Synthesis of bisphosphonate derivatives of ATP by T4 RNA ligase

Abstract

T4 RNA ligase catalyzes the synthesis of ATP b,cbisphosphonate analogues, using the following substrates with the relative velocity rates indicated between brackets: methylenebisphosphonate (pCH 2 p) (100), clodronate (pCCl 2 p) (52), and etidronate (pC(OH)(CH 3 )p) (4). The presence of pyrophosphatase about doubled the rate of these syntheses. Pamidronate (pC(OH)(CH 2 -CH 2 -NH 2 )p), and alendronate (pC(OH)(CH 2 -CH 2 -CH 2 -NH 2 )p) were not substrates of the reaction. Clodronate displaced the AMP moiety of the complex E-AMP in a concentration dependent manner. The K m values and the rate of synthesis (k cat ) determined for the bisphosphonates as substrates of the reaction were, respectively: methylenebisphosphonate, 0.26 ± 0.05 mM (0.28 ± 0.05 s À1 ); clodronate, 0.54 ± 0.14 mM (0.29 ± 0.05 s À1 ); and etidronate, 4.3 ± 0.5 mM (0.028 ± 0.013 s À1 ). In the presence of GTP, and ATP or AppCCl 2 p the relative rate of synthesis of adenosine 5 0 ,5 000 -P 1 ,P 4 -tetraphosphoguanosine (Ap 4 G) was around 100% and 33%, respectively; the methylenebisphosphonate derivative of ATP (AppCH 2 p) was a very poor substrate for the synthesis of Ap 4 G. To our knowledge this report describes, for the first time, the synthesis of ATP b,c-bisphosphonate analogues by an enzyme different to the classically considered aminoacyl-tRNA synthetases.

Bisphosphonamidate Clodronate Prodrug Exhibits Potent Anticancer Activity in Non-Small-Cell Lung Cancer Cells

“…T4 RNA ligase has been shown to effectively convert NNBPs to their ATP analogs. 35 Both bisphosphonate and clodronate are known to be substrates of T4 RNA ligase with clodronate being converted to AppCCl 2 p more efficiently than the corresponding conversion of the non-chlorinated bisphosphonate to AppCH 2 p. On this basis, we anticipate intracellular conversion of bisphosphonate to AppCH 2 p is also less efficient than the conversion of clodronate to AppCCl 2 p. The observed 3-fold decrease in IC 50 of 14 compared to 15 is consistent with this idea. Detailed studies to determine the kinetics of intracellular prodrug activation and clodronate metabolism are required to correlate these events with observed growth inhibitory activity.…”

Synthesis of ATP derivatives of compounds of the mevalonate pathway (isopentenyl di- and triphosphate; geranyl di- and triphosphate, farnesyl di- and triphosphate, and dimethylallyl diphosphate) catalyzed by T4 RNA ligase, T4 DNA ligase and other ligases

“…To investigate whether the ligases capable to synthesize bisphosphonate derivatives of ATP, as already shown in our laboratory [8,9], were able to synthesize as well, the ATP derivative of isopentenyl pyrophosphate (ApppI [16], here iso-pppA), the enzyme T4 RNA ligase was firstly tested. The 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 9 view, these results indicate that the synthesis of iso-pppA catalyzed by T4…”

“…Bisphosphonates (P-C(R1)(R2)-P) (BPs) are widely used in the treatment of osteoporosis and other bone diseases [1]. After being fixed to bone tissues [2][3][4], they exert a damaging effect on osteoclasts through two classically described mechanisms: a) as substrates of different ligases generating BP derivatives of ATP through reactions 1, 5; 1a, 5a (see Table 1); this effect is supposed to be carried out by the first generation, or not containing nitrogen, bisphosphonates (non-N-BPs) [5][6][7][8][9]; b) as inhibitors of the mevalonate pathway ( Fig. 1) by causing several pathological consequences such as: inhibition of the synthesis of cholesterol and its derivatives and, secondarily, inhibition of the post-translational modifications (isoprenylation, farnesylation) of important regulatory proteins [10][11][12][13][14][15].…”

“…B. Several ligases catalyze the transfer of AMP from the E-X-AMP or E-AMP complexes to a bisphosphonate, with formation of the corresponding derivative (reactions 5, 5a, Table 1) [8,9].…”

Effect of Bisphosphonates on Root Growth and on Chlorophyll Formation in Arabidopsis thaliana Seedlings

“…In fact, they have the ability of interfering with metabolic pathways located at the crossroads of essential processes for life. Some important examples of this crucial role are as follows: a) Bisphosphonates may act as analogs of PP in many of the reactions catalyzed by ligases and some transferases (reaction 1) in which derivatives of the type NRpp-CH 2 -p are synthesized in the reverse reaction (reaction 2) (Günther Sillero et al, 2008;Günther Sillero et al, 2006;Rogers et al, 1996;Russell, 2011).…”