The cystic fibrosis mutation G551D alters the non-Michaelis-Menten behavior of the cystic fibrosis transmembrane conductance regulator (CFTR) channel and abolishes the inhibitory Genistein binding site

Loss of cystic fibrosis transmembrane conductance regulator (CFTR) channel activity explains most of the manifestations of the cystic fibrosis (CF) disease. To understand the consequences of CF mutations on CFTR channel activity, we compared the pharmacological properties of wild-type (wt) and G551D-CFTR. Dose-dependent relationships of wt-CFTR activated by genistein follows a non-Michaelis-Menten behavior consistent with the presence of two binding sites. With phosphorylated CFTR, a high affinity site for genistein is the activator (K(s) approximately 3 microm), whereas a second site of low affinity (K(i) approximately 75 microm) is the inhibitor. With non-phosphorylated CFTR, K(s) was increased (K(s) approximately 12 microm), but K(i) was not affected (K(i) approximately 70 microm). In G551D-CFTR cells, channel activity was recovered by co-application of forskolin and genistein in a dose-dependent manner. A further stimulation of G551D-CFTR channel activity was measured at concentrations from 30 microm to 1 mm. The dose response is described by a classical Michaelis-Menten kinetics with only a single apparent site (K(m) approximately 11 microm). Our results suggest glycine 551 in NBD1 as an important location within the low affinity inhibitory site for genistein and offers new evidence for pharmacological alteration caused by an NBD1 mutation of CFTR. This study also reveals how a mutation of an ion channel converts a non-Michaelis-Menten behavior (two binding sites) into a classical Michaelis-Menten model (one binding site).     

The noncatalytic beta-propeller domain of prolyl oligopeptidase enhances the catalytic capability of the peptidase domain

Prolyl oligopeptidase, which is involved in memory disorders, is a member of a new family of serine peptidases. In addition to the peptidase domain, the enzyme contains a beta-propeller, which excludes large peptides from the active site. The enzyme is inhibited with thiol reagents, possibly by reacting with Cys-255 located close to the substrate binding site. This assumption was tested with the Cys-255 --> Thr, Cys-255 --> Ala, and Cys-255 --> Ser variants of prolyl oligopeptidase. In contrast to the wild type enzyme, the Cys-255 --> Thr variant was not inhibited with N-ethylmaleimide, indicating that Cys-255, of the 16 free cysteine residues, exclusively accounts for the enzyme inhibition. Unlike the wild type enzyme that showed a doubly bell-shaped pH rate profile, the modified enzyme displayed a single bell-shaped pH dependence with benzyloxycarbonyl-Gly-Pro-naphthylamide. It was the high pH form of the enzyme that virtually disappeared with all three enzyme variants. A substantial reduction was also observed in k(cat)/K(m) for the aminobenzoyl-Ser-Pro-Phe(NO(2))-Ala-OH substrate. The high pK(a) (9.77) of Cys-255 determined by titration with N-ethylmaleimide excluded the possibility that ionization of the thiol group was responsible for generation of the two active enzyme forms. The impaired activity of the enzyme variants could be rationalized in terms of weaker binding, which manifests itself in high K(m) for substrates and high K(i) for inhibitors, like benzyloxycarbonyl-Gly-Pro-OH and aminobenzoyl-Ser-d-Pro-Phe(NO(2))-Ala-OH. It was concluded that, besides selecting substrates by size, the beta-propeller domain containing Cys-255 remarkably contributed to catalysis of the peptidase domain.     

