Abstract
The approved drugs that target carbonic anhydrases (CA, EC 4.2.1.1), a family of zinc metalloenzymes, comprise almost exclusively of primary sulfonamides (R-SO2NH2) as the zinc binding chemotype. New clinical applications for CA inhibitors, particularly for hard-to-treat cancers, has driven a growing interest in the development of novel CA inhibitors. We recently discovered that the thiazolidinedione heterocycle, where the ring nitrogen carries no substituent, is a new zinc binding group and an alternate CA inhibitor chemotype. This heterocycle is curiously also a substructure of the glitazone class of drugs used in the treatment options for type 2 diabetes. Herein, we investigate and characterise three glitazone drugs (troglitazone 11, rosiglitazone 12 and pioglitazone 13) for binding to CA using native mass spectrometry, protein X-ray crystallography and hydrogen–deuterium exchange (HDX) mass spectrometry, followed by CA enzyme inhibition studies. The glitazone drugs all displayed appreciable binding to and inhibition of CA isozymes. Given that thiazolidinediones are not credited as a zinc binding group nor known as CA inhibitors, our findings indicate that CA may be an off-target of these compounds when used clinically. Furthermore, thiazolidinediones may represent a new opportunity for the development of novel CA inhibitors as future drugs.
Highlights
Fragment-based drug discovery (FBDD) continues to be widely applied in academia and the biotechnology and pharmaceutical sectors as an established drug lead-finding technology [1,2,3,4,5,6]
The calculated value for troglitazone is 86.4%, rosiglitazone is 75.1% and pioglitazone is 24.2%. These findings provide confirmation that the glitazone drug class bind to human CA II enzyme (hCA II)
Glitazone compound dry stocks were dissolved in DMSO to give 5 mM stock solutions. hCA II was concentrated and buffer exchanged into 10 mM NH4OAc pH 7.0 using Amicon Ultra 0.5 centrifugal filters (Merck Millipore, Sydney, NSW, Australia) and immediately prior to mass spectrometric analysis, described by us previously [19]
Summary
Fragment-based drug discovery (FBDD) continues to be widely applied in academia and the biotechnology and pharmaceutical sectors as an established drug lead-finding technology [1,2,3,4,5,6]. An essential component of the FBDD workflow is the screening of fragment libraries against a protein of interest to identify fragment hits. The screening hits are validated and triaged for development into lead compounds that may progress through the drug development pipeline. It is estimated that 10% of human proteins utilise zinc to support structure, function, or both [7], it is not surprising that fragment screening campaigns with zinc metalloproteins have been pursued. Cohen and colleagues have used fragment libraries with a composition bias toward known metal binding pharmacophores for screening with a range of zinc metalloproteins [8,9,10]. Our team has a long-standing interest in discovery of novel inhibitors of carbonic anhydrases (CA, EC 4.2.1.1), a family of zinc metalloenzymes that catalyse the reversible hydration
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