Cyclin F-mediated degradation of ribonucleotide reductase M2 controls genome integrity and DNA repair.

F-box proteins are the substrate binding subunits of SCF (Skp1-Cul1-F-box protein) ubiquitin ligase complexes. Using affinity purifications and mass spectrometry, we identified RRM2 (the ribonucleotide reductase family member 2) as an interactor of the F-box protein cyclin F. Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides (dNTPs), which are necessary for both replicative and repair DNA synthesis. We found that, during G2, following CDK-mediated phosphorylation of Thr33, RRM2 is degraded via SCF(cyclin F) to maintain balanced dNTP pools and genome stability. After DNA damage, cyclin F is downregulated in an ATR-dependent manner to allow accumulation of RRM2. Defective elimination of cyclin F delays DNA repair and sensitizes cells to DNA damage, a phenotype that is reverted by expressing a nondegradable RRM2 mutant. In summary, we have identified a biochemical pathway that controls the abundance of dNTPs and ensures efficient DNA repair in response to genotoxic stress.

CRL4AMBRA1 is a master regulator of D-type cyclins.

D-type cyclins are central regulators of the cell division cycle and are among the most frequently deregulated therapeutic targets in human cancer1, but the mechanisms that regulate their turnover are still being debated2,3. Here, by combining biochemical and genetics studies in somatic cells, we identify CRL4AMBRA1 (also known as CRL4DCAF3) as the ubiquitin ligase that targets all three D-type cyclins for degradation. During development, loss of Ambra1 induces the accumulation of D-type cyclins and retinoblastoma (RB) hyperphosphorylation and hyperproliferation, and results in defects of the nervous system that are reduced by treating pregnant mice with the FDA-approved CDK4 and CDK6 (CDK4/6) inhibitor abemaciclib. Moreover, AMBRA1 acts as a tumour suppressor in mouse models and low AMBRA1 mRNA levels are predictive of poor survival in cancer patients. Cancer hotspot mutations in D-type cyclins abrogate their binding to AMBRA1 and induce their stabilization. Finally, a whole-genome, CRISPR-Cas9 screen identified AMBRA1 as a regulator of the response to CDK4/6 inhibition. Loss of AMBRA1 reduces sensitivity to CDK4/6 inhibitors by promoting the formation of complexes of D-type cyclins with CDK2. Collectively, our results reveal the molecular mechanism that controls the stability of D-type cyclins during cell-cycle progression, in development and in human cancer, and implicate AMBRA1 as a critical regulator of the RB pathway.

AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity.

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway1,2. Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis.

Regioselective synthesis and molecular docking studies of functionalized imidazo [1,2-a]pyridine derivatives through MCRs.

A efficient protocol has been developed for the synthesis of regioselective imidazo[1,2-a]pyridine and imidazo[1,2-a]pyrimidine derivatives through cascade reaction between 2-aminopyridine, arylelglyoxal, and 4-hydroxypyran via three-component reaction to prepare targeted compounds with good to excellent yields. The advantages of this transformation are a catalyst-free reaction, green solvent, operationally simple, scalable, and eco-friendly. The product collects with simple filtration which avoided tedious and expensive purification techniques. In addition, computational studies like molecular docking were conducted to provide the theoretical possibilities of binding these types of synthesized compounds to the VEGFR2 receptors as potential key inhibitors of tumor cell growth and angiogenesis.

A one-pot ring-closure and ring-opening sequence for the cascade synthesis of dihydrofurofurans and functionalized furans.

We have developed a simple novel ring-closure and ring-opening pathway using an organo-base system for the synthesis of highly substituted dihydrofurofuran and furan frameworks via a triethylamine-catalyzed one-pot three-component reaction. The protocol involved a Knoevenagel and Michael adduct via Paal-Knorr cyclization with aromatic/aliphatic glyoxal and 2-cyanoacetophenone under mild and heating conditions with excellent yields through a simple filtration method. The merits of this methodology, including the use of easily available feedstocks and an inexpensive catalyst, Gram-scale synthesis, wide functional group tolerance, an open-air reaction setup, and no need for workup and column-chromatography procedures, make the developed methodology a practical way to access dihydrofurofurans and functionalized furans.

Regulation of the CRL4(Cdt2) ubiquitin ligase and cell-cycle exit by the SCF(Fbxo11) ubiquitin ligase.