Optimization of the Degenerated Interfacial ATP Binding Site Improves the Function of Disease-related Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, an ATP binding cassette (ABC) protein whose defects cause the deadly genetic disease cystic fibrosis (CF), encompasses two nucleotide binding domains (NBD1 and NBD2). Recent studies indicate that in the presence of ATP, the two NBDs coalesce into a dimer, trapping an ATP molecule in each of the two interfacial composite ATP binding sites (site 1 and site 2). Experimental evidence also suggests that CFTR gating is mainly controlled by ATP binding and hydrolysis in site 2, whereas site 1, which harbors several non-canonical substitutions in ATP-interacting motifs, is considered degenerated. The CF-associated mutation G551D, by introducing a bulky and negatively charged side chain into site 2, completely abolishes ATP-induced openings of CFTR. Here, we report a strategy to optimize site 1 for ATP binding by converting two amino acid residues to ABC consensus (i.e. H1348G) or more commonly seen residues in other ABC proteins (i.e. W401Y,W401F). Introducing either one or both of these mutations into G551D-CFTR confers ATP responsiveness for this disease-associated mutant channel. We further showed that the same maneuver also improved the function of WT-CFTR and the most common CF-associated ΔF508 channels, both of which rely on site 2 for gating control. Thus, our results demonstrated that the degenerated site 1 can be rebuilt to complement or support site 2 for CFTR function. Possible approaches for developing CFTR potentiators targeting site 1 will be discussed.     

Functional analysis of mutations in the putative binding site for cystic fibrosis transmembrane conductance regulator potentiators. Interaction between activation and inhibition

An increasing number of compounds able to potentiate the activity of mutants of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have been identified by high throughput screening or by individual search of derivatives of known active compounds. Several lines of evidence suggest that most CFTR potentiators act through the same mechanism, probably by binding to the nucleotide binding domains to promote the activity of the protein and then, with lower affinity, to an inhibitory site. With the aim of identifying the activating binding site, we recently modeled the nucleotide binding domain dimer and predicted a common binding site for potentiators in its interface. To validate this model experimentally, we mutated some of the residues involved in the putative binding site, i.e. Arg(553), Ala(554), and Val(1293). The activity of CFTR potentiators was measured as apical membrane currents on polarized cells stably expressing wild type or mutated proteins. CFTR activity was elicited by application of a membrane-permeable cAMP analogue followed by increasing concentrations of potentiators. We found that all three mutants responded to cAMP, although the affinity of R553Q was higher than that of wild type CFTR. In R553Q and V1293G mutants, the dissociation constant of potentiators for the activating site was increased, whereas the dissociation constant for the inhibitory site was reduced. Our results show that the mutated residues are part of the activating binding site for potentiators, as suggested by the molecular model. In addition, these results suggest that the activating and inhibitory sites are not independent of each other.     

Model of the cAMP activation of chloride transport by CFTR channel and the mechanism of potentiators

Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis, a hereditary lethal disease. CFTR is a chloride channel expressed in the apical membrane of epithelia. It is activated by cAMP dependent phosphorylation and gated by the binding of ATP. The impaired chloride transport of some types of cystic fibrosis mutations could be pharmacologically solved by the use of chemical compounds called potentiators. Here it is undertaken the construction of a model of the CFTR activation pathways, and the possible modification produced by a potentiator application. The model yields a novel mechanism for the potentiator action, describing the activatory and inhibitory activities on two different positions in the CFTR activation pathway.     

Design of Crotoxin-Based Peptides with Potentiator Activity Targeting the ΔF508NBD1 Cystic Fibrosis Transmembrane Conductance Regulator

We have previously shown that the CBb subunit of crotoxin, a β-neurotoxin with phospholipase A₂ (PLA₂) activity, targets the human ΔF508CFTR chloride channel implicated in cystic fibrosis (CF). By direct binding to the nucleotide binding domain 1 (NBD1) of ΔF508CFTR, this neurotoxic PLA₂ acts as a potentiator increasing chloride channel current and corrects the trafficking defect of misfolded ΔF508CFTR inside the cell.Here, for a therapeutics development of new anti-cystic fibrosis agents, we use a structure-based in silico approach to design peptides mimicking the CBb-ΔF508NBD1 interface. Combining biophysical and electrophysiological methods, we identify several peptides that interact with the ΔF508NBD1 domain and reveal their effects as potentiators on phosphorylated ΔF508CFTR. Moreover, protein-peptide interactions and electrophysiological studies allowed us to identify key residues of ΔF508NBD1 governing the interactions with the novel potentiators. The designed peptides bind to the same region as CBb phospholipase A₂ on ΔF508NBD1 and potentiate chloride channel activity. Certain peptides also show an additive effect towards the clinically approved VX-770 potentiator. The identified CF therapeutics peptides represent a novel class of CFTR potentiators and illustrate a strategy leading to reproducing the effect of specific protein–protein interactions.     

