Glaucio Valdameri

Substituted chromones as highly potent nontoxic inhibitors, specific for the breast cancer resistance protein.

A series of 13 disubstituted chromones was synthesized. Two types of substituents, on each side of the scaffold, contributed to both the potency of ABCG2 inhibition and the cytotoxicity. The best compound, 5-(4-bromobenzyloxy)-2-(2-(5-methoxyindolyl)ethyl-1-carbonyl)-4H-chromen-4-one (6g), displayed high-affinity inhibition and low cytotoxicity, giving a markedly high therapeutic index. The chromone derivative specifically inhibited ABCG2 versus other multidrug ABC transporters and was not transported. It constitutes a highly promising candidate for in vivo chemosensitization of ABCG2-expressing tumors.

Dual RNA-seq transcriptional analysis of wheat roots colonized by Azospirillum brasilense reveals up-regulation of nutrient acquisition and cell cycle genes

BackgroundThe rapid growth of the world’s population demands an increase in food production that no longer can be reached by increasing amounts of nitrogenous fertilizers. Plant growth promoting bacteria (PGPB) might be an alternative to increase nitrogenous use efficiency (NUE) in important crops such wheat. Azospirillum brasilense is one of the most promising PGPB and wheat roots colonized by A. brasilense is a good model to investigate the molecular basis of plant-PGPB interaction including improvement in plant-NUE promoted by PGPB.ResultsWe performed a dual RNA-Seq transcriptional profiling of wheat roots colonized by A. brasilense strain FP2. cDNA libraries from biological replicates of colonized and non-inoculated wheat roots were sequenced and mapped to wheat and A. brasilense reference sequences. The unmapped reads were assembled de novo. Overall, we identified 23,215 wheat expressed ESTs and 702 A. brasilense expressed transcripts. Bacterial colonization caused changes in the expression of 776 wheat ESTs belonging to various functional categories, ranging from transport activity to biological regulation as well as defense mechanism, production of phytohormones and phytochemicals. In addition, genes encoding proteins related to bacterial chemotaxi, biofilm formation and nitrogen fixation were highly expressed in the sub-set of A. brasilense expressed genes.ConclusionsPGPB colonization enhanced the expression of plant genes related to nutrient up-take, nitrogen assimilation, DNA replication and regulation of cell division, which is consistent with a higher proportion of colonized root cells in the S-phase. Our data support the use of PGPB as an alternative to improve nutrient acquisition in important crops such as wheat, enhancing plant productivity and sustainability.

Pharmacological inhibition of LIM Kinase stabilizes microtubules and inhibits neoplastic growth

The emergence of tumor resistance to conventional microtubule-targeting drugs restricts their clinical use. Using a cell-based assay that recognizes microtubule polymerization status to screen for chemicals that interact with regulators of microtubule dynamics, we identified Pyr1, a cell permeable inhibitor of LIM kinase, which is the enzyme that phosphorylates and inactivates the actin-depolymerizing factor cofilin. Pyr1 reversibly stabilized microtubules, blocked actin microfilament dynamics, inhibited cell motility in vitro and showed anticancer properties in vivo, in the absence of major side effects. Pyr1 inhibition of LIM kinase caused a microtubule-stabilizing effect, which was independent of any direct effects on the actin cytoskeleton. In addition, Pyr1 retained its activity in multidrug-resistant cancer cells that were resistant to conventional microtubule-targeting agents. Our findings suggest that LIM kinase functions as a signaling node that controls both actin and microtubule dynamics. LIM kinase may therefore represent a targetable enzyme for cancer treatment.

Involvement of catalase in the apoptotic mechanism induced by apigenin in HepG2 human hepatoma cells

Apigenin has been reported to inhibit proliferation of cancer cells; however, the mechanism underlying its action is not completely understood. Here, we evaluated the effects of apigenin on the levels of expression and activity of antioxidant enzymes, and the involvement of ROS in the mechanism of cell death induced by apigenin in HepG2 human hepatoma cells. Upon treatment with apigenin, HepG2 cells displayed a reduction in cell viability in a dose- and time-dependent manner, and some morphological changes. In addition, apigenin treatment induced ROS generation and significantly decreased the mRNA levels and activity of catalase and levels of intracellular GSH. On the other hand, apigenin treatment did not alter the expression or activity levels of other antioxidant enzymes. Addition of exogenous catalase significantly reduced the effects of apigenin on HepG2 cell death. We also demonstrated that HepG2 cells are more sensitive to apigenin-mediated cell death than are primary cultures of mouse hepatocytes, suggesting a differential toxic effect of this agent in tumor cells. Our results suggest that apigenin-induced apoptosis in HepG2 cells may be mediated by a H(2)O(2)-dependent pathway via reduction of the antioxidant defenses.

