejuvenescence in Paramecium Life History.

Fertilization-initiated development and adult-onset aging are standard features in the life history of eukaryotes. In Paramecium, the number of cell divisions after the birth of a new generation is an essential parameter of sexual phase transition and aging. However, the gene driving this process and its evolutionary origin have not yet been elucidated. Here we report several critical outcomes obtained by molecular genetics, immunofluorescence microscopy, transformation by microinjection, and enzymological analysis. The cloned immaturin gene induces sexual rejuvenation in both mature and senescent cells by microinjection. The immaturin gene originated from proteobacteria's glutathione-S-transferase (GST) gene. However, immaturin has been shown to lose GST activity and instead acquire nuclease activity. In vitro substrates for immaturin-nuclease are single- and double-stranded DNA, linear and circular DNA, and single-stranded viral genome RNA such as coronavirus. Anti-immaturin antibodies have shown that the subcellular localizations of immaturin are the macronucleus, cytoplasm, cell surface area, and cilia. The phase transition of sexuality is related to a decrease in the intracellular abundance of immaturin. We propose that sexual maturation and rejuvenation is a process programmed by the immaturin gene, and the sexual function of each age is defined by both the abundance and the intracellular localization mode of the immaturin-nuclease.

Rapid generation of gene disruption constructs by in vitro transposition and identification of a Dictyostelium protein kinase that regulates its rate of growth and development.

We describe a rapid method for creating Dictyo stelium gene disruption constructs, whereby the target gene is interrupted by a drug resistance cassette using in vitro transposition. A fragment of genomic DNA containing the gene to be disrupted is amplified by PCR, cloned into a plasmid vector using topoisomerase and then employed as the substrate in an in vitro Tn5 transposition reaction. The transposing species is a fragment of DNA containing a Dictyostelium blasticidin S resistance (bs(r)) cassette linked to a bacterial tetracycline resistance (tet(r)) cassette. After transposition the plasmid DNA is transformed into Escherichia coli and clones in which the bs(r)-tet(r) cassette is inserted into the Dictyostelium target DNA are identified. To demonstrate its utility we have employed the method to disrupt the gene encoding QkgA, a novel protein kinase identified from the Dictyostelium genome sequencing project. QkgA is structurally homologous to two previously identified Dictyostelium kinases, GbpC and pats1. Like them it contains a leucine-rich repeat domain, a small GTP-binding (ras) domain and a MEKK domain. Disruption of the qkgA gene causes a marked increase in growth rate and, during development, aggregation occurs relatively slowly to form abnormally large multicellular structures.

Cellular Differentiation in Submerged Monolayers of Dictyostelium discoideum: Possible Functions of Cytoplasmic Ca2+ and DIF: (cellular slime mold/differentiation/monolayer culture/Ca2+ /DIF).

Cytoplasmic calcium ion (Ca2+ ) has generally been proposed to be a key factor of numerous cellular processes. Among several agents which might be expected to alter cytoplasmic Ca2+ -concentration ([Ca2+ ]i ), unexpectedly Ca2+ -antagonist TMB-8 was found to raise considerably [Ca2+ ]i , and inhibited not only the formation of prespore cells, but also their maintenance in the monolayer cultures of Dictyostelium discoideum. This seems to indicate that higher [Ca2+ ]i is unfavorable to the prespore differentiation. In this study, we adopted the monolayer culture technique to monitor cell differentiation. However, in high density monolayers there arised a number of unique cells which was highly vacuolated and morphologically intermediate between the stalk and spore cells. These vacuolated cells having both cellulosic wall and spore coat were also induced by differentiation inducing factor (DIF). Thus the monolayer culture system used might be not necessarily qualified to monitor the terminal differentiation of Dictyostelium cells. Nevertheless, the data presented here have strongly suggested that DIF have two physiologically valued roles: 1) Induction of the membrane fusion of vesicles and/or vacuoles (vacuolization), and 2) Induction of the fusion between the cell membrane and vacuole (or vesicle) membrane (exocytosis).

The Dictyostelium prestalk cell inducer DIF regulates nuclear accumulation of a STAT protein by controlling its rate of export from the nucleus.

