Application of ProTide technology to Gemcitabine: A successful approach to overcome the key cancer resistance mechanisms leads to a new agent (NUC-1031) in clinical development

Gemcitabine is a nucleoside analogue commonly used in cancer therapy but with limited efficacy due to a high susceptibility to cancer cell resistance. The addition of a phosphoramidate motif to the gemcitabine can protect it against many of the key cancer resistance mechanisms. We have synthesized a series of gemcitabine phosphoramidate prodrugs and screened for cytostatic activity in a range of different tumor cell lines. Among the synthesized compounds, one in particular (NUC-1031, 6f) was shown to be potent in vitro. Importantly, compared with gemcitabine, 6f activation was significantly less dependent on deoxycytidine kinase and on nucleoside transporters, and it was resistant to cytidine deaminase-mediated degradation. Moreover, 6f showed a significant reduction in tumor volumes in vivo in pancreatic cancer xenografts. The ProTide 6f is now in clinical development with encouraging efficacy signals in a Phase I/II study, which strongly supports the ProTide approach to generate promising new anticancer agents

Regulation of the IKs current by PKA phosphorylation

The IKs current, which is composed of KCNQ1 and KCNE1 subunits, increases in size as a result of beta-adrenergic stimulation. This response is a result of the KCNQ1 subunit being phosphorylated by protein kinase A (PKA). This is an important physiological response at high heart rates that allows the ventricles adequate time to fill. Mutations in either of these subunits can cause impaired cardiac repolarization and result in long and short QT syndromes, as well as familial atrial fibrillation. The mechanism by which the channel complex reacts to this stimulation has not been fully elucidated. To investigate the mechanism behind this, both total internal reflection florescence microscopy (TIRF) and single-channel recording were used. 8-CPT-cAMP, a membrane-permeant analog of cAMP, was used to induce PKA phosphorylation of KCNQ1. TIRF studies found no significant change in the number of channels at the cell surface. Single-channel recordings of IKs had a reduced first latency to opening showing that the channel opened more quickly in response to 8-CPT-cAMP. The IKs current has multiple open states and, in the presence of 8-CPT-cAMP, occupied the higher subconducting states more frequently. An increase in the open probability of the channel complex was also seen. In response to phosphorylation triggered by 8-CPT-cAMP, the first latency to opening of the channel is reduced and the channel opens more quickly, more often and passes more current by increasing the higher conducting open states. This results in an increase in IKs current at the macroscopic level. Using enhanced gating mutant KCNQ1 channels, it is shown that the effect of phosphorylation is likely through further activation of the voltage sensor. KCNE1 is required for a functional response to PKA phosphorylation. Both whole-cell and single-channel recordings show that as the number of KCNE1 subunits is reduced, a graded effect is seen in response to 8-CPT-cAMP; the less KCNE1 subunits, the smaller the response. However, in single-channel recordings, there was also a KCNE1-independent effect of phosphorylation as the first latencies for all KCNQ1-KCNE1 complexes were reduced in a non-graded manner. This suggests that phosphorylation may have both KCNE1-dependent and independent effects on the channel.Medicine, Faculty ofAnesthesiology, Pharmacology and Therapeutics, Department ofGraduat

cAMP-dependent regulation of IKs single-channel kinetics

International audienceThe delayed potassium rectifier current, IKs, is composed of KCNQ1 and KCNE1 subunits and plays an important role in cardiac action potential repolarization. During β-adrenergic stimulation, 3′-5′-cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) phosphorylates KCNQ1, producing an increase in IKs current and a shortening of the action potential. Here, using cell-attached macropatches and single-channel recordings, we investigate the microscopic mechanisms underlying the cAMP-dependent increase in IKs current. A membrane-permeable cAMP analog, 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP), causes a marked leftward shift of the conductance–voltage relation in macropatches, with or without an increase in current size. Single channels exhibit fewer silent sweeps, reduced first latency to opening (control, 1.61 ± 0.13 s; cAMP, 1.06 ± 0.11 s), and increased higher-subconductance-level occupancy in the presence of cAMP. The E160R/R237E and S209F KCNQ1 mutants, which show fixed and enhanced voltage sensor activation, respectively, largely abolish the effect of cAMP. The phosphomimetic KCNQ1 mutations, S27D and S27D/S92D, are much less and not at all responsive, respectively, to the effects of PKA phosphorylation (first latency of S27D + KCNE1 channels: control, 1.81 ± 0.1 s; 8-CPT-cAMP, 1.44 ± 0.1 s, P < 0.05; latency of S27D/S92D + KCNE1: control, 1.62 ± 0.1 s; cAMP, 1.43 ± 0.1 s, nonsignificant). Using total internal reflection fluorescence microscopy, we find no overall increase in surface expression of the channel during exposure to 8-CPT-cAMP. Our data suggest that the cAMP-dependent increase in IKs current is caused by an increase in the likelihood of channel opening, combined with faster openings and greater occupancy of higher subconductance levels, and is mediated by enhanced voltage sensor activation