F-box proteins and DCAF proteins are the substrate binding subunits of the Skp1-Cul1-F-box protein (SCF) and Cul4-RING protein ligase (CRL4) ubiquitin ligase complexes, respectively. Using affinity purification and mass spectrometry, we determined that the F-box protein FBXO11 interacts with CDT2, a DCAF protein that controls cell-cycle progression, and recruits CDT2 to the SCF(FBXO11)complex to promote its proteasomal degradation. In contrast to most SCF substrates, which exhibit phosphodegron-dependent binding to F-box proteins, CDK-mediated phosphorylation of Thr464 present in the CDT2 degron inhibits recognition by FBXO11. Finally, our results show that the functional interaction between FBXO11 and CDT2 is evolutionary conserved from worms to humans and plays an important role in regulating the timing of cell-cycle exit.

An approach towards the synthesis of novel fused nitrogen tricyclic heterocyclic scaffolds via GBB reaction.

A concise and efficient one-pot synthesis of novel N-fused tricyclic derivatives has been developed by using the Groebke-Blackburn-Bienaymé (GBB) reaction, which involved the reaction of 3-amino-1H-indazoles, aldehydes and isonitriles to afford 2-aryl-5H-imidazo[1,2-b]indazol-3-amine derivatives via a formal [4 + 1] cycloaddition reaction. Furthermore, we describe an unprecedented reaction of chromone-3-carboxaldehydes with 3-amino-1H-indazoles to afford (2-hydroxyphenyl)(pyrimido[1,2-b]indazol-3-yl)methanones in one-pot at ambient temperature. This protocol features a robust method for the one-step construction of new tricyclic rings, column chromatography free methods with a clean reaction profile, high yields, operational simplicity and it tolerates a diverse collection of reactants.

Synthesis and Characterization of Multiwalled Carbon Nanotubes/Poly(HEMA-co-MMA) by Utilizing Click Chemistry.

The hybrid material consisting of multi walled carbon nanotubes (MWNTs) and poly(2-hydroxyethylmethacrylate-co-methylmethacrylate) [poly(HEMA-co-MMA)] was synthesized by a combination of RAFT and Click chemistry. In the primary stage, the copolymer poly(HEMA-co-MMA) was prepared by applying RAFT technique. Alkynyl side groups were incorporated onto the poly(HEMA-co-MMA) backbone by esterification reaction. Then, MWNTs-N3 was prepared by treating MWNTs with 4-azidobutylamine. The click coupling reaction between azide-functionalized MWNTs (MWNTs-N3) and the alkyne-functionalized random copolymer ((HEMA-co-MMA)-Alkyne) with the Cu(I)-catalyzed [3+2] Huisgen cycloaddition afforded the hybrid compound. The structure and properties of poly(MMA-co-HEMA)-g-MWNTs were investigated by FT-IR, EDX and TGA measurements. The copolymer brushes were observed to be immobilized onto the functionalized MWNTs by SEM and TEM analysis.

A metal-free tandem C-C/C-O bond formation approach to densely functionalized indolyl 4H-chromenes catalyzed by polystyrene-supported p-toluenesulfonic acid under solvent-free conditions.

A new environmentally benign and highly convergent protocol for the synthesis of indolyl 4H-chromene derivatives has been developed. This one-pot three-component condensation reaction of salicylaldehyde, cyclic 1,3-diketones, and indole is promoted by PS-PTSA as a reusable heterogeneous acid catalyst under solvent-free conditions. This protocol demonstrates several notable advantages such as that the catalyst is readily available and can be recovered and reused for at least five runs without any significant impact on product yields, high atom economy, excellent yields, and efficiency of producing three new bonds (two C-C and one C-O) and one stereo center in a single operation.

Preparation of ß-Cyclodextrin Multi-Decorated Halloysite Nanotubes as a Catalyst and Nanoadsorbent for Dye Removal.

