Tomáš Pospíšil

Jasmonates (JAs) are a family of oxylipin phytohormones regulating plant development and growth and mediating ‘defense versus growth’ responses. The upstream JA biosynthetic precursor cis-(+)-12-oxo-phytodienoic acid (cis-OPDA) acts independently of CORONATIVE INSENSITIVE 1 (COI1)-mediated JA signaling in several stress-induced and developmental processes. However, its perception and metabolism are only partially understood. A few years ago, a low abundant isoleucine analog of the biologically active JA-Ile, OPDA-Ile, was detected years ago in wounded leaves of flowering plants, opening up the possibility that conjugation of cis-OPDA to amino acids might be a relevant mechanism for cis-OPDA regulation. Here, we extended the analysis of amino acid conjugates of cis-OPDA and identified naturally occurring OPDA-Val, OPDA-Phe, OPDA-Ala, OPDA-Glu, and OPDA-Asp accumulating in response to biotic and abiotic stress in Arabidopsis (Arabidopsis thaliana). The OPDA-amino acid conjugates displayed cis-OPDA-related plant responses in a JA-Ile-dependent manner. We also showed that the synthesis and hydrolysis of cis-OPDA amino acid conjugates are mediated by members of the amidosynthetase GRETCHEN HAGEN 3 (GH3) and the amidohydrolase INDOLE-3-ACETYL-LEUCINE RESISTANT 1 (ILR1)/ILR1-like (ILL) families. Thus, OPDA amino acid conjugates function in the catabolism or temporary storage of cis-OPDA in stress responses instead of acting as chemical signals per se.

Caffeine is an alkaloid with a purine structure and has been well known for centuries due to its presence in popular drinks—tea and coffee. However, the structural and spectroscopic parameters of this compound, as well as its chemical and biological activities, are still not fully known. In this study, for the first time, we report on the measured oxygen-17 NMR spectra of this stimulant. To support the assignment of our experimental NMR data, extensive quantum chemical calculations of NMR parameters, including nuclear magnetic shielding constants and indirect spin–spin coupling constants, were performed. In a theoretical study, using nine efficient density functionals (B3LYP, BLYP, BP86, CAM-B3LYP, LC-BLYP, M06, PBE0, TPSSh, wB97x), and in combination with a large and flexible correlation-consistent aug-cc-pVTZ basis set, the structure and NMR parameters were predicted for a free molecule of caffeine and in chloroform, DMSO and water. A polarized continuum model (PCM) was used to include a solvent effect. As a result, an optimal methodology was developed for predicting reliable NMR data, suitable for studies of known, as well as newly discovered, purines and similar alkaloids. The results of the current work could be used in future basic and applied studies, including NMR identification and intermolecular interactions of caffeine in various raw materials, like plants and food, as well as in the structural and spectroscopic characterization of new compounds with similar structures.

Jasmonates (JAs) are a family of oxylipin phytohormones regulating plant development and growth and mediating ‘defense versus growth’ responses. The upstream JA biosynthetic precursor cis -(+)-12-oxo-phytodienoic acid ( cis -OPDA) has been reported to act independently of the COI1-mediated JA signaling in several stress-induced and developmental processes. However, its means of perception and metabolism are only partially understood. Furthermore, cis -OPDA, but not JA, occurs in non-vascular plant species, such as bryophytes, exhibiting specific functions in defense and development. A few years ago, a low abundant isoleucine analog of the biologically active JA-Ile, OPDA-Ile, was detected in wounded leaves of flowering plants, opening up to the possibility that conjugation of cis -OPDA to amino acids might be a relevant mechanism for cis -OPDA regulation. Here, we extended the analysis of amino acid conjugates of cis -OPDA and identified naturally occurring OPDA-Val, OPDA-Phe, OPDA-Ala, OPDA-Glu, and OPDA-Asp in response to biotic and abiotic stress in Arabidopsis. The newly identified OPDA-amino acid conjugates show cis -OPDA-related plant responses in a JAR1-dependent manner. We also discovered that the synthesis and hydrolysis of cis -OPDA amino acid conjugates are regulated by members of the amidosynthetase GH3 and the amidohydrolase ILR1/ILL families. Finally, we found that the cis -OPDA conjugative pathway already functions in non-vascular plants and gymnosperms. Thus, one level of regulation by which plants modulate cis -OPDA homeostasis is the synthesis and hydrolysis of OPDA-amino acid conjugates, which temporarily store cis -OPDA in stress responses.