Potentiation of ABCA3 lipid transport function by ivacaftor and genistein

ABCA3 is a phospholipid transporter implicated in pulmonary surfactant homoeostasis and localized at the limiting membrane of lamellar bodies, the storage compartment for surfactant in alveolar type II cells. Mutations in ABCA3 display a common genetic cause for diseases caused by surfactant deficiency like respiratory distress in neonates and interstitial lung disease in children and adults, for which currently no causal therapy exists. In this study, we investigated the effects of ivacaftor and genistein, two potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR), on ABCA3‐specific lipid transport function. Wild‐type (WT) and functional ABCA3 mutations N568D, F629L, G667R, T1114M and L1580P were stably expressed in A549 cells. Three‐dimensional modelling predicted functional impairment for all five mutants that was confirmed by in vitro experiments (all <14% of WT functional activity). Treatment with potentiators rescued the mutants N568D (up to 114% of WT), F629L (up to 47% of WT), and G667R (up to 60% of WT), the latter variation needing higher concentrations of genistein, showing reduced affinity of the potentiator to the mutant protein. Our results present a first proof that functional ABCA3 mutations are rescued by CFTR potentiators, making them a potential therapeutical option for patients suffering from surfactant deficiency due to ABCA3 mutations.     

Evidence for two different classes of redox-active cysteines in ribonucleotide reductase of Escherichia coli

The large subunit of ribonucleotide reductase from Escherichia coli contains redox-active cysteine residues. In separate experiments, five conserved and 2 nonconserved cysteine residues were substituted with alanines by oligonucleotide-directed mutagenesis. The activities of the mutant proteins were determined in the presence of three different reductants: thioredoxin, glutaredoxin, or dithiothreitol. The results indicate two different classes of redox-active cysteines in ribonucleotide reductase: 1) C-terminal Cys-754 and Cys-759 responsible for the interaction with thioredoxin and glutaredoxin; and 2) Cys-225 and Cys-439 located at the nucleotide-binding site. Our classification of redox-active cysteines differs from the location of the active site cysteines in E. coli ribonucleotide reductase suggested previously (Lin, A.-N. I., Ashley, G. W., and Stubbe, J. (1987) Biochemistry 26, 6905-6909).     

Potentiation of ΔF508- and G551D-CFTR-Mediated Cl- Current by Novel Hydroxypyrazolines

The most common mutation of CFTR, affecting approximately 90% of CF patients, is a deletion of phenylalanine at position 508 (F508del, ΔF508). Misfolding of ΔF508-CFTR impairs both its trafficking to the plasma membrane and its chloride channel activity. To identify small molecules that can restore channel activity of ΔF508-CFTR, we synthesized and evaluated eighteen novel hydroxypyrazoline analogues as CFTR potentiators. To elucidate potentiation activities of hydroxypyrazolines for ΔF508-CFTR, CFTR activity was measured using a halide-sensitive YFP assay, Ussing chamber assay and patch-clamp technique. Compounds 7p, 7q and 7r exhibited excellent potentiation with EC₅₀ value <10 μM. Among the compounds, 7q (a novel CFTR potentiator, CP7q) showed the highest potentiation activity with EC₅₀ values of 0.88 ± 0.11 and 4.45 ± 0.31 μM for wild-type and ΔF508-CFTR, respectively. In addition, CP7q significantly potentiated chloride conductance of G551D-CFTR, a CFTR gating mutant; its maximal potentiation activity was 1.9 fold higher than the well-known CFTR potentiator genistein. Combination treatment with CP7q and VX-809, a corrector of ΔF508-CFTR, significantly enhanced functional rescue of ΔF508-CFTR compared with VX-809 alone. CP7q did not alter the cytosolic cAMP level and showed no cytotoxicity at the concentration showing maximum efficacy. The hydroxypyrazolines may be potential development candidates for drug therapy of cystic fibrosis.     

