Potential Pendrin-Targeted Pharmacological Interventions

As detailed elsewhere in this book, both decreases and increases in pendrin function have been linked to distinct pathological conditions, namely Pendred syndrome and non-syndromic deafness in the case of pendrin loss-of-function (Parts I and II), and inflammatory and infectious lung diseases in the case of pendrin gain-of-function (Chap. 9). Furthermore, the established role of pendrin in regulating blood pressure (Wall 2015) recommends pendrin inhibition as a worthwhile strategy for the treatment of fluid overload states unresponsive to diuretics in current use (Soleimani

2012). These considerations suggest that pharmacological modulators of pendrin activity may have wide clinical utility.

Screening of Pendrin Ligands

For many years, pendrin seemed an “undraggable” target. Since the initial studies aimed at its functional characterization, pendrin has repeatedly exhibited low sensitivity to established inhibitors of ion transport, such as the anion exchange blocker 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS), furosemide, and probenecid (Scott et al. 1999) (Table 11.3). The anti-inflammatory drugs niflumic acid and tenidap (Dossena et al. 2006b; Reimold et al. 2011) remained for a while the only substances capable of a significant inhibitory effect on pendrin activity (Table 11.3), and a first published screening of a large library of compounds contributed no novel pendrin inhibitors (Pedemonte et al. 2007).

Table 11.3 Effect of known inhibitors of ion transport on pendrin

Test compound

Conventional target

Effect on pendrin

Acetazolamide

Carbonic anhydrase (Belsky 1953)

No effect (Pedemonte et al. 2007; Bernardinelli et al. 2016)

Amiloride

ENaC

(McDonald et al. 1994; Tamargo et al. 2014a)

No effect (Pedemonte et al. 2007; Bernardinelli et al. 2016)

Bumetanide

NKCC2

Tamargo et al. (2014a)

No effect (Pedemonte et al. 2007; Bernardinelli et al. 2016)

Chlorothiazide

NCC

(Monroy et al. 2000; Tamargo et al. 2014b)

No effect (Pedemonte et al. 2007; Bernardinelli et al. 2016)

Eplerenone

Aldosterone antagonist (Tamargo et al. 2014a; Frishman and Stier 2004)

No effect (10 pM) (Bernardinelli et al. 2016)

Furosemide

NKCC2

(Tamargo et al. 2014a; Garay et al. 1998)

No effect (Pedemonte et al. 2007; Bernardinelli et al. 2016)

  • 59 % inhibition (1 mM) (Scott et al. 1999)
  • 39 % inhibition (1 mM) (Bernardinelli et al. 2016)

Hydrochlorothiazide

NCC

(Monroy et al. 2000; Tamargo et al. 2014b)

No effect (Pedemonte et al. 2007; Bernardinelli et al. 2016)

Hydroflumethiazide

NKCC2

(Tamargo et al. 2014a)

No effect (Pedemonte et al. 2007; Bernardinelli et al. 2016)

Indapamide

NCC (?)

(Tamargo et al. 2014b)

No effect (Pedemonte et al. 2007; Bernardinelli et al. 2016)

Methazolamide

Carbonic anhydrase (Lindskog 1997)

No effect (Pedemonte et al. 2007; Bernardinelli et al. 2016)

Torsemide

NKCC2

(Tamargo et al. 2014a)

No effect (Bernardinelli et al. 2016)

Triamterene

ENaC

(Tamargo et al. 2014a)

No effect (Pedemonte et al. 2007; Bernardinelli et al. 2016)

Spironolactone

Aldosterone antagonist (Tamargo et al. 2014a; Frishman and Stier 2004)

No effect (Pedemonte et al. 2007; Bernardinelli et al. 2016)

CFTR-inhibitor

172

CFTR (Taddei et al. 2004)

No effect (Pedemonte et al. 2007) No effect (1.6 pM) (Bernardinelli et al. 2016)

Table 11.3 (continued)

Test compound

Conventional target

Effect on pendrin

DIDS

Anion transport

  • (Jessen et al. 1986; Zhang et al.
  • 2015; Shen et al. 2015)

No effect (Pedemonte et al. 2007; Bernardinelli et al. 2016)

No effect (0.5 mM) (Reimold et al. 2011)

No effect (0.5 mM) (Dossena et al. 2006b)

88 % inhibition (0.5 mM) (Soleimani et al. 2001)

Full inhibition (0.5 mM) (Azroyan et al. 2011)

  • 62 % inhibition (1 mM) (Scott et al. 1999)
  • 48 % inhibition (1 mM) (Bernardinelli et al. 2016)

DNDS

Anion transport

(Barzilay and Cabantchik 1979)

