We have developed and validated a novel LC-MS/MS method for simultaneously analyzing amino acids, biogenic amines, and their acetylated and methylated derivatives in plants. This method involves a one-step extraction of 2-5 mg of lyophilized plant material followed by fractionation of different biogenic amine forms, and exploits an efficient combination of hydrophilic interaction liquid chromatography (HILIC), reversed phase (RP) chromatography with pre-column derivatization, and tandem mass spectrometry (MS). This approach enables high-throughput processing of plant samples, significantly reducing the time needed for analysis and its cost. We also present a new synthetic route for deuterium-labeled polyamines. The LC-MS/MS method was rigorously validated by quantifying levels of nitrogen-related metabolites in seedlings of seven plant species, including Arabidopsis, maize, and barley, all of which are commonly used model organisms in plant science research. Our results revealed substantial variations in the abundance of these metabolites between species, developmental stages, and growth conditions, particularly for the acetylated and methylated derivatives and the various polyamine fractions. However, the biological relevance of these plant metabolites is currently unclear. Overall, this work contributes significantly to plant science by providing a powerful analytical tool and setting the stage for future investigations into the functions of these nitrogen-related metabolites in plants.

• Klíčová slova
• Acetylated amino acids, LC-MS/MS, acetylated biogenic amines, amino acids, biogenic amines, methylated amino acids, plant metabolism,
• MeSH
• Arabidopsis metabolismus   růst a vývoj   MeSH
• chromatografie kapalinová MeSH
• dusík * metabolismus   MeSH
• ječmen (rod) metabolismus   růst a vývoj   MeSH
• kapalinová chromatografie-hmotnostní spektrometrie MeSH
• kukuřice setá metabolismus   růst a vývoj   MeSH
• polyaminy metabolismus   analýza   MeSH
• rostliny metabolismus   MeSH
• tandemová hmotnostní spektrometrie * metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• dusík * MeSH
• polyaminy MeSH

Cytokinin oxidase/dehydrogenase (CKX) inhibitors reduce the degradation of cytokinins in plants and thereby may improve the efficiency of agriculture and plant tissue culture-based practices. Here, we report a synthesis and structure-activity relationship study of novel urea derivatives concerning their CKX inhibitory activity. The most active compounds showed sub-nanomolar IC50 values with maize ZmCKX1, the lowest value yet documented. Other CKX isoforms of maize and Arabidopsis were also inhibited very effectively. The binding mode of four compounds was characterized based on high-resolution crystal complex structures. Using the soil nematode Caenorhabditis elegans, and human skin fibroblasts, key CKX inhibitors with low toxicity were identified. These compounds enhanced the shoot regeneration of Lobelia, Drosera, and Plectranthus, as well as the growth of Arabidopsis and Brassica napus. At the same time, a key compound (identified as 82) activated a cytokinin primary response gene, ARR5:GUS, and a cytokinin sensor, TCSv2:GUS, without activating the Arabidopsis cytokinin receptors AHK3 and AHK4. This strongly implies that the effect of compound 82 is due to the up-regulation of cytokinin signalling. Overall, this study identifies highly effective and easily prepared CKX inhibitors with a low risk of environmental toxicity for further investigation of their potential in agriculture and biotechnology.

• Klíčová slova
• Agriculture, Arabidopsis, CKX inhibitor, biostimulant, biotechnology, cytokinin, cytokinin oxidase/dehydrogenase, diphenylurea, nutrient use efficiency, oilseed rape,
• MeSH
• Arabidopsis * účinky léků   genetika   MeSH
• Brassica napus genetika   účinky léků   MeSH
• cytokininy metabolismus   MeSH
• inhibitory enzymů farmakologie   MeSH
• kukuřice setá účinky léků   genetika   růst a vývoj   MeSH
• oxidoreduktasy * metabolismus   genetika   MeSH
• rostlinné proteiny metabolismus   genetika   chemie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zemědělství MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytokinin oxidase MeSH Prohlížeč
• cytokininy MeSH
• inhibitory enzymů MeSH
• oxidoreduktasy * MeSH
• rostlinné proteiny MeSH

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.