Characterization of a 7,8-Benzoflavone Double Effect on CFTR Cl- Channel Activity

The human cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the superfamily of adenosine triphosphate (ATP)-binding cassette (ABC) transporter ATPases. This protein forms a Cl^- channel with a complex regulation; gene mutations cause cystic fibrosis disease. We investigated the interaction between the protein and the flavone UCCF-029 using the patch-clamp technique in the excised inside-out configuration in order to study the molecular mechanism of action for this potentiator on completely phosphorylated channel (25 U/ml protein kinase A) and a relatively low level of ATP (0.3 mm). Low concentrations of UCCF-029 (<50 nm) increase the open probability (p _o), favoring the channel transition to an activated state, while high UCCF-029 (>50 nm) levels determine inhibition of the CFTR by a reduction of the total open time. Our data suggest that this drug can potentiate CFTR by binding to a specific site on the nucleotide binding domain, promoting dimer formation. The response of CFTR to variable concentrations of ATP is not modified by application of the potentiator UCCF-029 at either low, activatory, concentration or high, inhibitory, levels. Hence, we conclude that the potentiator may not interfere with binding of ATP but probably acts at an independent site in the protein, interacting directly with CFTR to modulate channel activity.     

Structure–activity analysis of a CFTR channel potentiator: Distinct molecular parts underlie dual gating effects

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette transporter superfamily that functions as an epithelial chloride channel. Gating of the CFTR ion conduction pore involves a conserved irreversible cyclic mechanism driven by ATP binding and hydrolysis at two cytosolic nucleotide-binding domains (NBDs): formation of an intramolecular NBD dimer that occludes two ATP molecules opens the pore, whereas dimer disruption after ATP hydrolysis closes it. CFTR dysfunction resulting from inherited mutations causes CF. The most common CF mutation, deletion of phenylalanine 508 (ΔF508), impairs both protein folding and processing and channel gating. Development of ΔF508 CFTR correctors (to increase cell surface expression) and potentiators (to enhance open probability, P_o) is therefore a key focus of CF research. The practical utility of 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), one of the most efficacious potentiators of ΔF508 CFTR identified to date, is limited by its pore-blocking side effect. NPPB-mediated stimulation of P_o is unique in that it involves modulation of gating transition state stability. Although stabilization by NPPB of the transition state for pore opening enhances both the rate of channel opening and the very slow rate of nonhydrolytic closure, because of CFTR’s cyclic gating mechanism, the net effect is P_o stimulation. In addition, slowing of ATP hydrolysis by NPPB delays pore closure, further enhancing P_o. Here we show that NPPB stimulates gating at a site outside the pore and that these individual actions of NPPB on CFTR are fully attributable to one or the other of its two complementary molecular parts, 3-nitrobenzoate (3NB) and 3-phenylpropylamine (3PP), both of which stimulate P_o: the pore-blocking 3NB selectively stabilizes the transition state for opening, whereas the nonblocking 3PP selectively slows the ATP hydrolysis step. Understanding structure–activity relationships of NPPB might prove useful for designing potent, clinically relevant CFTR potentiators.     

Targeting autophagy as a novel strategy for facilitating the therapeutic action of potentiators on ΔF508 cystic fibrosis transmembrane conductance regulator

Channel activators (potentiators) of cystic fibrosis (CF) transmembrane conductance regulator (CFTR), can be used for the treatment of the small subset of CF patients that carry plasma membrane-resident CFTR mutants. However, approximately 90% of CF patients carry the misfolded ΔF508-CFTR and are poorly responsive to potentiators, because ΔF508-CFTR is intrinsically unstable at the plasma membrane (PM) even if rescued by pharmacological correctors. We have demonstrated that human and mouse CF airways are autophagy deficient due to functional sequestration of BECN1 and that the tissue transglutaminase-2 inhibitor, cystamine, or antioxidants restore BECN1-dependent autophagy and reduce SQSTM1/p62 levels, thus favoring ΔF508-CFTR trafficking to the epithelial surface. Here, we investigated whether these treatments could facilitate the beneficial action of potentiators on ΔF508-CFTR homozygous airways. Cystamine or the superoxide dismutase (SOD)/catalase-mimetic EUK-134 stabilized ΔF508-CFTR at the plasma membrane of airway epithelial cells and sustained the expression of CFTR at the epithelial surface well beyond drug withdrawal, overexpressing BECN1 and depleting SQSTM1. This facilitates the beneficial action of potentiators in controlling inflammation in ex vivo ΔF508-CFTR homozygous human nasal biopsies and in vivo in mouse ΔF508-CFTR lungs. Direct depletion of Sqstm1 by shRNAs in vivo in ΔF508-CFTR mice synergized with potentiators in sustaining surface CFTR expression and suppressing inflammation. Cystamine pre-treatment restored ΔF508-CFTR response to the CFTR potentiators genistein, Vrx-532 or Vrx-770 in freshly isolated brushed nasal epithelial cells from ΔF508-CFTR homozygous patients. These findings delineate a novel therapeutic strategy for the treatment of CF patients with the ΔF508-CFTR mutation in which patients are first treated with cystamine and subsequently pulsed with CFTR potentiators.     