No effect (Bernardinelli et al. 2016)

NPPB

Chloride channels (Brown and Dudley 1996; Zhang et al. 2015)

No effect (Pedemonte et al. 2007)

59 % inhibition (Dossena et al. 2006b) 33 % inhibition (Bernardinelli et al. 2016)

Probenecid

OAT1

(Takeda et al. 2001)

No effect (Pedemonte et al. 2007; Bernardinelli et al. 2016)

37 % inhibition (1 mM) (Scott et al. 1999)

No effect (1 mM) (Bernardinelli et al. 2016)

Glybenclamide

KCNJ1

(Gribble and Reimann 2003)

No effect (Pedemonte et al. 2007; Bernardinelli et al. 2016)

Hydroxycinnamate

No effect (Pedemonte et al. 2007; Bernardinelli et al. 2016)

No effect (1 mM) (Reimold et al. 2011)

Niflumic acid

COX, PLA2

(Jabeen et al. 2005; Shen et al. 2015)

Inhibition (0.25 mM) (Nakao et al. 2008)

Inhibition (Pedemonte et al. 2007; Dossena et al. 2006b)

64 % inhibition (Bernardinelli et al. 2016)

Tenidap

COX, LOX (?)

(Moore et al. 1996) (Blackburn et al. 1991)

49 % inhibition (Reimold et al. 2011) 30 % inhibition (Bernardinelli et al. 2016)

Modified from Bernardinelli et al. (2016), with permission

The transporter or enzyme known to be blocked by each compound (“conventional target”) is indicated together with the effect observed on pendrin transport activity. Compounds were tested at 0.1 mM, unless otherwise specified

CFTR cystic fibrosis transmembrane conductance regulator, COX cyclooxygenase, DIDS 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid, DNDS 4,4'-dinitrostilbene-2,2'-disulfonate, DRA down- regulated in adenoma, ENaC epithelial sodium channel, KCNJ1 potassium channel, inwardly rectifying subfamily J, member 1, LOX lipooxygenase, NCC sodium chloride cotransporter, NKCC2 sodium potassium chloride cotransporter 2, NPPB 5-nitro-2-[(3-phenylpropyl)amino]benzoic acid, OAT1 organic anion transporter 1, PLA2 phospholipase A2, SLC26A3 solute carrier family 26, member A3

The search for potent and specific pendrin ligands has seen a breakthrough with the recently published studies of Verkman and colleagues (Haggie et al. 2016). These investigators used a semi-automated, high-throughput method to screen

36,000 commercially available small compounds from multiple chemical classes for the ability to inhibit pendrin-mediated Cl-/SCN- exchange in Fischer rat thyroid cells. Compounds identified from two chemical classes - tetrahydropyrazo- lopyridines and pyrazolothiophenesulfonamides - exhibited 50 % inhibition of pendrin transport activity in the low micromolar concentration range. Further characterization of two of these candidates (PDSinh-A01 and PDSinh-C01) revealed similar inhibitory potency and efficacy against pendrin-dependent Cl-/SCN-, Cl-/ I-, and Cl-/NO3- exchange activities. PDSinh-A01 showed slightly greater inhibitory potency for pendrin’s Cl-/HCO3- exchange activity, with 50 % inhibition at 2.5 pM, while exhibiting no inhibition of the pendrin homolog SLC26A3/DRA, or of NKCC1, ENaC, CFTR, CaCC, or epithelial K+ channels. The 5 minutes lag time for onset of inhibition and the kinetics of reversibility upon compound washout suggested an intracellular site of action and excluded possible effects on transcript and/or protein levels. PDSinh-A01 was also found to increase airway surface liquid (ASL) depth in IL-13-treated human bronchial epithelial cells from healthy subjects and also from patients with cystic fibrosis, thus suggesting that pendrin inhibition can be applied therapeutically to increase ASL hydration in cystic fibrosis and other pulmonary diseases (Haggie et al. 2016).

A more recent study by the same group showed that in vivo administration of PDSinh-C01 in the setting of short- or long-term furosemide treatment increased urine output by 30 % and 60 %, respectively. The increased urine output was associated with increased natriuresis and chloruresis. Interestingly, treatment of mice with similar concentrations of PDSinh-C01 as a sole agent had no effect on urine output, salt excretion, or acid-base balance. These results are consistent with earlier mouse knockout studies (Soleimani 2015; Wall 2015), and strengthen the hypothesis that addition of pendrin inhibitors to a regimen of multiple diuretics may offer substantial benefit in the treatment of hypertension, edema, and other fluid overload states resistant to combination therapy with currently available diuretics (Cil et al. 2016).

 
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