Converting potent indeno[1,2-b]indole inhibitors of protein kinase CK2 into selective inhibitors of the breast cancer resistance protein ABCG2.

A series of indeno[1,2-b]indole-9,10-dione derivatives were synthesized as human casein kinase II (CK2) inhibitors. The most potent inhibitors contained a N(5)-isopropyl substituent on the C-ring. The same series of compounds was found to also inhibit the breast cancer resistance protein ABCG2 but with totally different structure-activity relationships: a N(5)-phenethyl substituent was critical, and additional hydrophobic substituents at position 7 or 8 of the D-ring or a methoxy at phenethyl position ortho or meta also contributed to inhibition. The best ABCG2 inhibitors, such as 4c, 4h, 4i, 4j, and 4k, behaved as very weak inhibitors of CK2, whereas the most potent CK2 inhibitors, such as 4a, 4p, and 4e, displayed limited interaction with ABCG2. It was therefore possible to convert, through suitable substitutions of the indeno[1,2-b]indole-9,10-dione scaffold, potent CK2 inhibitors into selective ABCG2 inhibitors and vice versa. In addition, some of the best ABCG2 inhibitors, which displayed a very low cytotoxicity, thus giving a high therapeutic ratio, and appeared not to be transported, constitute promising candidates for further investigations.

ABCG2: recent discovery of potent and highly selective inhibitors

ABCG2 impacts oral availability, tissue distribution and excretion of its substrates, including anticancer and anti-infectious drugs. Highly expressed at physiological barriers, its secretion level significantly controls drug distribution. Furthermore, its increased content into many types of cancer may lead to cell chemoresistance. Owing to the clinical relevance of ABCG2 in the multidrug resistance phenomenon, ABCG2 constitutes an appealing therapeutic target to increase drug distribution. Development of ABCG2 inhibitors can be used in combination with anticancer drugs to block the drug secretion from cancer cells. Very recently, an alternative use of ABCG2 inhibitors in enhancing the bioavailability of ABCG2 substrates has emerged. Hence, it is important to investigate ABCG2 inhibitors with high selectivity, high potency and safety. New inhibitors discovered during the last 5 years will be presented and discussed.

Azaindole derivatives are inhibitors of microtubule dynamics, with anti-cancer and anti-angiogenic activities.

Drugs targeting microtubules are commonly used for cancer treatment. However, the potency of microtubule inhibitors used clinically is limited by the emergence of resistance. We thus designed a strategy to find new cell‐permeable microtubule‐targeting agents.

Comparative proteomics analysis of the rice roots colonized by Herbaspirillum seropedicae strain SmR1 reveals induction of the methionine recycling in the plant host.

Although the use of plant growth-promoting bacteria in agriculture is a reality, the molecular basis of plant-bacterial interaction is still poorly understood. We used a proteomic approach to study the mechanisms of interaction of Herbaspirillum seropedicae SmR1 with rice. Root proteins of rice seedlings inoculated or noninoculated with H. seropedicae were separated by 2-D electrophoresis. Differentially expressed proteins were identified by MALDI-TOF/TOF and MASCOT program. Among the identified proteins of H. seropedicae, the dinitrogenase reductase NifH and glutamine synthetase GlnA, which participate in nitrogen fixation and ammonium assimilation, respectively, were the most abundant. The rice proteins up-regulated included the S-adenosylmethionine synthetase, methylthioribose kinase, and acireductone dioxygenase 1, all of which are involved in the methionine recycling. S-Adenosylmethionine synthetase catalyzes the synthesis of S-adenosylmethionine, an intermediate used in transmethylation reactions and in ethylene, polyamine, and phytosiderophore biosynthesis. RT-qPCR analysis also confirmed that the methionine recycling and phytosiderophore biosynthesis genes were up-regulated, while ACC oxidase mRNA level was down-regulated in rice roots colonized by bacteria. In agreement with these results, ethylene production was reduced approximately three-fold in rice roots colonized by H. seropedicae. The results suggest that H. seropedicae stimulates methionine recycling and phytosiderophore synthesis and diminishes ethylene synthesis in rice roots.

