Discovery of Novel Haloalkane Dehalogenase Inhibitors

Abstract

bHaloalkane dehalogenases (HLDs) have recently been discovered in a number of bacteria, including symbionts and pathogens of both plants and humans. However, the biological roles of HLDs in these organisms are unclear. The development of efficient HLD inhibitors serving as molecular probes to explore their function would represent an important step toward a better understanding of these interesting enzymes. Here we report the identification of inhibitors for this enzyme family using two different approaches. The first builds on the structures of the enzymes' known substrates and led to the discovery of less potent nonspecific HLD inhibitors. The second approach involved the virtual screening of 150,000 potential inhibitors against the crystal structure of an HLD from the human pathogen Mycobacterium tuberculosis H37Rv. The best inhibitor exhibited high specificity for the target structure, with an inhibition constant of 3 M and a molecular architecture that clearly differs from those of all known HLD substrates. The new inhibitors will be used to study the natural functions of HLDs in bacteria, to probe their mechanisms, and to achieve their stabilization. Haloalkane dehalogenases (HLDs; EC 3.5.1.8) are enzymes that catalyze the hydrolytic cleavage of carbon-halogen bonds in alkyl halides ( Fig. 1) with a wide range of potential applications in biocatalysis, biodegradation, biosensing, decontamination, and cell imaging (1). In structural terms, they belong to the ␣/␤-hydrolase superfamily (2-4). The HLDs have broad substrate specificity, enabling them to catalyze conversion of diverse chlorinated, brominated and iodinated alkanes, alkenes, cycloalkanes, alcohols, epoxides, carboxylic acids, esters, ethers, amides, and nitriles (5, 6).The first known members of this family were isolated from the bacteria Xanthobacter autotrophicus GJ10, Sphinghomonas paucimobilis UT26, and Rhodococcus rhodochrous NCIMB13064 (3, 7, 8), which colonize environments that have been heavily contaminated with halogenated pollutants, such as 1,2-dichloroethane, 1,2,3,4,5,6-hexachlorocyclohexane, and 1-chlorobutane. In these microorganisms, the HLDs were found to be components of metabolic pathways that enable the microbes to utilize otherwise toxic haloalkanes as their sole source of carbon and energy. The HLDs have been recently discovered in a wider range of organisms, including symbiotic bacteria such as Bradyrhizobium japonicum USDA110 and Mesorhizobium loti MAF303099 (9, 10), pathogenic bacteria such as Agrobacterium tumefaciens C58 and Mycobacterium spp. (11,12), and the eukaryotic organism Strongylocentrotus purpuratus (13). Even though HLDs have been studied intensively over the last 25 years and isolated from many different environments and species, most of their biological functions remain elusive. For instance, it is undoubtedly interesting that the plant pathogen Agrobacterium tumefaciens C58 carries the HLDencoding gene datA on its tumor-inducing plasmid while there is no clear link between HLD activity and tumorigenesis (1...

From machine learning to deep learning: Advances in scoring functions for protein–ligand docking

“…Further assessment on the DUD dataset showed that NNScore 2.0 did not perform as well as NNScore 1.0 on average, but outperformed AutoDock, AutoDock Vina and Glide HTVS . In addition, NNScore was also involved in the discovery of inhibitors towards several important targets, which further testified its practicality …”

“…132 In addition, NNScore was also involved in the discovery of inhibitors towards several important targets, which further testified its practicality. [133][134][135][136][137] Ouyang et al 115 developed a method named CScore that was trained by using a modified Cerebellar Model Articulation Controller learning architecture for binding affinity prediction. Similar with RF-Score, this method also relied on simple element-element atom pairs but used fuzzy membership functions rather than directly counting the occurrence to represent each feature.…”

Bioremediation 3.0: Engineering pollutant-removing bacteria in the times of systemic biology

“…Tools similar to the EcoliTox server could be integrated into a computational framework for improved design of native or heterologous biodegradation pathways and used to fine-tune their expression in selected microbial hosts. A powerful virtual screening approach, currently applied predominantly in drug design studies (Buryska et al, 2016), could be used similarly…”

Dehalogenases: From Improved Performance to Potential Microbial Dehalogenation Applications

“…A major deviation in the catalytic groups of HLDs and other non-hydrolytic enzymes such as the 4-chlorobenzoyl-coenzyme A dehalogenase is seen with the essential tryptophan residues in which two Trp 175,125 residues form a binding pocket for the incipient halide ion [ 19 ]. Initially, carboxylate oxygen of the aspartate launches a nucleophilic attack on the carbon atom of the substrate that is bonded with halogen [ 14 , 18 , 19 , 20 , 21 , 61 , 69 , 70 ]. It can be clearly seen from that the aspartate acts as nucleophile and histidine as base for different type of haloalkane dehalogenases but the acid can be glutamate or aspartate and the two halide-stabilizing residues can be any combination formed by tryptophan and asparagine mostly.…”