• Klíčová slova
• Cyclin-dependent kinase 7, Inhibitor, Selectivity, pyrazolo[4,3-d]pyrimidine,
• MeSH
• buněčný cyklus MeSH
• cyklin-dependentní kinasy * genetika   MeSH
• fosforylace MeSH
• inhibitory proteinkinas farmakologie   chemie   MeSH
• kinasa aktivující cyklin dependentní kinasy * MeSH
• pyrimidiny farmakologie   chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cyklin-dependentní kinasy * MeSH
• inhibitory proteinkinas MeSH
• kinasa aktivující cyklin dependentní kinasy * MeSH
• pyrimidiny MeSH

Phenolic acids are known flavonoid metabolites, which typically undergo bioconjugation during phase II of biotransformation, forming sulfates, along with other conjugates. Sulfated derivatives of phenolic acids can be synthesized by two approaches: chemoenzymatically by 3'-phosphoadenosine-5'-phosphosulfate (PAPS)-dependent sulfotransferases or PAPS-independent aryl sulfotransferases such as those from Desulfitobacterium hafniense, or chemically using SO3 complexes. Both approaches were tested with six selected phenolic acids (2-hydroxyphenylacetic acid (2-HPA), 3-hydroxyphenylacetic acid (3-HPA), 4-hydroxyphenylacetic acid (4-HPA), 3,4-dihydroxyphenylacetic acid (DHPA), 3-(4-hydroxyphenyl)propionic acid (4-HPP), and 3,4-dihydroxyphenylpropionic acid (DHPP)) to create a library of sulfated metabolites of phenolic acids. The sulfates of 3-HPA, 4-HPA, 4-HPP, DHPA, and DHPP were all obtained by the methods of chemical synthesis. In contrast, the enzymatic sulfation of monohydroxyphenolic acids failed probably due to enzyme inhibition, whereas the same reaction was successful for dihydroxyphenolic acids (DHPA and DHPP). Special attention was also paid to the counterions of the sulfates, a topic often poorly reported in synthetic works. The products obtained will serve as authentic analytical standards in metabolic studies and to determine their biological activity.

• Klíčová slova
• aryl sulfotransferase, biotransformation, flavonoid metabolites, phenolic acids, sulfation,
• MeSH
• fosfoadenosinfosfosulfát * chemie   metabolismus   MeSH
• hydroxybenzoáty MeSH
• sírany metabolismus   MeSH
• sulfotransferasy * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fosfoadenosinfosfosulfát * MeSH
• hydroxybenzoáty MeSH
• phenolic acid MeSH Prohlížeč
• sírany MeSH
• sulfotransferasy * MeSH

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.

• MeSH
• cyklin-dependentní kinasa 2 MeSH
• cyklin-dependentní kinasy * MeSH
• cykliny metabolismus   MeSH
• inhibitory proteinkinas chemie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• protinádorové látky * chemie   farmakologie   MeSH
• pyrimidiny chemie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cyklin-dependentní kinasa 2 MeSH
• cyklin-dependentní kinasy * MeSH
• cykliny MeSH
• inhibitory proteinkinas MeSH
• protinádorové látky * MeSH
• pyrimidine MeSH Prohlížeč
• pyrimidiny MeSH

Kinetin (N6-furfuryladenine), a plant growth substance of the cytokinin family, has been shown to modulate aging and various age-related conditions in animal models. Here we report the synthesis of kinetin isosteres with the purine ring replaced by other bicyclic heterocycles, and the biological evaluation of their activity in several in vitro models related to neurodegenerative diseases. Our findings indicate that kinetin isosteres protect Friedreich́s ataxia patient-derived fibroblasts against glutathione depletion, protect neuron-like SH-SY5Y cells from glutamate-induced oxidative damage, and correct aberrant splicing of the ELP1 gene in fibroblasts derived from a familial dysautonomia patient. Although the mechanism of action of kinetin derivatives remains unclear, our data suggest that the cytoprotective activity of some purine isosteres is mediated by their ability to reduce oxidative stress. Further, the studies of permeation across artificial membrane and model gut and blood-brain barriers indicate that the compounds are orally available and can reach central nervous system. Overall, our data demonstrate that isosteric replacement of the kinetin purine scaffold is a fruitful strategy for improving known biological activities of kinetin and discovering novel therapeutic opportunities.