Hybrid materials of ß-cyclodextrin multi-decorated halloysite nanotubes (HNTs-g-ßCD) were prepared by a facile route, which showed high efficiency for catalysis and dye adsorption. Initially, the surface of halloysite nanotubes (HNTs) was modified with poly(glycidyl methacrylate) by the reversible addition fragmentation chain transfer (RAFT) polymerization of glycidyl methacrylate having epoxy groups as a monomer. Subsequently, ß-cyclodextrin was conjugated with the modified HNTs to produce HNTs-g-ßCD by the epoxide ring-opening reaction of mono-6-deoxy-6-hexanediamine-ß-cyclodextrin. The nanocomposites were characterized by FT-IR, TGA, SEM, and TEM. The HNTs-g-ßCD composites could be used as a nano adsorbent for methylene blue and a catalyst in the oxidation reaction of benzyl alcohol owing to the unique structure of ß-cyclodextrin. The HNTs-g-ßCD shows promiseas potential multi-functional materials by a combination of ß-cyclodextrin and HNTs properties.

FeF(3) catalyzed cascade C-C and C-N bond formation: synthesis of differentially substituted triheterocyclic benzothiazole functionalities under solvent-free condition.

A series of diverse polyfunctionalized triheterocyclic benzothiazoles were easily prepared in excellent yields via the Biginelli reaction of 2-aminobenzothiazole with substituted benzaldehydes and α-methylene ketones using FeF(3) as an expeditious catalyst under solvent-free conditions. The protocol provides a practical and straightforward approach toward highly functionalized triheterocyclic benzothiazole derivatives in excellent yields. The reaction was conveniently promoted by FeF(3) and the catalyst could be recovered easily after the reaction and reused without any loss of its catalytic activity. The advantageous features of this methodology are high atom economy, operational simplicity, shorter reaction time, convergence, and facile automation.

Preparation and characterization of graphene/poly(diphenylamine) composites.

Nanocomposites of graphene nanosheets and poly(diphenylamine) (graphene-PDPA) were synthesized via the in-situ oxidative polymerization of diphenylamine in a sulphuric acid medium. First, graphite oxide (GO) was prepared by oxidation of natural graphite using the modified Hummer's method and subsequently reduced using hydrazine monohydrate. The as-prepared graphene sheets were noncovalently grafted with PDPA using ammonium peroxydisulphate as an oxidant. During the polymerization, graphene sheets were homogeneously dispersed in the PDPA matrix. The formation of the hybrid material was confirmed by FTIR, XPS, TGA, HRTEM, FESEM and XRD measurements. XPS analysis revealed the removal of oxygen functionality from the GO surface after reduction and the bonding structure of the reduced hybrids. In addition, the nanocomposites showed better thermal properties due to the intrinsic property of the graphene sheets.

(Z)-Ethyl 2-cyano-3-(1H-imidazol-2-yl)acrylate.

The crystal structure of the title compound, C9H9N3O2, features N-H⋯N and C-H⋯O inter-actions. The N-H⋯N inter-action generates a chain running along the a axis and the C-H⋯O inter-action generates a chain along the c axis. An intra-molecular C-H⋯O inter-action is also observed.

1-(4-Chloro-phen-yl)-1H-1,2,3,4-tetra-zole.

There are two independent mol-ecules in the asymmetric unit of the title compound, C(7)H(5)ClN(4), in which the tetra-zole and benzene rings are twisted by dihedral angles of 12.9 (1) and 39.8 (1)°. In the crystal, the independent mol-ecules are connected into a tetra-mer by C-H⋯N hydrogen bonds, generating an R(4) (4)(12) graph-set motif.

(E)-Ethyl 2-cyano-3-(furan-2-yl)acrylate.

There are two independent mol-ecules in the asymmetric unit of the title compound, C(10)H(9)NO(3), in both of which, all non-H atoms except for the methyl C atom lie nearly in the same plane [maximum deviations = 0.094 (3) and 0.043 (2) Å]. In the crystal, each independent mol-ecules is linked by pairs of C-H⋯O inter-actions, generating inversion dimers with R(2) (2)(10) ring motifs.

1-(4-Methyl-phen-yl)-1H-1,2,3,4-tetra-zole.

In the title compound, C(8)H(8)N(4), the dihedral angle between the tetra-zole and benzene rings is 21.6 (1)°. An inter-molecular C-H⋯π inter-action is observed.

1-(3,5-Dichloro-phen-yl)-1H-1,2,3,4-tetra-zole.

In the title compound, C(7)H(4)Cl(2)N(4), the dihedral angle between the tetra-zole and benzene rings is 17.2 (2)°. In the crystal, C-H⋯N inter-actions link the mol-ecules into a flattened helical chain along the b axis.

