Network Architecture Predisposes an Enzyme to Either Pharmacologic or Genetic Targeting

Abstract

SUMMARY Chemical inhibition and genetic knockdown of enzymes are not equivalent in cells, but network-level mechanisms that cause discrepancies between knockdown and inhibitor perturbations are not understood. Here we report that enzymes regulated by negative feedback are robust to knockdown but susceptible to inhibition. Using the Raf–MEK–ERK kinase cascade as a model system, we find that ERK activation is resistant to genetic knockdown of MEK but susceptible to a comparable degree of chemical MEK inhibition. We demonstrate that negative feedback from ERK to Raf causes this knockdown-versus-inhibitor discrepancy in vivo. Exhaustive mathematical modeling of three-tiered enzyme cascades suggests that this result is general: negative autoregulation or feedback favors inhibitor potency, whereas positive autoregulation or feedback favors knockdown potency. Our findings provide a rationale for selecting pharmacologic versus genetic perturbations in vivo and point out the dangers of using knockdown approaches in search of drug targets.

Dual-specificity phosphatase 5 controls the localized inhibition, propagation, and transforming potential of ERK signaling

“…4 and 6). The notion that DUSP5 (and likely other MKP/DUSPs) should mediate effects through 'feedback relief' is perhaps to be expected in light of the number of studies demonstrating that pharmacological inhibitors of the ERK pathway cause similar effects (25,30,36,37,45,46). Our results indicate that the nuclear sequestration of inactive ERK by DUSP5 evokes compensatory increases in upstream RAF and MEK activation through feedback relief ( Fig.…”

“…A major mechanism by which ERK self-limits its activity is through direct phosphorylation and inhibition of upstream components, such as RAS exchange factors (7), RTKs (8,9), and RAF kinases, which prevent RAS-mediated RAF dimerization and activation (4)(5)(6). This rapid, negative feedback confers homeostatic control, such that pharmacological inhibition or a reduction in total ERK concentration causes strong compensatory increases in upstream kinase activity, which acts to maintain near constant p-ERK output levels (30,36,37). We therefore hypothesized that the nuclear sequestration and inactivation of ERK by DUSP5 may cause similar compensatory increases in upstream kinase activity, and that this may explain why cytoplasmic p-ERK levels are maintained or even prolonged in the presence of DUSP5.…”

“…S5A, and Table S1; SI Materials and Methods). Previous studies indicate that ERK-mediated inhibition of RAF is chiefly responsible for homeostatic control of the ERK cascade, and is highly nonlinear in nature (30,36,37). We therefore modeled the relationship between ERK* and K* as highly cooperative by using a Hill function with a Hill coefficient of 4 (Table S1; SI Materials and Methods).…”

“…The oncogenic V600E substitution in BRAF causes constitutive kinase activation, irrespective of RAS activation or RAF dimerization status (5). As such, BRAF V600E is refractory to feedback inhibition by ERK (26,30,36,37). In contrast, RAS mut oncoproteins constitutively activate the ERK pathway, but leave ERK-RAF negative feedback intact.…”

The complexities and versatility of the RAS-to-ERK signalling system in normal and cancer cells

“…This negative loop (i) speeds up the response time, (ii) converts intrinsic switch-like activation kinetics into graded linear responses, (iii) conveys robustness to changes in rates of reactions within the NFA module, (iv) stabilises outputs in response to drug-induced perturbations, and also has therapeutic implications ( Figure 4). It was shown that the ERK negative feedback diminishes the effectiveness of RAF and MEK inhibitors [107], but also that the drug-effect is quite different depending on the inhibitor's mechanism of action [108]. In negative feedback configurations, competitive inhibitors tend to be more effective compared to gene knockdowns [108].…”