ABCMdb

PMID: 18945216

Pasyk S, Li C, Ramjeesingh M, Bear CE
Direct interaction of a small-molecule modulator with G551D-CFTR, a cystic fibrosis-causing mutation associated with severe disease.
Biochem J. 2009 Feb 15;418(1):185-90., 2009-02-15 [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp CFTR is a member of the ABC (ATP-binding-cassette) superfamily of membrane proteins and a disease-causing missense mutation within the ABC signature sequence; G551D-CFTR exhibits defective phosphorylation and ATP-dependent channel gating. Login to comment 2 ABCC7 p.Gly551Asp Studies of the purified and reconstituted G551D-CFTR protein revealed that faulty gating is associated with defective ATP binding and ATPase activity, reflecting the key role of G551 in these functions. Login to comment 3 ABCC7 p.Gly551Asp Recently, high-throughput screens of chemical libraries led to identification of modulators that enhance channel activity of G551D-CFTR. Login to comment 6 ABCC7 p.Gly551Asp First, we confirmed that VRT-532 causes a significant increase in channel activity of G551D-CFTR using a novel assay of CFTR function in inside-out membrane vesicles. Login to comment 7 ABCC7 p.Gly551Asp Biochemical studies of purified and reconstituted G551D-CFTR revealed that potentiation of the ATPase activity of VRT-532 is mediated by enhancing the affinity of the mutant for ATP. Login to comment 9 ABCC7 p.Gly551Asp To summarize, these studies provide direct evidence that this compound binds to G551D-CFTR to rescue its specific defect in ATP binding and hydrolysis. Login to comment 15 ABCC7 p.Gly551Asp The CF-causing missense mutation G551D-CFTR is associated with severe disease [2]. Login to comment 16 ABCC7 p.Gly551Asp Unlike the more common F508-CFTR, G551D-CFTR is not mistrafficked and exhibits normal expression on the cell surface [1]. Login to comment 19 ABCC7 p.Gly551Asp Therefore it has been suggested that the disease-causing mutation G551D will interfere with ATP binding, NBD1 and NBD2 heterodimerization and the ATPase activity that results from this domain-domain interaction [9]. Login to comment 20 ABCC7 p.Gly551Asp In fact, we showed in studies of purified and reconstituted G551D-CFTR that it is severely defective as an ATPase [10,11]. Login to comment 23 ABCC7 p.Gly551Asp A subgroup of these molecules is effective in modulating the functional expression of two disease-causing mutants: F508-CFTR and G551D-CFTR [13]. Login to comment 24 ABCC7 p.Gly551Asp As we have optimized the purification and functional reconstitution of G551D-CFTR, we have an important reagent with which to evaluate whether certain of these pharmacological compounds act directly to modify the mutant CFTR protein. Login to comment 27 ABCC7 p.Gly551Asp EXPERIMENTAL Vesicle-based flux assay of Wt (wild-type) CFTR and G551D-CFTR function The measurement of iodide efflux from membrane vesicles was adapted from a previously described cellular iodide efflux method [14]. Login to comment 38 ABCC7 p.Gly551Asp Purification and reconstitution of Wt CFTR and G551D-CFTR Detailed protocols regarding the generation of Wt and mutant CFTR-His proteins are described elsewhere [15]. Login to comment 44 ABCC7 p.Gly551Asp ATPase assay of purified Wt CFTR and G551D-CFTR protein ATPase activity was measured as the production of [γ -32 P]Pi from [γ -32 P]ATP as described by Gross et al. [17a]. Login to comment 51 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp RESULTS A novel iodide efflux assay reports potentiating activity of VRT-532 on G551D-CFTR VRT-532 has been shown to potentiate cAMP-activated chloride ion flux through G551D-CFTR in Fischer rat thyroid epithelial cells grown in monolayers and studied in voltage-clamp experiments conducted in the Ussing chamber apparatus [13]. Login to comment 52 ABCC7 p.Gly551Asp The increase in chloride flux described above could report changes in the function of other membrane proteins that have an impact on the driving force for chloride ion flux through G551D-CFTR