Screening-based Discovery and Structural Dissection of a Novel Family 18 Chitinase Inhibitor

Abstract

Family 18 chitinases play key roles in the life cycles of a variety of organisms ranging from bacteria to man. Very recently it has been shown that one of the mammalian chitinases is highly overexpressed in the asthmatic lung and contributes to the pathogenic process through recruitment of inflammatory cells. Although several potent natural product chitinase inhibitors have been identified, their chemotherapeutic potential or their use as cell biological tools is limited due to their size, complex chemistry, and limited availability. We describe a virtual screening-based approach to identification of a novel, purine-based, chitinase inhibitor. This inhibitor acts in the low micromolar (K i ‫؍‬ 2.8 ؎ 0.2 M) range in a competitive mode. Dissection of the binding mode by x-ray crystallography reveals that the compound, which consists of two linked caffeine moieties, binds in the active site through extensive and not previously observed stacking interactions with conserved, solvent exposed tryptophans. Such exposed aromatics are also present in the structures of many other carbohydrate processing enzymes. The compound exhibits favorable chemical properties and is likely to be useful as a general scaffold for development of pan-family 18 chitinase inhibitors.Family 18 chitinases (CAZy GH 18) hydrolyze chitin, a homopolymer of ␤-(1,4)-linked N-acetylglucosamine. Chitin is a key component of the fungal cell wall, and chitinases are thought to be required for fungal cell separation and morphogenesis (2-4). Strikingly, whereas chitin does not occur in humans, it is known that we possess two chitinases (5, 6). Human macrophage chitotriosidase (HCHT) 4 is secreted by activated macrophages and is able to degrade chitin from the cell wall of the fungal pathogen Candida albicans. Although its precise role has not been defined, it has been shown to be upregulated during fungal or bacterial infection (7) and Gaucher disease, a lysosomal storage disorder (8). A second chitinase, termed acidic mammalian chitinase was subsequently discovered and shown to possess an unusually low pH optimum (Ͻ3) (6), and its overexpression in the lung has been linked to the progression of asthma.Most known family 18 chitinase inhibitors are natural products (reviewed in Ref. 9), including the cyclic dipeptide CI-4 and related compounds (10, 11), the cyclic pentapeptides argifin and argadin (12)(13)(14), and the pseudotrisaccharide allosamidin, a nanomolar inhibitor isolated from Streptomyces sp. (15). Their poor synthetic accessibility and limited availability from natural sources has so far hampered the use of these inhibitors for in vivo studies, whereas their chemical complexity makes them unsuitable as starting points for ligand design efforts. Nevertheless, numerous crystallographic and biochemical studies have elucidated the interactions between these compounds and family 18 chitinases (e.g. Refs. 11,14,[16][17][18].Recently, a high throughput screen identified three xanthine derivatives, theophylline, caffeine, and pentoxifylline, as competi...

Legionella pneumophila type II secretome reveals unique exoproteins and a chitinase that promotes bacterial persistence in the lung

“…In a second scenario, the effect of ChiA is not directly due to a bacterial enzymatic activity but involves the host response to the protein; when ChiA is present, the host response is compromised sufficiently to allow progression to pneumonia, but when ChiA is absent, a more effective response is mounted. That chitinases can modulate host response is recently evident from studies of the asthmatic lung in which overexpression of mammalian chitinases contribute to pathogenesis by the recruitment of inflammatory cells (56). Incidentally, this type of observation may help explain the detrimental effect that overexpression of cloned chiA had on legionellae in the lung.…”

Fully Deacetylated Chitooligosaccharides Act as Efficient Glycoside Hydrolase Family 18 Chitinase Inhibitors

“…FO-7314, respectively (19 -21), have IC 50 values in the nanomolar to micromolar range (21)(22)(23)(24)(25). Other molecules, such as chitobiose and chitotriose thiazolines (26), xanthine derivatives (27), and CI-4 (28, 29), exhibit nanomolar to millimolar level inhibitory activities. Unfortunately, complex synthesis and/or limited availability of the starting materials limit the practical application of most of these potent molecules.…”

Human YKL-39 is a pseudo-chitinase with retained chitooligosaccharide-binding properties

“…Rationally designed dixanthine derivatives, bisdionins, have been reported as micromolar range inhibitors of family 18 chitinases [49] (Figure 4). Structure-guided modifications to either xanthine ring can be readily incorporated to fine-tune the affinity and selectivity of these ligands, as illustrated by the recently reported bisdionin F derivative (Figure 4), that possesses submicromolar activity against hAMCase with selectivity over hCHT [10].…”