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1.
J Enzyme Inhib Med Chem ; 36(1): 1248-1257, 2021 Dec.
Article in English | MEDLINE | ID: mdl-34107832

ABSTRACT

In certain cancers, such as breast, prostate and some lung and skin cancers, the gene for the enzyme catalysing the second and last step in proline synthesis, δ1-pyrroline-5-carboxylate (P5C) reductase, has been found upregulated. This leads to a higher proline content that exacerbates the effects of the so-called proline-P5C cycle, with tumour cells effectively using this method to increase cell survival. If a method of reducing or inhibiting P5C reductase could be discovered, it would provide new means of treating cancer. To address this point, the effect of some phenyl-substituted derivatives of aminomethylene-bisphosphonic acid, previously found to interfere with the catalytic activity of plant and bacterial P5C reductases, was evaluated in vitro on the human isoform 1 (PYCR1), expressed in E. coli and affinity purified. The 3.5-dibromophenyl- and 3.5-dichlorophenyl-derivatives showed a remarkable effectiveness, with IC50 values lower than 1 µM and a mechanism of competitive type against both P5C and NADPH. The actual occurrence in vivo of enzyme inhibition was assessed on myelogenous erythroleukemic K562 and epithelial breast cancer MDA-MB-231 cell lines, whose growth was progressively impaired by concentrations of the dibromo derivative ranging from 10-6 to 10-4 M. Interestingly, growth inhibition was not relieved by the exogenous supply of proline, suggesting that the effect relies on the interference with the proline-P5C cycle, and not on proline starvation.


Subject(s)
Cell Proliferation/drug effects , Diphosphonates/pharmacology , Neoplasms/metabolism , Proline/biosynthesis , Pyrroline Carboxylate Reductases/antagonists & inhibitors , Humans , Neoplasms/pathology , delta-1-Pyrroline-5-Carboxylate Reductase
2.
Forensic Sci Int Genet ; 52: 102464, 2021 05.
Article in English | MEDLINE | ID: mdl-33461105

ABSTRACT

The availability of molecular markers able to distinguish drug-type from fiber-type Cannabis sativa cultivars would allow fast and cheap analysis of any plant specimen, including seeds and leaves. Several approaches to this issue have been described, mainly using polymorphisms in the genes coding for tetrahydrocannabinol acid synthase or cannabidiolic acid synthase. Some studies reported sequencing of these genes from small groups of hemp varieties belonging to both chemotypes, showing the occurrence of specific DNA signatures. However, the effectiveness of the corresponding primers to discriminate among chemotypes has been validated on a limited number of cultivars, or not tested at all. Here we report a thorough in silico analysis of available gene sequences for both synthases, showing the existence of hypervariable regions at 3' and 5' ends. This notwithstanding, some possible signatures were identified, and 12 putatively specific primer pairs were designed and tested on 16 fiber-type and 11 drug-type varieties. In most cases inconsistent results were obtained, further strengthening the high genetic variability of these genes in hemp germplasm, yet some highly informative polymorphisms were identified. Potentiality and perspectives of this approach are discussed.


Subject(s)
Cannabis/genetics , Dronabinol/analogs & derivatives , Genetic Markers , Intramolecular Oxidoreductases/genetics , Humans , Polymerase Chain Reaction , Polymorphism, Single Nucleotide , Sequence Analysis, DNA
3.
Amino Acids ; 43(1): 493-7, 2012 Jul.
Article in English | MEDLINE | ID: mdl-21938400

ABSTRACT

The streptococcal enzyme that catalyzes the last step in proline biosynthesis was heterologously expressed and the recombinant protein was purified to electrophoretic homogeneity and characterized thoroughly. As for δ1-pyrroline-5-carboxylate reductases from other sources, it was able to use either NADH or NADPH as the electron donor in vitro. However, with NADH the activity was markedly inhibited by physiological levels of NADP+. Results also strengthen the possibility that an unusual ordered substrate binding occurs, in which the dinucleotide binds last.