• Klíčová slova
• Cytokinin, Kinetin, Mitoprotection – familial dysautonomia, Neuroprotection, bioisostery – Friedreich́s ataxia,
• MeSH
• cytoprotekce MeSH
• kinetin chemická syntéza   chemie   farmakologie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• molekulární struktura MeSH
• oxidační stres účinky léků   MeSH
• puriny chemická syntéza   chemie   farmakologie   MeSH
• viabilita buněk účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kinetin MeSH
• purine MeSH Prohlížeč
• puriny MeSH

Increasing crop productivity is our major challenge if we are to meet global needs for food, fodder and fuel. Controlling the content of the plant hormone cytokinin is a method of improving plant productivity. Cytokinin oxidase/dehydrogenase (CKO/CKX) is a major target in this regard because it degrades cytokinins. Here, we describe the synthesis and biological activities of new CKX inhibitors derived mainly from diphenylurea. They were tested on four CKX isoforms from maize and Arabidopsis, where the best compounds showed IC50 values in the 10-8 M concentration range. The binding mode of the most efficient inhibitors was characterized from high-resolution crystal complexed structures. Although these compounds do not possess intrinsic cytokinin activity, we have demonstrated their tremendous potential for use in the plant tissue culture industry as well as in agriculture. We have identified a key substance, compound 19, which not only increases stress resistance and seed yield in Arabidopsis, but also improves the yield of wheat, barley and rapeseed grains under field conditions. Our findings reveal that modulation of cytokinin levels via CKX inhibition can positively affect plant growth, development and yield, and prove that CKX inhibitors can be an attractive target in plant biotechnology and agriculture.

• Klíčová slova
• Agriculture, CKX inhibitor, biotechnology, crystal structure, cytokinin, cytokinin oxidase/dehydrogenase, diphenylurea, plant tissue culture, stress, yield,
• MeSH
• Arabidopsis * MeSH
• biotechnologie MeSH
• cytokininy MeSH
• oxidoreduktasy * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokinin oxidase MeSH Prohlížeč
• cytokininy MeSH
• oxidoreduktasy * MeSH

Cyclin-dependent kinases are therapeutic targets frequently deregulated in various cancers. By convenient alkylation of the 5-sulfanyl group, we synthesized 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2) H-pyrazolo[4,3- d]pyrimidines with various substitutions at position 5 with potent antiproliferative activity in non-Hodgkin lymphoma cell lines. The most potent derivative 4.35 also displayed activities across more than 60 cancer cell lines. The kinase profiling confirmed high selectivity of 4.35 toward cyclin-dependent kinases (CDKs) 2, 5, and 9, and the cocrystal with CDK2/cyclin A2 revealed its binding in the active site. Cultured lymphoma cell lines treated with 4.35 showed dephosphorylation of CDK substrates, cleavage of PARP-1, downregulation of XIAP and MCL-1, and activation of caspases, which collectively confirmed ongoing apoptosis. Moreover, 4.35 demonstrated significant activity in various cell line xenograft and patient-derived xenograft mouse models in vivo both as a monotherapy and as a combination therapy with the BCL2-targeting venetoclax. These findings support further studies of combinatorial treatment based on CDK inhibitors.