rather than changes in the intrinsic activity of the mutant protein. Login to comment 54 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Therefore, as the first step towards understanding the mechanism of action of this small molecule in G551D-CFTR function, we were prompted to re-evaluate the effect of this modulator on the channel activity of G551D-CFTR in a more simplified system. Login to comment 55 ABCC7 p.Gly551Asp Typically, activation of the channel function of CFTR and CF-causing mutants (including G551D-CFTR) by membrane-permeable agonists of cAMP-dependent PKA has been studied in intact cells using a convenient method known as the iodide efflux assay [18-20]. Login to comment 58 ABCC7 p.Gly551Asp At the other end of the spectrum, patch-clamp studies of single channels in isolated membrane patches provide exquisite resolution of the kinetics of channel gating; however, as the channel activity of certain mutants, including G551D-CFTR, is severely impaired, acquisition of sufficient data to draw conclusions regarding the pharmacological effects of interesting compounds is time-consuming and often limited to a small number of channel proteins. Login to comment 60 ABCC7 p.Gly551Asp Vesicles were prepared from Sf9 cells infected with baculovirus containing cDNA coding for Wt CFTR or G551D-CFTR using methods that have been described previously [21]. Login to comment 78 ABCC7 p.Gly551Asp Figure 2 shows an example of the regulated efflux from membrane vesicles expressing G551D-CFTR. Login to comment 81 ABCC7 p.Gly551Asp VRT-532 directly binds reconstituted G551D-CFTR and modifies its ATPase activity It has been shown that ATP-dependent heterodimerization of the two NBDs of CFTR is required for their activity as an ATPase and is permissive for subsequent conformational changes leading to opening of the chloride channel gate [22-24]. Login to comment 82 ABCC7 p.Gly551Asp Further, it has been suggested that the failure of G551D-CFTR to bind ATP accounts for its defective ATPase activity and channel gating [5]. Login to comment 83 ABCC7 p.Gly551Asp These findings prompted us to test the hypothesis that VRT-532 potentiates channel opening of G551D-CFTR by directly binding to enhance ATP affinity and hydrolysis. Login to comment 84 ABCC7 p.Gly551Asp As previously determined, purified and reconstituted G551D-CFTR exhibits a very low level of ATPase activity relative to the Wt protein (0.1 +- 0.1 versus 1.4 +- 0.1 nmol·μg·h-1 respectively) in the presence of 1 mM MgATP [15]. Login to comment 88 ABCC7 p.Gly551Asp Although the ATPase activity of G551D-CFTR was significantly enhanced by the addition of VRT-532, the activity of the treated mutant protein failed to achieve the level of activity exhibited by the Wt CFTR protein (measured at 1 mM MgATP) as shown in Figure 4(A). Login to comment 90 ABCC7 p.Gly551Asp In Figure 5 and Table 1, the apparent ATP dependence of the ATPase activity exhibited by G551D-CFTR after treatment with VRT-532 (5 μM) is shown. Login to comment 91 ABCC7 p.Gly551Asp In contrast with the dose response obtained for untreated G551D-CFTR, which failed to exhibit saturable ATP dependence, the data corresponding to the VRT-532-treated protein could be fitted using a Michaelis-Menten function (r2 = 0.9). Login to comment 92 ABCC7 p.Gly551Asp These findings suggest that VRT-532 acts to enhance the ATPase activity of G551D-CFTR by increasing the apparent affinity for ATP. Login to comment 93 ABCC7 p.Gly551Asp Figure 2 VRT-532 enhances channel activity in G551D-CFTR (A)Asexpected,iodideeffluxmediatedbyvesiclesexpressingG551D-CFTRwasnotsignificantly enhanced by PKA and MgATP plus valinomycin. Login to comment 96 ABCC7 p.Gly551Asp (B) The histogram compares iodide efflux rates of membrane vesicles containing G551D-CFTR treated with MgATP (1 mM) and PKA (200 nM) before and after VRT-532 treatment (n = 5). Login to comment 101 ABCC7 p.Gly551Asp In these 'proof of principle` studies, we revealed the direct action of one such