Enhanced sialylation of recombinant human erythropoietin in Chinese hamster ovary cells by combinatorial engineering of selected genes.

Therapeutic glycoproteins with exposed galactose (Gal) residues are cleared rapidly from the bloodstream by asialoglycoprotein receptors in hepatocytes. Various approaches have been used to increase the content of sialic acid, which occupies terminal sites of N- or O-linked glycans and thereby increases the half-life of therapeutic glycoproteins. We enhanced sialylation of human erythropoietin (EPO) by genetic engineering of the sialylation pathway in Chinese hamster ovary (CHO) cells. The enzyme GNE (uridine diphosphate-N-acetyl glucosamine 2-epimerase)/MNK (N-acetyl mannosamine kinase), which plays a key role in the initial two steps of sialic acid biosynthesis, is regulated by cytidine monophosphate (CMP)-sialic acid through a feedback mechanism. Since sialuria patient cells fail in regulating sialic acid biosynthesis by feedback mechanism, various sialuria-like mutated rat GNEs were established and subjected to in vitro activity assay. GNE/MNK-R263L-R266Q mutant showed 93.6% relative activity compared with wild type and did not display feedback inhibition. Genes for sialuria-mutated rat GNE/MNK, Chinese hamster CMP-sialic acid transporter and human α2,3-sialyltransferase (α2,3-ST) were transfected simultaneously into recombinant human (rh) EPO-producing CHO cells. CMP-sialic acid concentration of engineered cells was significantly (>10-fold) increased by sialuria-mutated GNE/MNK (R263L-R266Q) expression. The sialic acid content of rhEPO produced from engineered cells was 43% higher than that of control cells. Ratio of tetra-sialylated glycan of rhEPO produced from engineered cells was increased ∼32%, but ratios of asialo- and mono-sialylated glycans were decreased ∼50%, compared with control. These findings indicate that sialuria-mutated rat GNE/MNK effectively increases the intracellular CMP-sialic acid level. The newly constructed host CHO cell lines produced more highly sialylated therapeutic glycoproteins through overexpression of sialuria-mutated GNE/MNK, CMP-SAT and α2,3-ST.

Synthesis, stereochemistry and in vitro antimicrobial evaluation of novel 2-[(2,4-diaryl-3-azabicyclo[3.3.1]nonan-9-ylidene)hydrazono]-4-phenyl-2,3-dihydrothiazoles.

2-[(2,4-Diaryl-3-azabicyclo[3.3.1]nonan-9-ylidene)hydrazono]-4-phenyl-2,3-dihydrothiazoles (3a-3k) have been synthesized by the cyclization of 2-[(2,4-diaryl-3-azabicyclo[3.3.1]nonan-9-one thiosemicarbazones with phenacyl bromide and characterized by analytical (melting point and elemental analysis) and spectral (IR, (1)H NMR, (13)C NMR, D(2)O exchange, NOESY and mass) techniques. The novel Hantzsch products (3a-3k) were screened for their in vitro antibacterial and antifungal activities against some selected microorganisms. Structure activity relationship (SAR) for the reported compounds was studied by comparing their MIC values with standard drugs (Streptomycin and Amphotericin B). The results show that 3e against Escherichia coli and Cryptococcus neoformans3i against Bacillus Subtilis, 3b against Aspergillus flavus, and 3k against Rhizopus sp. were found to show significant growth inhibition.

Synthesis of polyfunctionalized piperidone oxime ethers and their cytotoxicity on HeLa cells.

A series of twenty 2,6-diarylpiperidin-4-one O-methyloximes were synthesized with fluoro/chloro/bromo/methyl/methoxy/ethoxy/isopropyl substituents on various positions of the phenyl at C-2 and C-6 in association with/without methyl substituent on the secondary amino group and methyl/ethyl/isopropyl substituents on the active methylene centers. Regardless of their substitution all compounds predominantly exist in the chair conformation except 3m, which adopts a twist-boat conformation. All the synthesized compounds were evaluated for their in vitro antiproliferative activity against human cervical carcinoma (HeLa) cell line. The cytotoxicity of the test compounds was determined by measuring the number of live cells after 24 h of treatment by MTT assay method. This preliminary SAR suggests some lead molecules 3c-f, 3j-k, 4d-g, and 4i with a scope of further structural optimization of the piperidone pharmacophore toward the development of anticancer drug synthesis.