A credit-card library approach for disrupting protein–protein interactions

Xu, Yuanfu; Jin, Shi; Yamamoto, Noboru; Moss, Jason A.; Vogt, Peter K.; Janda, Kim D.

doi:10.1016/j.bmc.2005.11.052

Cited by 84 publications

(72 citation statements)

References 66 publications

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“…The study of small molecules that disrupt protein-protein interactions has evolved into a rich area, with molecules demonstrated to interrupt numerous systems of clinical significance (20)(21)(22)(23). It generally is accepted that the structural stability of protein-protein interactions derives from large, but relatively shallow, interfaces (24)(25)(26)(27) and that the difficulty in antagonizing interactions on such a large molecular scale has been linked to the size of the buried hydrophobic surfaces.…”

Section: Resultsmentioning

confidence: 99%

An in vitro and in vivo disconnect uncovered through high-throughput identification of botulinum neurotoxin A antagonists

Eubanks

Hixon

Jin

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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117

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Among the agents classified as “Category A” by the U.S. Centers for Disease Control and Prevention, botulinum neurotoxin (BoNT) is the most toxic protein known, with microgram quantities of the protein causing severe morbidity and mortality by oral or i.v. routes. Given that this toxin easily could be used in a potential bioterrorist attack, countermeasures urgently are needed to counteract the pathophysiology of BoNT. At a molecular level, BoNT exerts its paralytic effects through intracellular cleavage of vesicle docking proteins and subsequent organism-wide autonomic dysfunction. In an effort to identify small molecules that would disrupt the interaction between the light-chain metalloprotease of BoNT serotype A and its cognate substrate, a multifaceted screening effort was undertaken. Through the combination of in vitro screening against an optimized variant of the light chain involving kinetic analysis, cellular protection assays, and in vivo mouse toxicity assays, molecules that prevent BoNT/A-induced intracellular substrate cleavage and extend the time to death of animals challenged with lethal toxin doses were identified. Significantly, the two most efficacious compounds in vivo showed less effective activity in cellular assays intended to mimic BoNT exposure; indeed, one of these compounds was cytotoxic at concentrations three orders of magnitude below its effective dose in animals. These two lead compounds have surprisingly simple molecular structures and are readily amenable to optimization efforts for improvements in their biological activity. The findings validate the use of high-throughput screening protocols to define previously unrecognized chemical scaffolds for the development of therapeutic agents to treat BoNT exposure.

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Section: Resultsmentioning

confidence: 99%

An in vitro and in vivo disconnect uncovered through high-throughput identification of botulinum neurotoxin A antagonists

Eubanks

Hixon

Jin

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

117

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“…We postulate that P34 and P37 as well as other trypanosomespecific proteins interact with these expansion segments and perform critical functions that could be targeted. The FRET assay has been described as an ideal method for the discovery and validation of drugs targeting protein-protein and protein-RNA interactions (44)(45)(46). The assay we present here provides a method for highthroughput screening for inhibitors of this protein-protein interaction.…”

Section: Discussionmentioning

confidence: 99%

Association of a Novel Preribosomal Complex in Trypanosoma brucei Determined by Fluorescence Resonance Energy Transfer

Wang¹,

Ciganda²,

Williams³

2013

Eukaryot Cell

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We have previously reported that the trypanosome-specific proteins P34 and P37 form a unique preribosomal complex with ribosomal protein L5 and 5S rRNA in the nucleoplasm. We hypothesize that this novel trimolecular complex is necessary for stabilizing 5S rRNA in Trypanosoma brucei and is essential for the survival of the parasite. In vitro quantitative analysis of the association between the proteins L5 and P34 is fundamental to our understanding of this novel complex and thus our ability to exploit its unique characteristics. Here we used in vitro fluorescence resonance energy transfer (FRET) to analyze the association between L5 and P34. First, we demonstrated that FRET can be used to confirm the association between L5 and P34. We then determined that the binding constant for L5 and P34 is 0.60 ؎ 0.03 M, which is in the range of protein-protein binding constants for RNA binding proteins. In addition, we used FRET to identify the critical regions of L5 and P34 involved in the protein-protein association. We found that the N-terminal APK-rich domain and RNA recognition motif (RRM) of P34 and the L18 domain of L5 are important for the association of the two proteins with each other. These results provide us with the framework for the discovery of ways to disrupt this essential complex.A frican trypanosomes are responsible for trypanosomiasis in humans and nagana in domestic animals. Trypanosoma brucei has a complicated life cycle that requires adaptation to life within a mammalian host and an insect vector, the tsetse fly (1). Throughout this life cycle, RNA binding proteins (RBPs) play the central roles in many adaptive processes, including regulation of gene expression and protein synthesis (2, 3), making them particularly important in this organism.RBPs play essential roles in the many aspects of ribosomal biogenesis. Ribosomal biogenesis in eukaryotes is an essential and conserved process that requires the processing and assembly of four rRNAs (18, 28, 5.8, and 5S rRNAs) with over 80 ribosomal proteins. Much of this process occurs in the nucleolus (4-6). However, unlike the other three rRNAs, 5S rRNA is independently transcribed by RNA polymerase III within the nucleoplasm and must be transported into the nucleolus by the L5 ribosomal protein (7,8). L5 is the only known eukaryotic ribosomal protein that forms a preribosomal L5-5S rRNA ribonucleoprotein (RNP) complex to stabilize and facilitate the trafficking of 5S rRNA to the nucleolus (7, 9, 10). Mutations in critical residues within L5 lead to loss of 5S rRNA and ribosomal biogenesis defects, indicating that it is essential to cell viability. T. brucei L5 is comprised of a predicted RanBP1_WASP domain in its N-terminal region, an L18 domain in the central region, and a C-terminal domain that possesses residues critical for 5S rRNA binding.Our laboratory has previously identified two trypanosomespecific RBPs, P34 and P37, which are essential for the viability of T. brucei (11). P34 and P37 are highly similar, the major difference between them being the pre...