Subject(s)
NADP/metabolism , NAD/metabolism , Pyrroline Carboxylate Reductases/metabolism , Streptococcus pyogenes/enzymology , Catalysis , Kinetics , NAD/chemistry , NADP/chemistry , Proline/biosynthesis , Proline/metabolism , Pyrroline Carboxylate Reductases/chemistry , Streptococcus pyogenes/metabolism , Substrate Specificity , delta-1-Pyrroline-5-Carboxylate Reductase
4.
Amino Acids ; 42(6): 2283-91, 2012 Jun.
Article in English | MEDLINE | ID: mdl-21744012

ABSTRACT

Compounds able to interfere with amino acid biosynthesis have the potential to inhibit cell growth. In both prokaryotic and eukaryotic microorganisms, unless an ornithine cyclodeaminase is present, the activity of δ1-pyrroline-5-carboxylate (P5C) reductase is mandatory to proline production, and the enzyme inhibition should result in amino acid starvation, blocking in turn protein synthesis. The ability of some substituted derivatives of aminomethylenebisphosphonic acid and its analogues to interfere with the activity of the enzyme from the human pathogen Streptococcus pyogenes was investigated. Several compounds were able to suppress activity in the micromolar range of concentrations, with a mechanism of uncompetitive type with respect to the substrate P5C and non-competitive with respect to the electron donor NAD(P)H. The actual occurrence of enzyme inhibition in vivo was supported by the effects of the most active derivatives upon bacterial growth and free amino acid content.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Diphosphonates/pharmacology , Enzyme Inhibitors/pharmacology , Pyrroline Carboxylate Reductases/antagonists & inhibitors , Streptococcus pyogenes/drug effects , Amino Acids/analysis , Anti-Bacterial Agents/chemical synthesis , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Cloning, Molecular , Diphosphonates/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Escherichia coli , Humans , Inhibitory Concentration 50 , Kinetics , Microbial Viability , Molecular Structure , Pyrroles/chemistry , Pyrroles/metabolism , Pyrroline Carboxylate Reductases/chemistry , Pyrroline Carboxylate Reductases/metabolism , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Streptococcus pyogenes/enzymology
5.
J Agric Food Chem ; 55(11): 4340-7, 2007 May 30.
Article in English | MEDLINE | ID: mdl-17474756

ABSTRACT

A series of N-substituted derivatives of aminomethylenebisphosphonic acid were evaluated as potential inhibitors of delta1-pyrroline-5-carboxylate reductase (EC 1.5.1.2), the enzyme that catalyzes the last step in proline biosynthesis, partially purified from Arabidopsis thaliana suspension cultured cells. At millimolar concentrations, three compounds out of 26 were found to interfere with the catalytic mechanism. One of them, namely, 3,5-dichloropyridyl-aminomethylenebisphosphonic acid, retained such inhibitory activity in the micromolar range. Kinetic analyses ruled out the possibility that the inhibition could simply rely upon the chelating properties of bisphosphonates and showed mechanisms of a noncompetitive type against NADH and an uncompetitive type against delta1-pyrroline-5-carboxylic acid, with KI values of 199 +/- 6 and 10.3 +/- 1.5 microM, respectively. A computer-aided docking analysis, performed on the basis of the crystal structure of the enzyme from Streptococcus pyogenes, suggested that this phosphonate may interact with amino acid residues near the binding site of delta1-pyrroline-5-carboxylic acid, thus blocking the substrate in a pocket and preventing its interaction with NADH. Because in higher plants the step catalyzed by delta1-pyrroline-5-carboxylate reductase is shared by all pathways leading to proline synthesis, such a compound may represent a lead structure to be exploited for the design of new substances endowed with herbicidal activity.


Subject(s)
Arabidopsis/enzymology , Diphosphonates/pharmacology , Enzyme Inhibitors/pharmacology , Pyrroline Carboxylate Reductases/metabolism , Amino Acid Sequence , Kinetics , Models, Molecular , Molecular Sequence Data , Pyrroline Carboxylate Reductases/antagonists & inhibitors , delta-1-Pyrroline-5-Carboxylate Reductase
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