• MeSH
• apoptóza účinky léků   MeSH
• bicyklické sloučeniny heterocyklické farmakologie   MeSH
• cyklin-dependentní kinasy antagonisté a inhibitory   MeSH
• inhibitory proteinkinas chemická syntéza   farmakologie   terapeutické užití   MeSH
• lidé MeSH
• lymfom farmakoterapie   MeSH
• molekulární struktura MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• proliferace buněk účinky léků   MeSH
• protinádorové látky chemická syntéza   farmakologie   terapeutické užití   MeSH
• pyrazoly chemická syntéza   farmakologie   terapeutické užití   MeSH
• pyrimidiny chemická syntéza   farmakologie   terapeutické užití   MeSH
• sulfonamidy farmakologie   MeSH
• synergismus léků MeSH
• vztahy mezi strukturou a aktivitou MeSH
• xenogenní modely - testy protinádorové aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bicyklické sloučeniny heterocyklické MeSH
• cyklin-dependentní kinasy MeSH
• inhibitory proteinkinas MeSH
• protinádorové látky MeSH
• pyrazoly MeSH
• pyrimidiny MeSH
• sulfonamidy MeSH
• venetoclax MeSH Prohlížeč

Cytokinins (CKs) and their metabolites and derivatives are essential for cell division, plant growth regulation and development. They are typically found at minute concentrations in plant tissues containing very complicated biological matrices. Therefore, defined standards labelled with stable isotopes are required for precise metabolic profiling and quantification of CKs, as well as in vivo elucidation of CK biosynthesis in various plant species. In this work, 11 [15N]-labelled C6-purine derivatives were prepared, among them 5 aromatic (4, 5, 6, 7, 8) and 3 isoprenoid (9, 10, 11) CKs. Compared to current methods, optimized syntheses of 6-amino-9H-[15N5]-purine (adenine) and 6-chloro-9H-[15N4]-purine (6-chloropurine) were performed to achieve more effective, selective and generally easier approaches. The chemical identity and purity of prepared compounds were confirmed by physico-chemical analyses (TLC; HRMS; HPLC-MS; 1H, 13C, 15N NMR). The presented approach is applicable for the synthesis of any other desired [15N4]-labelled C6-substituted purine derivatives.

• Klíčová slova
• 15N-labelled, cytokinin, purine, synthesis,
• Publikační typ
• časopisecké články MeSH

A series of amphiphilic derivatives of (3β,17β)-3-hydroxyandrost-5-ene-17-carboxylic acid (1) with the polyamine spermine and three other diamines, 1,2-diaminoethane, piperazine and cadaverine, were synthesized and their antimicrobial activity and cytotoxicity were investigated. Among the target compounds, several ones showed antimicrobial activity on Gram positive and Gram negative microorganisms. The most active compounds were 20 (Streptococcus mutans CCM 7409, 3.125 µM), 16 (Streptococcus mutans CCM 7409, 12.5 µM) and 10d (Escherichia coli CCM 3954, 12.5 µM). In addition, compounds 5d, 10d, 13 and 20 displayed cytotoxicity on CEM (12.1 ± 2.1 µM, 7.6 ± 1.0 µM, 19.0 ± 0.4 µM and 5.9 ± 0.7 µM, respectively). Two additional compounds displayed medium cytotoxicity on CEM, 5a (34.6 ± 5.2 µM) and 5c (37.7 ± 5.9 µM). The compound 13 and 20 displayed high toxicity also on normal fibroblasts.

• Klíčová slova
• Amphiphile, Antimicrobial activity, Cytotoxicity, Diamine, Polyamine, Steroid,
• MeSH
• androsteny chemická syntéza   chemie   farmakologie   MeSH
• antibakteriální látky chemická syntéza   chemie   farmakologie   MeSH
• Escherichia coli účinky léků   patogenita   MeSH
• kyseliny karboxylové chemická syntéza   chemie   farmakologie   MeSH
• mikrobiální testy citlivosti MeSH
• polyaminy chemická syntéza   chemie   farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• androsteny MeSH
• antibakteriální látky MeSH
• kyseliny karboxylové MeSH
• polyaminy MeSH