A quantitative description of the activation and inhibition of CFTR by potentiators: Genistein

The CFTR, encoded by the gene mutated in cystic fibrosis (CF) patients, is responsible for cAMP dependent chloride transport in epithelia. Substances that activate CFTR have been suggested as possible CF therapy. Most substances investigated so far exert a dual effect on the CFTR: low concentrations stimulate CFTR, whereas higher concentrations inhibit CFTR. Besides, the CFTR phosphorylation level determines the apparent affinity of the drug. We have studied the properties of genistein, the well known CFTR potentiator, by measuring apical membrane current on epithelia formed by cells stably transfected with CFTR and stimulated with different concentrations of CPTcAMP. We propose a quantitative model to describe the activatory and inhibitory effect of genistein, accounting also for the cAMP dependent activation.     

Introduction of the most common cystic fibrosis mutation (Delta F508) into human P-glycoprotein disrupts packing of the transmembrane segments

The most common mutation in cystic fibrosis (deletion of phenylalanine 508 (DeltaF508) in the cystic fibrosis conductance transmembrane regulator (CFTR) gene) causes defective synthesis of CFTR protein. To understand how this deletion interferes with protein folding, we made the equivalent deletion (DeltaY490) in P-glycoprotein (P-gp). A Cys-less P-gp with cysteines in transmembrane (TM) 4 or TM5 can be cross-linked with a cysteine in TM12. Deleting Tyr(490) in P-gp resulted in an inactive and defectively processed mutant in which no cross-linking between TM4 or TM5 and TM12 was detected. Expression of the DeltaY490 mutant in the presence of a chemical chaperone corrected the processing defect and yielded active P-gp mutants that could be cross-linked between TM4 or TM5 and TM12. Cross-linking between TM4 or TM5 and TM12 was also detected when residues (483)TIAENIRYG(491) in P-gp were replaced with residues (501)TIKENIIFG(509) from CFTR (P-gp/CFTR). Deleting Phe(508) in the P-gp/CFTR chimera, however, caused defective processing of the mutant protein and no detectable cross-linking between TM4 or TM5 and TM12. The processing defect was corrected with a chemical chaperone and yielded active P-gp/CFTR mutant proteins that could be cross-linked. These results show that deletion at residue 490 disrupts packing of the TM segments possibly by affecting interaction between the first nucleotide-binding domain (Tyr(490)) and the first cytoplasmic loop (Glu(184)).     

Targeting autophagy as a novel strategy for facilitating the therapeutic action of potentiators on ΔF508 cystic fibrosis transmembrane conductance regulator

