Oscar A. Roveri

Inhibition by suramin of mitochondrial ATP synthesis.

Suramin, a drug intensively used in the chemotherapy of African trypanosomiasis and onchocerciasis, is currently being tested in clinical trials for AIDS treatment. Its effects on mitochondrial energy metabolism in mammals were studied. At low concentrations it inhibited ATP synthesis and ATPase activity in submitochondrial particles, as well as ADP-stimulated oxygen consumption and the uncoupler-stimulated ATPase activity in intact rat liver mitochondria. At higher concentrations it also inhibited uncoupled electron transport in both submitochondrial particles and intact mitochondria. From comparison of the kinetic patterns of those inhibitions, evidence suggesting that the adenine nucleotide translocase may be another target for the action of suramin was obtained. The relevance of these findings to the understanding of the biochemical basis of suramin toxicity is discussed.

Steady-state kinetics of F1-ATPase: Mechanism of anion activation

The kinetic behaviour of the ATPase activity of beef heart F1 depends largely on the exposure of the enzyme to some anionic ligands such as sulphate and/or EDTA. F1 prepared in the presence of such anions exhibited a triphasic kinetic pattern whereas F1 from which those anions were removed by dialysis exhibited only two K m values for ATP. Conversely to what has been previously reported, bicarbonate did not linearize F2‐ATPase kinetics. Moreover, anion activation cannot be simply explained by promotion of ADP release but mainly by an increase in affinity of the third catalytic site for ATP.

Inhibition of mitochondrial energy-linked reactions by 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (dimboa), a hydroxamic acid from gramineae

DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) is the main hydroxamic acid isolated from maize extracts. It inhibited reversibly ATP synthesis, Pi-ATP exchange reaction and ATPase activity in submitochondrial particles from bovine heart. Half-maximal effects were obtained with 4, 2, and 6 mM DIMBOA respectively. At higher concentrations it also inhibited mitochondrial electron transport (I50 = 11 mM). Irreversible inactivation of mitochondrial electron transport, Pi-ATP exchange reaction and 8-anilino-1-naphthalene sulfonate energy-dependent fluorescence enhancement was also observed. These effects of DIMBOA on energy-linked mitochondrial reactions may explain the inhibitory action of DIMBOA on several aerobic organisms.

Spegazzinine, a new inhibitor of mitochondrial oxidative phosphorylation

The effects of spegazzinine, a dihydroindole alkaloid, on mitochondrial oxidative phosphorylation were studied. Spegazzinine inhibited coupled respiration and phosphorylation in rat liver mitochondria. The I50 was 120 microM. Uncouplers released the inhibition of coupled respiration. Arsenate-stimulated mitochondrial respiration was partially inhibited by spegazzinine. The stimulation of mitochondrial respiration by Ca2+ and the proton ejection associated with the ATP-dependent Ca2+ uptake were not affected by the alkaloid. Oxidative phosphorylation and the Pi-ATP exchange reaction of phosphorylating beef heart submitochondrial particles were strongly inhibited by spegazzinine (I50, 50 microM) while the ATP-dependent reactions, reduction of NAD+ by succinate and the pyridine nucleotides transhydrogenase were less sensitive (I50, 125 microM). Oxygen uptake by submitochondrial particles was not affected. The 2,4-dinitrophenol-stimulated ATPase activity of rat liver mitochondria was not affected by 300 microM spegazzinine, a concentration of alkaloid that completely inhibited phosphorylation. However, higher concentrations of spegazzinine did partially inhibit it. The ATPase activities of submitochondrial particles, insoluble and soluble ATPases were also partially inhibited by high concentrations of spegazzinine. The inhibitory properties of spegazzinine on energy transfer reactions are compared with those of oligomycin, aurovertin and dicyclohexylcarbodiimide. It is concluded that spegazzinine effects are very similar to the effects of aurovertin and that its site of action may be the same or near the site of aurovertin.

Effects of gramine on energy metabolism of rat and bovine mitochondria

The indole alkaloid gramine is found in several plant families. Its effects on mammalian mitochondria and submitochondrial particles were studied. Low concentrations of gramine slightly stimulated basal electron transport, totally inhibited the Ca2+-induced respiratory control and partially abolished the enhancement of 8-anilino-1-naphthalene sulfonate fluorescence induced by proton translocation coupled to ATP hydrolysis or to succinate oxidation. At higher concentrations gramine inhibited specifically the electron transport at the level of Complex I of the respiratory chain. The I50 values (2-6 mM) were dependent on the presence of uncouplers. Higher concentrations of the alkaloid also inhibited coupled succinate oxidation and ATP hydrolysis (I50 = 10 mM). Possible explanations for these effects are discussed.

