Anticancer activity profiling of parthenolide analogs generated via P450-mediated chemoenzymatic synthesis

The plant-derived sesquiterpene lactone parthenolide (PTL) was recently found to possess promising anticancer activity but elaboration of this natural product scaffold for optimization of its pharmacological properties has proven challenging via available chemical methods. In this work, P450-catalyzed C–H hydroxylation of positions C9 and C14 in PTL was coupled to carbamoylation chemistry to yield a panel of novel carbamate-based PTL analogs (‘parthenologs’). These compounds, along with a series of other C9- and C14-functionalized parthenologs obtained via O–H acylation, alkylation, and metal-catalyzed carbene insertion, were profiled for their cytotoxicity against a diverse panel of human cancer cell lines. These studies led to the discovery of several parthenologs with significantly improved anticancer activity (2–14-fold) compared to the parent molecule. Most interestingly, two PTL analogs with high cytotoxicity (LC₅₀ ～ 1–3 μM) against T cell leukemia (Jurkat), mantle cell lymphoma (JeKo-1), and adenocarcinoma (HeLa) cells as well as a carbamate derivative with potent activity (LC₅₀ = 0.6 μM) against neuroblastoma cells (SK-N-MC) were obtained. In addition, these analyses resulted in the identification of parthenologs featuring both a broad spectrum and tumor cell-specific anticancer activity profile, thus providing valuable probes for the future investigation of biomolecular targets that can affect cell viability across multiple as well as specific types of human cancers. Altogether, these results highlight the potential of P450-mediated chemoenzymatic C–H functionalization toward tuning and improving the anticancer activity of the natural product parthenolide.     

Screening for mutations causing X-linked severe combined immunodeficiency in the IL-2Rγ chain gene by single-strand conformation polymorphism analysis

Mutations in the common gamma chain (_c or IL2RG) of the interleukin-2, –4, –7, –9 and –15 receptors have been found to cause X-linked severe combined immunodeficiency (SCIDX1). We report here on the mutations identified in a further ten families. Two of the mutations identified have occurred twice in unrelated families, indicating two possible mutational hotspots. Seven of the mutations, which were identified by single-strand conformational polymorphism (SSCP) analysis, are point mutations, and the eighth is a small deletion. We also report on the first use of assays based on these mutations within IL2RG for unambiguous carrier determination. The consequences for the _c proteins produced as a result of these mutations are discussed.     

The SPP1 connection

Abstract: The connector of the virulent Bacillus subtilis bacteriophage SPP1 (Styloviridae) is a structure localized at the phage head vertex which attaches the tail. It is formed by oligomerization of SPP1 gene product 6 (gp6; portal protein). The purified protein is found in solution essentially as a homo-tredecamer. Its assembly pattern resembles the turbine-like organization found for other portal proteins and has a defined handedness (Dube et al. (1993) EMBO J. 12, 1303–1309). A preliminary reconstruction of the structure shows that gp6 is composed of a lower ring connected by a narrow region to the upper area consisting of 13 lobes radiating from an inner ring. The assembly is organized around a central channel which spans its full height. A functional characterization of gp6 mutants showed that substitutions of defined amino acids by more basic residues lead to packaging of reduced amounts of DNA into the phage head (Tavares et al. (1992) J. Mol. Biol. 225, 81–92). Since SPP1 encapsidates its DNA by a headful mechanism, these mutations ( siz ) affect most probably a function on the headful sensor—signal transduction—headful cut system. Combination of siz alleles has severe effects in packaging. The resulting gp6 versions lead to the encapsidation of shorter DNA molecules at a lower efficiency than single siz mutants. Gene 6 is expressed late during SPP1 infection. Interestingly, the mass of portal protein inside the cell then increases continuously until lysis, reaching a level several fold higher than the amount required to accomplish its role as a structural component of the virion.     

Variable region V1 ofSaccharomyces cerevisiae 18S rRNA participates in biogenesis and function of the small ribosomal subunit

The role of helix 6, which forms the major portion of the most 5′-located expansion segment ofSaccharomyces cerevisiae 18S rRNA, was studied by in vivo mutational analysis. Mutations that increased the size of the helical part and/or the loop, even to a relatively small extent, abolished 18S rRNA formation almost completely. Concomitantly, 35S pre-rRNA and an abnormal 23S precursor species accumulated. rDNA units containing these mutations did not support cell growth. A deletion removing helix 6 almost completely, on the other hand, had a much less severe effect on the formation of 18S rRNA, and cells expressing only the mutant rRNA remained able to grow, albeit at a much reduced rate. Disruption of the apical A·U base pair by a single point mutation did not cause a noticeable reduction in the level of 18S rRNA but did result in a twofold lower growth rate of the cells. This effect could not be reversed by introduction of a second point mutation that restores base pairing. We conclude that both the primary and the secondary structure of helix 6 play an important role in the formation and the biological function of the 40S subunit. Edited by: S.A. Gerbi     

Are the characteristics of betanidin glucosyltransferases from cell-suspension cultures of Dorotheanthus bellidiformis indicative of their phylogenetic relationship with flavonoid glucosyltransferases?