Channel activators (potentiators) of cystic fibrosis (CF) transmembrane conductance regulator (CFTR), can be used for the treatment of the small subset of CF patients that carry plasma membrane-resident CFTR mutants. However, approximately 90% of CF patients carry the misfolded ΔF508-CFTR and are poorly responsive to potentiators, because ΔF508-CFTR is intrinsically unstable at the plasma membrane (PM) even if rescued by pharmacological correctors. We have demonstrated that human and mouse CF airways are autophagy deficient due to functional sequestration of BECN1 and that the tissue transglutaminase-2 inhibitor, cystamine, or antioxidants restore BECN1-dependent autophagy and reduce SQSTM1/p62 levels, thus favoring ΔF508-CFTR trafficking to the epithelial surface. Here, we investigated whether these treatments could facilitate the beneficial action of potentiators on ΔF508-CFTR homozygous airways. Cystamine or the superoxide dismutase (SOD)/catalase-mimetic EUK-134 stabilized ΔF508-CFTR at the plasma membrane of airway epithelial cells and sustained the expression of CFTR at the epithelial surface well beyond drug withdrawal, overexpressing BECN1 and depleting SQSTM1. This facilitates the beneficial action of potentiators in controlling inflammation in ex vivo ΔF508-CFTR homozygous human nasal biopsies and in vivo in mouse ΔF508-CFTR lungs. Direct depletion of Sqstm1 by shRNAs in vivo in ΔF508-CFTR mice synergized with potentiators in sustaining surface CFTR expression and suppressing inflammation. Cystamine pre-treatment restored ΔF508-CFTR response to the CFTR potentiators genistein, Vrx-532 or Vrx-770 in freshly isolated brushed nasal epithelial cells from ΔF508-CFTR homozygous patients. These findings delineate a novel therapeutic strategy for the treatment of CF patients with the ΔF508-CFTR mutation in which patients are first treated with cystamine and subsequently pulsed with CFTR potentiators.     

Evidence that cysteine 298 is in the active site of tryptophan indole-lyase

Escherichia coli tryptophan indole-lyase (tryptophanase) mutants, with cysteine residues 294 and 298 selectively replaced by serines, have been prepared by site-directed mutagenesis. Both mutant enzymes are highly active for beta-elimination reactions measured with both L-tryptophan and S-(o-nitrophenyl)-L-cysteine. The Cys-294----Ser mutant enzyme is virtually identical to the wild type with respect to pyridoxal phosphate binding (KCO = 2 microM), cofactor absorption spectrum (lambda max = 420 and 337 nm) and pH dependence (pK alpha = 7.3), pH profile for catalysis, and rate of bromopyruvic acid inactivation. In contrast, the Cys-298----Ser mutant enzyme exhibits a reduced affinity for pyridoxal phosphate (KCO = 6 microM), a shift in the cofactor absorption spectrum to 414 nm and an altered pK alpha = 8.5, an alkaline shift in the pH profile for catalysis, and resistance to inactivation of the apoenzyme by bromopyruvic acid. The C298S mutant enzyme (wherein cysteine 298 is altered to serine) also undergoes an isomerization to an unreactive state upon storage at 4 degrees C. These results demonstrate that the sulfhydryl groups of Cys-294 and Cys-298 are catalytically nonessential. However, these data suggest that Cys-298 is located within or very near the active site of the enzyme and is the reactive cysteine residue previously observed by others.     

The molecular chaperone, ClpA, has a single high affinity peptide binding site per hexamer

Substrate recognition by Clp chaperones is dependent on interactions with motifs composed of specific peptide sequences. We studied the binding of short motif-bearing peptides to ClpA, the chaperone component of the ATP-dependent ClpAP protease of Escherichia coli in the presence of ATPgammaS and Mg2+ at pH 7.5. Binding was measured by isothermal titration calorimetry (ITC) using the peptide, AANDENYALAA, which corresponds to the SsrA degradation motif found at the C terminus of abnormal nascent polypeptides in vivo. One SsrA peptide was bound per hexamer of ClpA with an association constant (K(A)) of 5 x 10(6) m(-1). Binding was also assayed by changes in fluorescence of an N-terminal dansylated SsrA peptide, which bound with the same stoichiometry of one per ClpA hexamer (K(A) approximately 1 x 10(7) m(-1)). Similar results were obtained when ATP was substituted for ATPgammaS at 6 degrees C. Two additional peptides, derived from the phage P1 RepA protein and the E. coli HemA protein, which bear different substrate motifs, were competitive inhibitors of SsrA binding and bound to ClpA hexamers with K(A)' > 3 x 10(7) m(-1). DNS-SsrA bound with only slightly reduced affinity to deletion mutants of ClpA missing either the N-terminal domain or the C-terminal nucleotide-binding domain, indicating that the binding site for SsrA lies within the N-terminal nucleotide-binding domain. Because only one protein at a time can be unfolded and translocated by ClpA hexamers, restricting the number of peptides initially bound should avoid nonproductive binding of substrates and aggregation of partially processed proteins.     