Synthesis and β-lactamase inhibitory evaluation of novel 6α-halo-2β-chloromethyl-2α-methylpenam-3α-carboxylic acids and their sulfones and 6α-halo-2β-mercaptobenzothiazolylmethyl-2α-methylpenam-3α-carboxylic acids

Abstract The synthesis and the inhibitory activity against β-lactamase I from Bacillus cereus is described for a new series of 6α-bromo, chloro or fluoro-2β-chloromethyl-2α-methylpenam-3α-carboxylic acids (5a-c) and their corresponding sulfones 6a-c and 6α-bromo, chloro or fluoro-2β-mercaptobenzothiazolylmethyl-2α-methylpenam-3α-carboxylic acids (8a-c). Among the novel compounds, the highest activity was expressed by 6α-chloro-2β-chloromethyl-2α-methylpenam-3α-carboxylic acid 1,1-dioxide (6b).

Inhibition of steady-state mitochondrial ATP synthesis by bicarbonate, an activating anion of ATP hydrolysis.

Bicarbonate, an activating anion of ATP hydrolysis, inhibited ATP synthesis coupled to succinate oxidation in beef heart submitochondrial particles but diminished the lag time and increased the steady-state velocity of the (32)Pi-ATP exchange reaction. The latter effects exclude the possibility that bicarbonate is inducing an intrinsic uncoupling between ATP hydrolysis and proton translocation at the level of F(1)F(o) ATPase. The inhibition of ATP synthesis was competitive with respect to ADP at low fixed [Pi], mixed at high [Pi] and non-competitive towards Pi at any fixed [ADP]. From these results we can conclude that (i) bicarbonate does not bind to a Pi site in the mitochondrial F(1); (ii) it competes with the binding of ADP to a low-affinity site, likely the low-affinity non-catalytic nucleotide binding site. It is postulated that bicarbonate stimulates ATP hydrolysis and inhibits ATP synthesis by modulating the relative affinities of the catalytic site for ATP and ADP.

Inhibition of energy metabolism by benzoxazolin-2-one.

The effects of the title compound (BOA) on energy-linked reactions in mitochondria were studied. BOA inhibited electron transfer between the flavin and ubiquinone in Complex I, and ATP synthesis at the F1 moiety of the ATPase complex. These results are discussed in relation to the toxicity of BOA towards a wide range of aerobic organisms.

Synthesis and elastase inhibitory activity of 6α-chloro-2,2-dimethyl-3α-(pivaloyloxy)methylpenam sulfone, 6α-chloro-2,2-dimethyl-3-exo-methylenepenam sulfone, benzyl and methyl 6α-substituted penicillanate sulfones

The triflates and pivalates of 3 alpha-hydroxymethyl-6-substituted-2,2-dimethylpenam sulfones 3, 5; methyl and benzyl 6-substituted penicillanates 6-9 and 3-exo-methylene-6-substituted-2,2-dimethylpenam sulfone 4 were synthesized. These novel compounds were evaluated as elastase inhibitors using porcine pancreatic elastase. The effects that structural modifications of substituents on C-3 and C-6 in the penam nucleus have on elastase activity were examined and several similarities and distinctions were identified when compared to the reported penicillin esters and amides elastase inhibitors.

Synthesis and porcine pancreatic elastase inhibitory evaluation of 6α-(sulfonyl)oxy-and 6α-chloropenicillanate sulfone esters and 3α-(acyloxy)methyl-6α-chloropenam sulfones

Abstract The synthesis of 6α-chloropenicillanate sulfone esters 4a-c, 9, the acetate and benzoate of 3α-hydroxymethyl-6α-chloropenam sulfones 6a-b and pivaloyloxymethyl and benzyl esters of several 6α-(sulfonyl)oxypenicillanate sulfones 12, 15a1-a3, 15b1-b3 are reported. When tested as inhibitors of porcine pancreatic elastase, the acetate of 3α-hydroxymethylpenam 6a proved to be more active in comparison with the esters of 3α-carboxylic acid counterparts 4a-c and 9. Compounds with diverse 6α-(sulfonyl)oxy substituents showed elastase inhibitory activity improved over the corresponding 6α-chloro derivatives 4a-c and 9; among those, compounds 15a2 and 15b2 were rather unstable, but compounds 15a1, 15a3, 15b1, 15b3 combined fair activity with better stability.