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.
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.
Aiming to improve their effectiveness, three modifications were introduced into the structure of the natural phytotoxins nostoclides, leading to the synthesis of novel 3-benzyl-4-isopropyl-5-(arylmethylene)furan-2(5H)-ones, 3-benzyl-5-(furan-2-ylmethylene)furan-2(5H)-ones, and 3,4-dihalo-5-arylidenefuran-2(5H)-ones. All compounds were characterized by IR, 1 H and 13 C NMR, NOEDIF, COSY, HETCOR and MS spectrometry. Increasing the length of the molecule was found to reduce the ability to interfere with ferricyanide reduction by isolated chloroplasts. The addition of an isopropyl moiety into the heterocyclic ring, as in naturally-occurring nostoclides, did not influence the inhibitory potential. Also the replacement of the electron-drawing phenyl substituent with two halogen residues did not improve the resulting activity. However, both latter modifications influenced the interaction with the photosynthetic machinery. These analogues could therefore represent novel leads to be explored toward the development of new herbicides targeting the chloroplastic electron transport chain.
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.
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, d 1 -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 IC 50 values lower than 1 mM 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 À6 to 10 À4 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.
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