Metabolic Resistance: The Protection of Enzymes against Drugs which are Tight‐Binding Inhibitors by the Accumulation of Substrate

Abstract

Blockade of a metabolic pathway by interaction of a drug with a particular 'target enzyme' results in depletion of essential end-products of the pathway and accumulation of intermediates prior to the blockade. Metubolic resistance to a particular drug can arise if the substrate of the inhibited enzyme accumulates to levels sufficiently high to compete effectively with the inhibitor, leading to restoration of full activity of the metabolic pathway after a transitory delay.Such resistance has recently been demonstrated in vitro for the interaction of the tight-binding inhibitor N-phosphonacetyl-L-aspartate (PAcAsp) with the aspartate transcarbamoylase activity of the trifunctional protein which initiates pyrimidine biosynthesis in mammals [Christopherson, R. I. and Jones, M. E. (1980) J . Biol. Chem. 255, 11 381 -11 3951. Carbamoyl phosphate, the product of the carbamoyl phosphate synthetase activity of this trifunctional protein, accumulates to a sufficiently high concentration that the inhibitory effect of PAcAsp is effectively abolished. We have developed a theoretical model for metabolic resistance which quantitatively accounts for these experimental data. This model has been used to simulate the interaction between the following potential or proven anti-cancer drugs and their target enzyme, under conditions similar to those which would occur in vivo: PAcAsp with aspartate transcarbamoylase; various OMP analogues [the 5'-monophosphates of 6-azauridine, pyrazofurin and I-(P-D-ribofuranosy1)-barbituric acid] with OMP decarboxylase; 5-fluorodeoxyUMP with thymidylate synthase; methotrexate with dihydrofolate reductase; and deoxycoformycin with adenosine deaminase.

Assessment of Mycobacterium tuberculosis Pantothenate Kinase Vulnerability through Target Knockdown and Mechanistically Diverse Inhibitors

“…These data clearly indicate that with lowered expression levels, the inhibitor is able to engage the target and bring about M. tuberculosis growth inhibition, suggesting that MtPanK is poorly or weakly vulnerable. The observations reported here match earlier reports in E. coli (26)(27)(28), where it was shown that an adverse effect on bacterial growth required Ͼ95% enzyme inhibition against a few enzymes of the CoA biosynthetic pathway, sustained over several generations.…”

“…According to earlier reports, a reduction in PanK activity of more than 95% was required to achieve growth inhibition since the steady-state levels of CoA are far in excess of what is critical for cell survival (24,27,28). Results from the current study are in concurrence with these earlier observations and were further substantiated by the inability to recover colonies on solid me-dium.…”

Cloning and Expression of Malarial Pyrimidine Enzymes

“…[4] We showed that exposure of P. falciparum in erythrocytic culture to TDHO or atovaquone induces accumulation of N-carbamyl-L-aspartate (CA-asp), and CA-asp and L-dihydroorotate (DHO), respectively, [2] with depletion of dTTP but not dCTP [5] resulting in inhibition of DNA synthesis and antimalarial activity. Combination chemotherapy with several potent inhibitors of the pyrimidine pathway would overcome metabolic resistance [6] which results from accumulation of the substrate for an inhibited enzyme to levels sufficient to out-compete the inhibitor. HDDP and TDHO are effective inhibitors of DHOase, but more potent inhibitors will be required for clinical antimalarial activity.…”

Metabolic resistance to tight-binding inhibitors of enzymes involved in the de novo pyrimidine pathway Simulation of time-dependent effects. Simulation of time-dependent effects

“…The accumulating substrate(s) can reach a concentration which is sufficiently high that the inhibition is effectively reversed, with restoration of the original flux through the inhibited reaction. We have recently developed a theoretical model for this phenomenon which we have called 'metabolic resistance' [R. I. Christopherson and R. G. Duggleby (1983) This simulation shows that on addition of BMP, there is a sequential depletion of all pyrimidine intermediates between UMP and dCDP and a concomitant accumulation of OMP. Eventually, OMP reaches a new steady-state concentration and the concentrations of the depleted intermediates rise to their original levels.…”

“…The accumulating substrate(s) can reach a concentration which is sufficiently high that the inhibition is effectively reversed, with restoration of the original flux through the inhibited reaction. We have recently developed a theoretical model for this phenomenon which we have called 'metabolic resistance' [R. I. Christopherson and R. G. Duggleby (1983) Eur. J .…”