Cis-(+)-12-oxophytodienoic acid (cis-(+)-OPDA) is a bioactive jasmonate, a precursor of jasmonic acid, which also displays signaling activity on its own. Modulation of cis-(+)-OPDA actions may be carried out via biotransformation leading to metabolites of various functions, similar to other phytohormones. This work introduces a methodology for the synthesis of racemic cis-OPDA conjugates with amino acids (OPDA-aa) and their deuterium-labeled analogs, which enables the identification and accurate quantification of these compounds in plants. We have developed a highly sensitive liquid chromatography-tandem mass spectrometry-based method for the reliable determination of seven OPDA-aa (OPDA-Alanine, OPDA-Aspartate, OPDA-Glutamate, OPDA-Glycine, OPDA-Isoleucine, OPDA-Phenylalanine, and OPDA-Valine) from minute amount of plant material. The extraction from 10 mg of fresh plant tissue by 10% aqueous methanol followed by single-step sample clean-up on hydrophilic–lipophilic balanced columns prior to final analysis was optimized. The method was validated in terms of accuracy and precision, and the method parameters such as process efficiency, recovery and matrix effects were evaluated. In mechanically wounded 30-day-old Arabidopsis thaliana leaves, five endogenous (+)-OPDA-aa were identified and their endogenous levels reached a maximum of pmol/g. The time-course accumulation revealed a peak 60 min after the wounding, roughly corresponding to the accumulation of cis-(+)-OPDA. Current synthetic and analytical methodologies support studies on cis-(+)-OPDA conjugation with amino acids and research into the biological significance of these metabolites in plants.

Targeting cyclin-dependent kinase 7 (CDK7) provides an interesting therapeutic option in cancer therapy because this kinase participates in regulating the cell cycle and transcription. Here, we describe a new trisubstituted pyrazolo[4,3- d ]pyrimidine derivative, LGR6768, that inhibits CDK7 in the nanomolar range and displays favourable selectivity across the CDK family. We determined the structure of fully active CDK2/cyclin A2 in complex with LGR6768 at 2.6 Å resolution using X-ray crystallography, revealing conserved interactions within the active site. Structural analysis and comparison with LGR6768 docked to CDK7 provides an explanation of the observed biochemical selectivity, which is linked to a conformational difference in the biphenyl moiety. In cellular experiments, LGR6768 affected regulation of the cell cycle and transcription by inhibiting the phosphorylation of cell cycle CDKs and the carboxy-terminal domain of RNA polymerase II, respectively. LGR6768 limited the proliferation of several leukaemia cell lines, triggered significant changes in protein and mRNA levels related to CDK7 inhibition and induced apoptosis in dose- and time-dependent experiments. Our work supports previous findings and provides further information for the development of selective CDK7 inhibitors. Graphical

3,5,7-Trisubstituted pyrazolo[4,3-d]pyrimidines have been identified as potent inhibitors of cyclin-dependent kinases (CDKs), which are established drug targets. Herein, we describe their further structural modifications leading to novel nanomolar inhibitors with strong antiproliferative activity. We determined the crystal structure of fully active CDK2/A2 with 5-(2-amino-1-ethyl)thio-3-cyclobutyl-7-[4-(pyrazol-1-yl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidine (24) at 1.7 Å resolution, confirming the competitive mode of inhibition. Biochemical and cellular assays in lymphoma cell lines confirmed the expected mechanism of action through dephosphorylation of retinoblastoma protein and RNA polymerase II, leading to induction of apoptosis. Importantly, we also revealed an interesting ability of compound 24 to induce proteasome-dependent degradation of cyclin K both in vitro and in a patient-derived xenograft in vivo. We propose that 24 has a dual mechanism of action, acting as a kinase inhibitor and as a molecular glue inducing an interaction between CDK12 and DDB1 that leads to polyubiquitination of cyclin K and its subsequent degradation.

Strigolactones (SLs) are natural compounds occurring in plants which have a numerous functions in plant development; therefore, they are plant hormones. Unfortunately, their natural abundance is very low and because of their structure complexity it is difficult to prepare them in big quantities; alternatives with simpler structures and similar biological activity was developed. SLs mimics are compounds with simple synthesis. Methods for preparation of basic SLs mimics are described here.

Strigolactones are phytohormones involved in shoot branching and hypocotyl elongation. The latter phenomenon was addressed herein by the exogenous application of a synthetic strigolactone GR24 and an inhibitor of strigolactone biosynthesis TIS108 on hypocotyls of wild type Arabidopsis and a strigolactone signalling mutant max2-1 (more axillary growth 2-1) . Owing to the interdependence between light and strigolactone signalling, the present work was extended to seedling cultivation under a standard light/dark regime, or under continuous darkness. Given the essential role of the cortical microtubules in cell elongation, their organization and dynamics were characterized under the conditions of altered strigolactone signalling using fluorescence microscopy methods with different spatiotemporal capacities such as confocal laser scanning microscopy and structured illumination microscopy. It was found that the strigolactone-dependent inhibition of hypocotyl elongation correlated with changes in cortical microtubule organization and dynamics, visualized in living wild type and max2-1 seedlings stably expressing genetically-encoded fluorescent molecular markers for microtubules. Quantitative analysis of microscopic datasets revealed that chemical and/or genetic manipulation of strigolactone signalling affected microtubule remodelling, especially under light conditions. The application of GR24 and TIS108 in dark conditions partially alleviated cytoskeletal rearrangement, suggesting a new mechanistic connection between the cytoskeletal behaviour and the light-dependence of strigolactone signalling. Highlight: Strigolactones regulate organization and dynamics of cortical microtubules in hypocotyl cells, which contributes to the light-mediated inhibition of hypocotyl growth in Arabidopsis seedlings.