Structural insight into exosite binding and discovery of novel exosite inhibitors of botulinum neurotoxin serotype A through in silico screening

Hu, Xin; Legler, Patricia M.; Southall, Noel; Maloney, David J.; Simeonov, Anton; Jadhav, Ajit

doi:10.1007/s10822-014-9758-7

Cited by 16 publications

(11 citation statements)

References 37 publications

(50 reference statements)

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“…5. In corroboration with a previous computational study on lomofungin-BoNT/LC binding, 36 the key interaction of both molecules appears to be a hydrogen bond (H-bond) with the phenol OH of Tyr250. Furthermore, the conformation of the Tyr250 loop has been shown to be profoundly influenced by substrate binding to the LC; 35 therefore, a potentially stronger interaction of CBIP relative to lomofungin with Tyr250 may disrupt SNAP-25 binding, resulting in the superior inhibitory activity of CBIP.…”

supporting

confidence: 86%

Picolinic acids as β-exosite inhibitors of botulinum neurotoxin A light chain

2016

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In developing small-molecule inhibitors of botulinum neurotoxin serotype A light chain (BoNT/A LC), substituted picolinic acids were identified. Extensive investigation into the SAR of the picolinic acid scaffold revealed 5-(1-butyl-4-chloro-1H-indol-2-yl)picolinic acid (CBIP), which possessed low micromolar activity against BoNT/A. Kinetic and docking studies demonstrated binding of CBIP to the β-exosite: a largely unexplored site on the LC that holds therapeutic relevance for botulism treatment.

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supporting

confidence: 86%

Picolinic acids as β-exosite inhibitors of botulinum neurotoxin A light chain

2016

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“…13 Clustering analysis of docking poses showed that the small molecule binds to the 7TM site of hRXFP1 in a similar manner as other agonists bound to different GPCRs. As shown in Figure 3, the core 2-acetamido-N-phenylbenzamide is accommodated in the typical ligand-binding pocket by forming extensive van der Waals and hydrophobic interactions with residues mainly from TM5 and TM7.…”

Section: Resultsmentioning

confidence: 96%

“…The binding mode of ML290 with the active RXFP1 was predicted using a step-wised ensemble-docking approach 13, 14 Ensembles of the active RXFP1 structure were generated from the MD simulations. A total of five cluster representatives were selected from the trajectory clustering analysis and used for the following docking study.…”

Section: Methodsmentioning

confidence: 99%

Structural Insights into the Activation of Human Relaxin Family Peptide Receptor 1 by Small-Molecule Agonists

Myhr²,

Huang³

et al. 2016

Biochemistry

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The GPCR relaxin family peptide receptor 1 (RXFP1) mediates the action of relaxin peptide hormone including its tissue remodeling and antifibrotic effects. The peptide hormone has a short half-life in plasma, limiting its therapeutic utility. However, small molecule agonists of human RXFP1 can overcome this limitation and may provide a useful therapeutic approach especially for chronic diseases such as heart failure and fibrosis. The first small molecule agonists of RXFP1 were recently identified from a high throughput screen using a homogenous cell-based cAMP assay. Optimization of the hit compounds resulted in a series of highly potent and RXFP1 selective agonists with low cytotoxicity, and excellent in vitro ADME and pharmacokinetic properties. Here we undertook extensive site-directed mutagenesis studies in combination with computational modeling analysis to probe the molecular basis of the small molecule binding to RXFP1. The results showed that the agonists bind to an allosteric site of RXFP1 in a manner that closely interacts with the seven transmembrane domain (TM7) and the third extracellular loop (ECL3). A number of residues were found to play an important role in the agonist binding and receptor activation including a hydrophobic region at TM7 consisting of W664, F668 and L670. The G659/T660 motif within ECL3 is crucial to the observed species selectivity of the agonists for RXFP1. The receptor binding and activation effects by the small molecule agonist ML290 were compared with the cognate ligand relaxin peptide, providing valuable insights on the structural basis and molecular mechanism of activation and selectivity of the agonists for RXFP1.

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“…We found that the CFP-YFP substrates were able to tolerate all of the tested human protein sequences; however, yields varied. In similar assays, substrates containing human protein sequences as long as 63 amino acids were successfully expressed, purified, and utilized for kinetic analyses and inhibitor screening 49,50,51 . Since only small amounts of the substrate are needed for the discontinuous assay, a large number of targets can be explored.…”

Section: Notementioning

confidence: 99%

Analysis of Group IV Viral SSHHPS Using In Vitro and In Silico Methods

Compton

Legler

2019

JoVE

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Alphaviral enzymes are synthesized in a single polypeptide. The nonstructural polyprotein (nsP) is processed by its nsP2 cysteine protease to produce active enzymes essential for viral replication. Viral proteases are highly specific and recognize conserved cleavage site motif sequences (~6-8 amino acids). In several Group IV viruses, the nsP protease(s) cleavage site motif sequences can be found in specific host proteins involved in generating the innate immune responses and, in some cases, the targeted proteins appear to be linked to the virus-induced phenotype. These viruses utilize short stretches of homologous host-pathogen protein sequences (SSHHPS) for targeted destruction of host proteins. To identify SSHHPS the viral protease cleavage site motif sequences can be inputted into BLAST and the host genome(s) can be searched. Cleavage initially can be tested using the purified nsP viral protease and fluorescence resonance energy transfer (FRET) substrates made in E. coli. The FRET substrates contain cyan and yellow fluorescent protein and the cleavage site sequence (CFP-sequence-YFP). This protease assay can be used continuously in a plate reader or discontinuously in SDS-PAGE gels. Models of the bound peptide substrates can be generated in silico to guide substrate selection and mutagenesis studies. CFP/YFP substrates have also been utilized to identify protease inhibitors. These in vitro and in silico methods can be used in combination with cell-based assays to determine if the targeted host protein affects viral replication.

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Structural insight into exosite binding and discovery of novel exosite inhibitors of botulinum neurotoxin serotype A through in silico screening

Cited by 16 publications

References 37 publications

Picolinic acids as β-exosite inhibitors of botulinum neurotoxin A light chain

Picolinic acids as β-exosite inhibitors of botulinum neurotoxin A light chain

Structural Insights into the Activation of Human Relaxin Family Peptide Receptor 1 by Small-Molecule Agonists

Analysis of Group IV Viral SSHHPS Using In Vitro and In Silico Methods

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