Dd-STATc becomes tyrosine phosphorylated, dimerises and accumulates in the nuclei of Dictyostelium cells exposed to DIF, the chlorinated hexaphenone that directs prestalk cell differentiation. By performing cytoplasmic photobleaching of living cells, we show that DIF inhibits the nuclear export of Dd-STATc. Within Dd-STATc there is a 50 amino acid region containing several consensus CRM1 (exportin 1)-dependent nuclear export signals (NESs). Deletion of this region causes Dd-STATc to accumulate in the nucleus constitutively and, when coupled to GFP, the same region directs nuclear export. We show that the N-terminal-proximal 46 amino acids are necessary for nuclear accumulation of Dd-STATc and sufficient to direct constitutive nuclear accumulation when fused to GFP. Combining the photobleaching and molecular analyses, we suggest that DIF-induced dimerisation of Dd-STATc functionally masks the NES-containing region and that this leads to nett nuclear accumulation, directed by the N-terminal-proximal import signals. These results show that the regulated nuclear accumulation of a STAT protein can be controlled at the level of nuclear export and they also provide a better understanding of the mechanism whereby DIF directs cell type divergence.

Dd-STATb, a Dictyostelium STAT protein with a highly aberrant SH2 domain, functions as a regulator of gene expression during growth and early development.

Dictyostelium, the only known non-metazoan organism to employ SH2 domain:phosphotyrosine signaling, possesses STATs (signal transducers and activators of transcription) and protein kinases with orthodox SH2 domains. Here, however, we describe a novel Dictyostelium STAT containing a remarkably divergent SH2 domain. Dd-STATb displays a 15 amino acid insertion in its SH2 domain and the conserved and essential arginine residue, which interacts with phosphotyrosine in all other known SH2 domains, is substituted by leucine. Despite these abnormalities, Dd-STATb is biologically functional. It has a subtle role in growth, so that Dd-STATb-null cells are gradually lost from the population when they are co-cultured with parental cells, and microarray analysis identified several genes that are either underexpressed or overexpressed in the Dd-STATb null strain. The best characterised of these, discoidin 1, is a marker of the growth-development transition and it is overexpressed during growth and early development of Dd-STATb null cells. Dimerisation of STAT proteins occurs by mutual SH2 domain:phosphotyrosine interactions and dimerisation triggers STAT nuclear accumulation. Despite its aberrant SH2 domain, the Dd-STATb protein sediments at the size expected for a homodimer and it is constitutively enriched in the nucleus. Moreover, these properties are retained when the predicted site of tyrosine phosphorylation is substituted by phenylalanine. These observations suggest a non-canonical mode of activation of Dd-STATb that does not rely on orthodox SH2 domain:phosphotyrosine interactions.

A STAT-regulated, stress-induced signalling pathway in Dictyostelium.

The Dictyostelium stalk cell inducer differentiation-inducing factor (DIF) directs tyrosine phosphorylation and nuclear accumulation of the STAT (signal transducer and activator of transcription) protein Dd-STATc. We show that hyperosmotic stress, heat shock and oxidative stress also activate Dd-STATc. Hyperosmotic stress is known to elevate intracellular cGMP and cAMP levels, and the membrane-permeant analogue 8-bromo-cGMP rapidly activates Dd-STATc, whereas 8-bromo-cAMP is a much less effective inducer. Surprisingly, however, Dd-STATc remains stress activatable in null mutants for components of the known cGMP-mediated and cAMP-mediated stress-response pathways and in a double mutant affecting both pathways. Also, Dd-STATc null cells are not abnormally sensitive to hyperosmotic stress. Microarray analysis identified two genes, gapA and rtoA, that are induced by hyperosmotic stress. Osmotic stress induction of gapA and rtoA is entirely dependent on Dd-STATc. Neither gene is inducible by DIF but both are rapidly inducible with 8-bromo-cGMP. Again, 8-bromo-cAMP is a much less potent inducer than 8-bromo-cGMP. These data show that Dd-STATc functions as a transcriptional activator in a stress-response pathway and the pharmacological evidence, at least, is consistent with cGMP acting as a second messenger.