A Bidirectional System for the Dynamic Small Molecule Control of Intracellular Fusion Proteins

Neklesa, Taavi K.; Noblin, Devin J.; Kuzin, A.; Lew, Scott; Seetharaman, J.; Acton, Thomas; Kornhaber, G.; Xiao, Rong; Montelione, Gaetano T.; Tong, Liang; Crews, Craig M.

doi:10.1021/cb400569k

Cited by 42 publications

(36 citation statements)

References 26 publications

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“…Additionally, the best structure-based inhibitor showed strong stabilization effects, raising the melting temperature of DmbA by 6°C. Similar effects have been observed with HaloTag stabilizers (42) and with DatA enzyme during crystallization experiments (44). Increased thermostability of the enzyme, while retaining its activity, represents an interesting strategy for extension of its half-life during industrial biocatalysis.…”

Section: Discussionsupporting

confidence: 69%

“…S4 in the supplemental material). A similar binding motif has been observed in the crystal structure of the HLD-based HaloTag with its stabilizer, where the molecule has a tetrazole part located at the similar position (42). Analysis of the enzyme-inhibitor complex by ITC indicates the single binding site model with an n of 0.91 Ϯ 0.01, K d of 3.37 Ϯ 0.12 M, ⌬H of Ϫ5.24 Ϯ 0.12 kcal · mol Ϫ1 , and ⌬S of 8.14 Ϯ 0.12 cal · mol Ϫ1 ·°C Ϫ1 , where n is number of binding sites, K d is dissociation constant, ⌬H is a change in enthalpy, and ⌬S is a change in entropy (Fig.…”

Section: Figsupporting

confidence: 67%

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Discovery of Novel Haloalkane Dehalogenase Inhibitors

Buryška

Daniel

Kunka

et al. 2016

Appl Environ Microbiol

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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...

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Section: Discussionsupporting

confidence: 69%

Section: Figsupporting

confidence: 67%

Discovery of Novel Haloalkane Dehalogenase Inhibitors

Buryška

Daniel

Kunka

et al. 2016

Appl Environ Microbiol

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“…This stabilization, reminiscent of the Shield system described above, allows for small molecule induced degradation and stabilization of the same system at once. [100] …”

Section: Inducers Of Protein Degradationmentioning

confidence: 99%

Small‐Molecule Control of Intracellular Protein Levels through Modulation of the Ubiquitin Proteasome System

2014

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Traditionally, biological probes and drugs have targeted the activities of proteins (such as enzymes and receptors) that can be easily controlled by small molecules. The remaining majority of the proteome has been deemed “undruggable”. By using small molecule modulators of the ubiquitin proteasome, protein levels, rather than protein activities can be targeted instead, increasing the number of druggable targets. While targeting the proteasome itself can lead to a global increase in protein levels, targeting other components of the UPS (e.g., the hundreds of E3 ubiquitin ligases) can lead to an increase in protein levels in a more targeted fashion. Alternatively, multiple strategies for inducing protein degradation with small molecule probes are emerging. With the ability to induce and inhibit the degradation of targeted proteins, small molecule modulators of the UPS have the potential to significantly expand the druggable portion of the proteome beyond traditional targets such as enzymes and receptors.

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“…Immunoblotting revealed a geldanamycin-dependent increase in Hsp70 levels, consistent with the finding that Hsp90 inhibition leads to activation of Heat Shock Factor 1 (HSF1) and its target genes, including HSP70 [25] . Hsp70 and the associated E3 ubiquitin ligase CHIP play a key role in targeting intractably misfolded proteins to the UPS [26] , and we have shown that labeling HaloTag with hydrophobic tags leads to enhanced association with Hsp70 [27] . Together, this suggests that the Hsp70/CHIP complex mediates SARD-induced AR degradation and that elevated Hsp70 levels underlie the increase in SARD279 activity in the context of Hsp90 inhibition.…”

mentioning

confidence: 99%

Small‐Molecule‐Mediated Degradation of the Androgen Receptor through Hydrophobic Tagging

et al. 2015

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Androgen Receptor (AR)-dependent transcription is a major driver of prostate tumor cell proliferation. Consequently, it is the target of several antitumor chemotherapeutic agents, including the AR antagonist MDV3100/enzalutamide. Recent studies have shown that a single AR mutation (F876L) converts MDV3100 action from an antagonist to an agonist. Here we describe the generation of a novel class of Selective Androgen Receptor Degraders (SARDs) to address this resistance mechanism. Molecules containing hydrophobic degrons linked to small molecule Androgen Receptor (AR) ligands induce AR degradation, reduce expression of AR target genes and inhibit proliferation in androgen-dependent prostate cancer cell lines. These results suggest that selective AR degradation may be an effective therapeutic prostate tumor strategy in the context of AR mutations that confer resistance to third generation AR antagonists.

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A Bidirectional System for the Dynamic Small Molecule Control of Intracellular Fusion Proteins

Cited by 42 publications

References 26 publications

Discovery of Novel Haloalkane Dehalogenase Inhibitors

Discovery of Novel Haloalkane Dehalogenase Inhibitors

Small‐Molecule Control of Intracellular Protein Levels through Modulation of the Ubiquitin Proteasome System

Small‐Molecule‐Mediated Degradation of the Androgen Receptor through Hydrophobic Tagging

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