Phenolic indeno[1,2- b ]indoles as ABCG2-selective potent and non-toxic inhibitors stimulating basal ATPase activity

Ketonic indeno[1,2-b]indole-9,10-dione derivatives, initially designed as human casein kinase II (CK2) inhibitors, were recently shown to be converted into efficient inhibitors of drug efflux by the breast cancer resistance protein ABCG2 upon suited substitutions including a N5-phenethyl on C-ring and hydrophobic groups on D-ring. A series of ten phenolic and seven p-quinonic derivatives were synthesized and screened for inhibition of both CK2 and ABCG2 activities. The best phenolic inhibitors were about threefold more potent against ABCG2 than the corresponding ketonic derivatives, and showed low cytotoxicity. They were selective for ABCG2 over both P-glycoprotein and MRP1 (multidrug resistance protein 1), whereas the ketonic derivatives also interacted with MRP1, and they additionally displayed a lower interaction with CK2. Quite interestingly, they strongly stimulated ABCG2 ATPase activity, in contrast to ketonic derivatives, suggesting distinct binding sites. In contrast, the p-quinonic indenoindoles were cytotoxic and poor ABCG2 inhibitors, whereas a partial inhibition recovery could be reached upon hydrophobic substitutions on D-ring, similarly to the ketonic derivatives.

6‐Halogenochromones Bearing Tryptamine: One‐Step Access to Potent and Highly Selective Inhibitors of Breast Cancer Resistance Protein

Most anticancer drugs are rendered less efficacious due to cell resistance to chemotherapy related to various mechanisms. A major mechanism is associated with the overexpression of ATP binding cassette (ABC) transporters, especially P-glycoprotein (Pgp/ABCB1), multidrug resistance-associated protein 1 (MRP1/ ABCC1) and breast cancer resistance protein (BCRP/ABCG2), which traffic chemotherapeutic agents out of cancer cells. ABCG2 was simultaneously discovered by three research groups and named ABCP for its abundance in placenta, BCRP for its identification in breast cancer cell lines, and MXR for its resistance to mitoxantrone. ABCG2 constitutes an important target for the design of efflux inhibitors that would, when co-administered with an anticancer agent, give increased intracellular drug concentrations and hence greater cytotoxicity. While several types of ABCG2 inhibitors have been evaluated in vitro, very few have entered preclinical trials. We recently discovered that some substituted chromones are selective and potent ABCG2 inhibitors. These compounds were synthesized in five steps, and the overall yields were quite low. In pursuing our efforts toward structurally simple and easily accessible specific inhibitors of BCRP, we investigated 6-halogenochromones linked to a tryptamine unit, obtained in only one step, as new potent inhibitors (Scheme 1). The choice of C-6 as the site of halogenation was motivated by a number of considerations: 1) the presence of a hydrophobic halogen at the C-6 position fulfills the previously identified need for a hydrophobic substituent in this part of the molecule; 2) halogens, especially bromine and iodine, have a positive contribution to inhibitory activity ; 3) halogens could open interesting opportunities for the generation of further potential inhibitors, as they can be easily replaced by a number of chemical entities. Access to target compounds 3–7 was achieved in one step by coupling 6-substituted-4-oxo-4H-chromene-2-carboxylic acid (1) with tryptamine (2) in the presence of bis(2-oxo-3-oxazolidinyl)phosphonic chloride (BOP-Cl) as the coupling agent (Scheme 2; full details are given in the Supporting Information). 6-Iodo-4-oxo-4H-chromene-2-carboxylic acid (R= I) was not commercially available, but was easily obtained by hydrolysis of the commercially available corresponding ethyl ester with sodium hydrogen carbonate (20% in water) at 80 8C. The test compounds were first screened by flow cytometry for their effects on the inhibition of mitoxantrone efflux in Scheme 1. Retrosynthetic rationale for the synthesis of targeted BCRP inhibitors, and the structures of the commercially available starting materials 1 and 2.