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“…Because c-Myc must heterodimerize with its obligatory bHLH-ZIP partner Max to exhibit transcriptional activity (Walhout et al, 1997), several groups have designed smallmolecule inhibitors to interfere with this protein-protein dimerization and thereby inactivate c-Myc function (Berg et al, 2002;Yin et al, 2003;Kiessling et al, 2006;Xu et al, 2006;Wang et al, 2007;Hammoudeh et al, 2009;Shi et al, 2009). Berg et al (2002) showed that small molecules can interfere with c-Myc/Max dimerization and inhibit c-Mycdependent transformation of chicken embryo fibroblasts.…”

Section: Introductionmentioning

confidence: 99%

In Vitro Cytotoxicity and In Vivo Efficacy, Pharmacokinetics, and Metabolism of 10074-G5, a Novel Small-Molecule Inhibitor of c-Myc/Max Dimerization

Clausen

Guo²,

Parise³

et al. 2010

J Pharmacol Exp Ther

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The c-Myc oncoprotein is overexpressed in many tumors and is essential for maintaining the proliferation of transformed cells. To function as a transcription factor, c-Myc must dimerize with Max via the basic helix-loop-helix leucine zipper protein (bHLH-ZIP) domains in each protein. The small molecule 7-nitro-N-(2-phenylphenyl)-2,1,3-benzoxadiazol-4-amine (10074-G5) binds to and distorts the bHLH-ZIP domain of c-Myc, thereby inhibiting c-Myc/Max heterodimer formation and inhibiting its transcriptional activity. We report in vitro cytotoxicity and in vivo efficacy, pharmacodynamics, pharmacokinetics, and metabolism of 10074-G5 in human xenograft-bearing mice. In vitro, 10074-G5 inhibited the growth of Daudi Burkitt's lymphoma cells and disrupted c-Myc/Max dimerization. 10074-G5 had no effect on the growth of Daudi xenografts in C.B-17 SCID mice that were treated with 20 mg/kg 10074-G5 intravenously for 5 consecutive days. Inhibition of c-Myc/Max dimerization in Daudi xenografts was not seen 2 or 24 h after treatment. Concentrations of 10074-G5 in various matrices were determined by high-performance liquid chromatography-UV, and metabolites of 10074-G5 were identified by liquid chromatography/tandem mass spectrometry. The plasma half-life of 10074-G5 in mice treated with 20 mg/kg i.v. was 37 min, and peak plasma concentration was 58 M, which was 10-fold higher than peak tumor concentration. The lack of antitumor activity probably was caused by the rapid metabolism of 10074-G5 to inactive metabolites, resulting in tumor concentrations of 10074-G5 insufficient to inhibit c-Myc/Max dimerization. Our identification of 10074-G5 metabolites in mice will help design new, more metabolically stable small-molecule inhibitors of c-Myc.

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A credit-card library approach for disrupting protein–protein interactions

Cited by 84 publications

References 66 publications

An in vitro and in vivo disconnect uncovered through high-throughput identification of botulinum neurotoxin A antagonists

An in vitro and in vivo disconnect uncovered through high-throughput identification of botulinum neurotoxin A antagonists

Association of a Novel Preribosomal Complex in Trypanosoma brucei Determined by Fluorescence Resonance Energy Transfer

In Vitro Cytotoxicity and In Vivo Efficacy, Pharmacokinetics, and Metabolism of 10074-G5, a Novel Small-Molecule Inhibitor of c-Myc/Max Dimerization

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