Abstract
Cystathionine β-synthase (CBS) is a key enzyme in the production of the signaling molecule hydrogen sulfide, deregulation of which is known to contribute to a range of serious pathological states. Involvement of hydrogen sulfide in pathways of paramount importance for cellular homeostasis renders CBS a promising drug target. An in-house focused library of heteroaromatic compounds was screened for CBS modulators by the methylene blue assay and a pyrazolopyridine derivative with a promising CBS inhibitory potential was discovered. The compound activity was readily comparable to the most potent CBS inhibitor currently known, aminoacetic acid, while a promising specificity over the related cystathionine γ-lyase was identified. To rule out any possibility that the inhibitor may bind the enzyme regulatory domain due to its high structural similarity with cofactor s-adenosylmethionine, differential scanning fluorimetry was employed. A sub-scaffold search guided follow-up screening of related compounds, providing preliminary structure-activity relationships with respect to requisites for efficient CBS inhibition by this group of heterocycles. Subsequently, a hypothesis regarding the exact binding mode of the inhibitor was devised on the basis of the available structure-activity relationships (SAR) and a deep neural networks analysis and further supported by induced-fit docking calculations.
Highlights
Generated hydrogen sulfide, H2S, is a signaling molecule of pivotal importance and its biosynthesis in mammalian cells is facilitated by three enzymes, cystathionine β-synthase (CBS), cystathionineMolecules 2020, 25, 3739; doi:10.3390/molecules25163739 www.mdpi.com/journal/moleculesMolecules 2020, 25, 3739 γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST) [1,2,3,4]
While there is no determined cocrystal structure of CBS with any of the published inhibitors, the available crystal structures and previous screening attempts regarding CBS have offered adequate structural data to support the notion that the protein could be characterized as a druggable target
The analysis afforded a high score (SiteScore: 1.012; Dscore: 0.973), suggesting a predominantly promising cavity and further strengthening the hypothesis that, apart from molecules the size of the natural substrates, the enzyme might interact with larger molecules that could function as modulators
Summary
Generated hydrogen sulfide, H2S, is a signaling molecule of pivotal importance and its biosynthesis in mammalian cells is facilitated by three enzymes, cystathionine β-synthase (CBS), cystathionine. CBS is a unique heme-containing pyridoxal 50 -phosphate (PLP)-dependent enzyme that is allosterically activated by s-adenosylmethionine (AdoMet or SAM) [24,25]. AOAA is not selective for CBS, as it inhibits CSE and interacts with other PLP-dependent enzymes as well [9]. Such a simple organic molecule could marginally be considered as a tractable lead for drug development. In this direction, exploring the available chemical space with the objective to discover new scaffolds that can act as promising leads for achieving potent and selective CBS is deemed a highly interesting screening endeavor [38]. The main results are derived from a series of orthogonal biochemical and biophysical assays and additional support is offered by theoretical approaches, including artificial intelligence modeling and molecular simulations
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