Application of ProTide technology to gemcitabine: A successful approach to overcome the key cancer resistance mechanisms leads to a new agent (NUC-1031) in clinical development

Gemcitabine is a nucleoside analogue commonly used in cancer therapy but with limited efficacy due to a high susceptibility to cancer cell resistance. The addition of a phosphoramidate motif to the gemcitabine can protect it against many of the key cancer resistance mechanisms. We have synthesized a series of gemcitabine phosphoramidate prodrugs and screened for cytostatic activity in a range of different tumor cell lines. Among the synthesized compounds, one in particular (NUC-1031, 6f) was shown to be potent in vitro. Importantly, compared with gemcitabine, 6f activation was significantly less dependent on deoxycytidine kinase and on nucleoside transporters, and it was resistant to cytidine deaminase-mediated degradation. Moreover, 6f showed a significant reduction in tumor volumes in vivo in pancreatic cancer xenografts. The ProTide 6f is now in clinical development with encouraging efficacy signals in a Phase I/II study, which strongly supports the ProTide approach to generate promising new anticancer agents.status: publishe

Application of ProTide Technology to Gemcitabine: A Successful Approach to Overcome the Key Cancer Resistance Mechanisms Leads to a New Agent (NUC-1031) in Clinical Development

Gemcitabine is a nucleoside analogue commonly used in cancer therapy but with limited efficacy due to a high susceptibility to cancer cell resistance. The addition of a phosphoramidate motif to the gemcitabine can protect it against many of the key cancer resistance mechanisms. We have synthesized a series of gemcitabine phosphoramidate prodrugs and screened for cytostatic activity in a range of different tumor cell lines. Among the synthesized compounds, one in particular (NUC-1031, <b>6f</b>) was shown to be potent <i>in vitro</i>. Importantly, compared with gemcitabine, <b>6f</b> activation was significantly less dependent on deoxycytidine kinase and on nucleoside transporters, and it was resistant to cytidine deaminase-mediated degradation. Moreover, <b>6f</b> showed a significant reduction in tumor volumes <i>in vivo</i> in pancreatic cancer xenografts. The ProTide <b>6f</b> is now in clinical development with encouraging efficacy signals in a Phase I/II study, which strongly supports the ProTide approach to generate promising new anticancer agents

Application of ProTide Technology to Gemcitabine: A Successful Approach to Overcome the Key Cancer Resistance Mechanisms Leads to a New Agent (NUC-1031) in Clinical Development

Gemcitabine is a nucleoside analogue commonly used in cancer therapy but with limited efficacy due to a high susceptibility to cancer cell resistance. The addition of a phosphoramidate motif to the gemcitabine can protect it against many of the key cancer resistance mechanisms. We have synthesized a series of gemcitabine phosphoramidate prodrugs and screened for cytostatic activity in a range of different tumor cell lines. Among the synthesized compounds, one in particular (NUC-1031, 6f) was shown to be potent in vitro. Importantly, compared with gemcitabine, 6f activation was significantly less dependent on deoxycytidine kinase and on nucleoside transporters, and it was resistant to cytidine deaminase-mediated degradation. Moreover, 6f showed a significant reduction in tumor volumes in vivo in pancreatic cancer xenografts. The ProTide 6f is now in clinical development with encouraging efficacy signals in a Phase I/II study, which strongly supports the ProTide approach to generate promising new anticancer agents