compound, VRT-532, to enhance the ATP affinity of the CF-causing mutant, G551D-CFTR. Login to comment 102 ABCC7 p.Gly551Asp As previously mentioned, VRT-532 is a particularly interesting small-molecule modulator as it enhances the channel activity of at least two different CFTR genotypes, including F508 and G551D-CFTR, suggesting that it may have a general role in repairing the molecular defects in multiple disease variants [13]. Login to comment 106 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Therefore the findings of the present Figure 3 VRT-532 enhances intrinsic ATPase activity of G551D-CFTR (A) ATPase activity of purified, reconstituted and phosphorylated G551D-CFTR measured as the production of radioactive Pi from radioactive ATP. Login to comment 109 ABCC7 p.Gly551Asp (B) The activation of G551D-CFTR is dependent on the dose of VRT-532, and this relationship can be fitted with a sigmoidal function (r2 = 0.94), yielding an EC50 of 2.7 μM (mean of duplicate measurements, from two different protein preparations; bars indicate range). Login to comment 110 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Figure 4 VRT-532 significantly enhances the ATPase activity of G551D-CFTR but not Wt CFTR (A) VRT-532 (5 μM) significantly enhances ATPase activity of G551D-CFTR in the presence of 500 μM MgATP (n = 3, P < 0.05) but this activity stimulated by G551D-CFTR is less than the activity of the Wt protein measured at the same MgATP concentration, as indicated by the broken line [15]. Login to comment 112 ABCC7 p.Gly551Asp study suggest that VRT-532 directly binds to purified reconstituted G551D-CFTR to modulate its activity, providing the impetus for future studies to determine whether it binds directly to F508-CFTR and to define the site for binding on these two mutants. Login to comment 113 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Both the chloride channel gating and ATPase functions of CFTR likely report multiple dynamic conformational changes occurring throughout the whole molecule and, hence, the specific site at 5 VRT-532 enhances the ATPase activity of G551D-CFTR by increasing the apparent affinity for ATP The graph shows the effect of VRT-532 (5 μM) on ATP dose dependence of the ATPase activity of G551D-CFTR. Login to comment 124 ABCC7 p.Gly551Asp Since the VRT-532 compound exerts a profound effect on ATPase activity by the G551D-CFTR protein rather than on the Wt protein, we speculate that it may be binding to or indirectly stabilizing an intramolecular interaction which is important for ATPase activity and is altered in the mutant protein. Login to comment 128 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp We are currently optimizing the purification and reconstitution of Table 1 Kinetic parameters obtained from analyses of ATP dependence of the ATPase activity of Wt CFTR and VRT-532-treated G551D-CFTR The ATPase activity of G551D-CFTR cannot be fitted using the Michaelis-Menten equation (see Figure 5 and [10,15]). Login to comment 131 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Protein Vmax (nmol · μg · h-1 ) Km ATP (mM) CFTR [10,15] 4.6 0.7 G551D-CFTR N.A. NA G551D-CFTR + VRT-532 1.6 3.6 the major CF mutant for the purpose of testing this hypothesis directly. Login to comment 132 ABCC7 p.Gly551Asp While G551D-CFTR is not a common mutation in the CF patient population, it does account for disease in approx. Login to comment 134 ABCC7 p.Gly551Asp Hence, the insights regarding the molecular mechanisms for functional repair of the G551D-CFTR mutant by the small molecule, VRT-532, generated in the present study will provide the template for future drug development and potentially improvement in the clinical care of these severely affected patients. Login to comment 135 ABCC7 p.Gly551Asp Currently, a clinical trial is in progress that is investigating the efficacy of a different molecule (VX-770) in patients bearing the G551D-CFTR mutation (http://www.cff.org/research/ClinicalResearch/ FAQs/VX-770). Login to comment