Mapping the binding site of colchicinoids on beta -tubulin. 2-Chloroacetyl-2-demethylthiocolchicine covalently reacts predominantly with cysteine 239 and secondarily with cysteine 354

2-Chloroacetyl-2-demethylthiocolchicine (2CTC) and 3-chloroacetyl-3-demethylthiocolchicine (3CTC) resemble colchicine in binding to tubulin and react covalently with beta-tubulin, forming adducts with cysteine residues 239 and 354. The adducts at Cys-239 are less stable than those at Cys-354 during formic acid digestion. Extrapolating to zero time, the Cys-239 to Cys-354 adduct ratio is 77:23 for 2CTC and 27:73 for 3CTC. Using energy minimization modeling to dock colchicinoids into the electron crystallographic model of beta-tubulin in protofilaments (Nogales, E. , Wolf, S. G., and Downing, K. H. (1998) Nature 391, 199-203), we found two potential binding sites. At one, entirely encompassed within beta-tubulin, the C2- and C3-oxygen atoms of 2CTC and 3CTC overlapped poorly with those of colchicine and thiocolchicine, but distances from the reactive carbon atoms of the analogs to the sulfur atoms of the cysteine residues were qualitatively consistent with reactivity. The other potential binding site was located at the alpha/beta interface. Here, the oxygen atoms of the analogs overlapped well with those of colchicine, but relative distances of the reactive carbons to the cysteine sulfur atoms did not correlate with the observed reactivity. A significant conformational change must occur in the colchicine binding site of tubulin in the transition from the unpolymerized to the polymerized state.     

Design and synthesis of a hybrid potentiator–corrector agonist of the cystic fibrosis mutant protein ΔF508-CFTR

A developing therapy of cystic fibrosis caused by the ΔF508 mutation in CFTR employs correction of defective CFTR chloride channel gating by a ‘potentiator’ and of defective CFTR protein folding by a ‘corrector’. Based on SAR data for phenylglycine-type potentiators and bithiazole correctors, we designed a hybrid molecule incorporating an enzymatic hydrolysable linker to deliver the potentiator (PG01) fragment 2 and the corrector (Corr-4a) fragment 13. The hybrid molecule 14 contained PG01-OH and Corr-4a–linker–CO₂H moieties, linked with an ethylene glycol spacer through an ester bond. The potentiator 2 and corrector 13 fragments (after cleavage) had low micromolar potency for restoration of ΔF508-CFTR channel gating and cellular processing, respectively. Cleavage of hybrid molecule 14 by intestinal enzymes under physiological conditions produced the active potentiator 2 and corrector fragments 13, providing proof-of-concept for small-molecule potentiator–corrector hybrids as a single drug therapy for CF caused by the ΔF508 mutation.     

Synthesis and biological evaluation of thiazole derivatives on basic defects underlying cystic fibrosis

Cystic fibrosis is a genetic disease caused by loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator gene, encoding for CFTR protein. The most frequent mutation is the deletion of phenylalanine at position 508 (F508del), which leads to distinct defects in channel gating and cellular processing. In last years, several thiazole containing small molecules, endowed with dual F508del-CFTR modulator activity, proved to be able to target these defects. In search of new chemical entities able to restore CFTR function, we designed and synthesized a small series of sixteen thiazole derivatives. The designed compounds were studied as correctors and potentiators of F508del-CFTR. Although none of the molecules showed significant corrector activity, compounds 10 and 11 exhibited potentiator effects, thus allowing to determine some basic structural features which enable to obtain F508del-CFTR potentiator activity. In silico ADME studies showed that these derivatives obey Lipinski’s rule of five and are expected to be orally bioavailable. Therefore, these molecules may represent a good starting point for the design of analogues endowed with improved CFTR potentiator activity and a good pharmacokinetic profile.