Uridine 5′-diphosphoglucose:betanidin 5-O- and 6-O-glucosyltransferases (5-GT and 6-GT; EC 2.4.1) catalyze the regiospecific formation of betanin (betanidin 5-O-β-glucoside) and gomphrenin I (betanidin 6-O-β-glucoside), respectively. Both enzymes were purified to near homogeneity from cell-suspension cultures of Dorotheanthus bellidiformis, the 5-GT by classical chromatographic techniques and the 6-GT by affinity dye-ligand chromatography using UDP-glucose as eluent. Data obtained with highly purified enzymes indicate that 5-GT and 6-GT catalyze the indiscriminate transfer of glucose from UDP-glucose to hydroxyl groups of betanidin, flavonols, anthocyanidins and flavones, but discriminate between individual hydroxyl groups of the respective acceptor compounds. The 5-GT catalyzes the transfer of glucose to the C-4′ hydroxyl group of quercetin as its best substrate, and the 6-GT to the C-3 hydroxyl group of cyanidin as its best substrate. Both enzymes also catalyze the formation of the respective 7-O-glucosides, but to a minor extent. Although the enzymes were not isolated to homogeneity, chromatographic, electrophoretic and kinetic properties proved that the respective enzyme activities were based on the presence of single enzymes, i.e. 5-GT and 6-GT. The N terminus of the 6-GT revealed high sequence identity to a proposed UDP-glucose:flavonol 3-O-glucosyltransferase (UF3GT) of Manihot esculenta. In addition to the 5-GT and 6-GT, we isolated a UF3GT from D. bellidiformis cell cultures that preferentially accepted myricetin and quercetin, but was inactive with betanidin. The same result was obtained with a UF3GT from Antirrhinum majus and a flavonol 4′-O-glucosyltransferase from Allium cepa. Based on these results, the main question to be addressed reads: Are the characteristics of the 5-GT and 6-GT indicative of their phylogenetic relationship with flavonoid glucosyltransferases? Received: 11 February 1997 / Accepted: 18 April 1997     

Theoretical Study of the Water Exchange Reaction on Divalent Zinc Ion using Density Functional Theory

Recent ab initio studies reported in the literature have challenged the mechanistic assignments made on the basis of volume of activation data [1,2]. In addition to that ab initio molecular orbital calculations on hydrated zinc(II)-ions were used to elucidate the general role of this ion in metalloproteins [3]. Due to our interest in both inorganic reaction mechanisms and enzymatic catalysis we started a systematic investigation of solvent exchange processes on divalent zinc-ion using density functional calculations. Our investigations cover aqua complexes of the general form [Zn(H₂O)_n]²⁺·mH₂0 with n=3-6 and m=0-2, where n and m represent the number of water molecules in the coordination and solvation sphere, respectively.The complexes [Zn(H₂O)₅]²⁺·2H₂O and [Zn(H₂O)₄]²⁺·2H₂O turnend out to be the most stable zinc complexes with seven and six water molecules, respectively. This implies that a heptacoordinated zinc(II) complex, where all water molecules are located in the co-ordination sphere, should be energetically highly unfavorable and that [Zn(H₂O)₆]²⁺ can quite readily push two coordinated water molecules into the solvation sphere. For the pentaqua complex [Zn(H₂O)₅]²⁺ only one water molecule is easily lost to the solvation sphere, which makes the [Zn(H2O)₄]²⁺·H₂O complex the most favorable in order to consider the limiting dissociative and associative water exchange process of hexacoordinated zinc(II). The dehydration and hydration energies using the most stable zinc(II) complexes [Zn(H₂O)₄]²⁺·2H₂O, [Zn(H₂O)₅]²⁺·2H₂O and [Zn(H₂O)₄]²⁺·H₂O were calculated to be 24.1 and -21.0 kcal/mol, respectively.     

A20 Deficiency in Lung Epithelial Cells Protects against Influenza A Virus Infection

A20 negatively regulates multiple inflammatory signalling pathways. We here addressed the role of A20 in club cells (also known as Clara cells) of the bronchial epithelium in their response to influenza A virus infection. Club cells provide a niche for influenza virus replication, but little is known about the functions of these cells in antiviral immunity. Using airway epithelial cell-specific A20 knockout (A20^AEC-KO) mice, we show that A20 in club cells critically controls innate immune responses upon TNF or double stranded RNA stimulation. Surprisingly, A20^AEC-KO mice are better protected against influenza A virus challenge than their wild type littermates. This phenotype is not due to decreased viral replication. Instead host innate and adaptive immune responses and lung damage are reduced in A20^AEC-KO mice. These attenuated responses correlate with a dampened cytotoxic T cell (CTL) response at later stages during infection, indicating that A20^AEC-KO mice are better equipped to tolerate Influenza A virus infection. Expression of the chemokine CCL2 (also named MCP-1) is particularly suppressed in the lungs of A20^AEC-KO mice during later stages of infection. When A20^AEC-KO mice were treated with recombinant CCL2 the protective effect was abrogated demonstrating the crucial contribution of this chemokine to the protection of A20^AEC-KO mice to Influenza A virus infection. Taken together, we propose a mechanism of action by which A20 expression in club cells controls inflammation and antiviral CTL responses in response to influenza virus infection.