Effective cell-free drug screening protocol for protein-protein interaction

Abstract

Specific protein-protein interaction (PPI) is an essential feature of many cellular processes however, targeting these interactions by small molecules is highly challenging due to the nature of the interaction interface. Thus, screening for PPI inhibitors requires enormous number of compounds. Here we describe a simple and improved protocol designed for a search of direct PPI inhibitors. We engineered a bacterial expression system for the split-Renilla luciferase (RL) complementation assay that monitors PPI. This enables production of large quantities of the RL fusion proteins in a simple and cost effective manner that is suitable for very large screens. Subsequently, inhibitory compounds are analyzed in a similar complementation assay in living cultured mammalian cells to select for those that can penetrate cells. We applied this method to NF-κB, a family of dimeric transcription factors that plays central roles in immune responses, cell survival and aging, and its dysregulation is linked to many pathological states. This strategy led to the identification of several direct NF-κB inhibitors. As the described protocol is very straightforward and robust it may be suitable for many pairs of interacting proteins.

Targeting Spt5-Pol II by Small-Molecule Inhibitors Uncouples Distinct Activities and Reveals Additional Regulatory Roles

“…Spt5 is a large protein that contains several conserved functional domains (Werner, 2012), including NusG N-terminal homology domain (NGN), which mediates the interaction with Pol II; 5 KOW domains, which are also involved in the interaction with Pol II; a repetitive heptapeptide motif in the C-terminal region called CTR, implicated in the positive elongation activity of Spt5 (Chen et al, 2009;Ivanov et al, 2000;Yamada et al, 2006); and a C-terminal domain with an unknown function. We applied the split Renilla luciferase (RL) complementation assay to detect the Spt5 interaction with Pol II, as previously described (Ashkenazi et al, 2016(Ashkenazi et al, , 2017. In this assay, RL is split into 2 inactive N-and C-terminal fragments and fused to target proteins.…”

“…For this purpose, the region spanning NGN and KOW 1 and 2 of Spt5 was fused to N-terminal Renilla luciferase (N-RL), and the coiled-coil (CC) domain of Rpb1 (the large subunit of Pol II) was fused to C-terminal Renilla luciferase (C-RL) (Figure 1A). These constructs were tested for specific interactions in mammalian cells and then co-expressed in Escherichia coli from a single plasmid, as described by Ashkenazi et al (2017). A major advantage of the use of recombinant protein fragments of large proteins such as Spt5 and Rpb1 is the potential to identify specific compounds that directly bind to these protein domains.…”

“…Approximately half of the compounds also inhibited the full-length RL at the high concentration, leaving 148 compounds that specifically inhibited the activity of the Spt5-Pol II split-RL pair. To select compounds that can enter mammalian cells, they were further tested in a live-cell split-RL assay, as previously described (Ashkenazi et al, 2017), and 140 retained their inhibitory activity. After filtering out 44 potentially non-specific compounds, as they also appeared in other screens of these libraries, we selected 41 Spt5-Pol II inhibitors (SPIs) displaying a half-maximal inhibitory concentration (IC 50 ) % 40 mM for further analysis.…”

“…The HTS was carried out in 1536 well plate format as previously described (Ashkenazi et al, 2017), with the following modifications: the final concentration of each drug was 15 mM and the incubation time with the Coelenterazine (GOLDBIO, CZ5) RL substrate was 30 min at RT. Compounds that diminished the luminescence activity by > 30% were subjected for an additional screen with bacterial lysate expressing recombinant full-length RL (diluted 1:500 in phosphate buffer) that served as a control for false-positive compounds that inhibit the RL enzymatic activity itself.…”

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications

“…As a sensory component of hybrids, various biospecific polypeptides were applied—antibodies or their fragments [ 111 , 112 , 113 , 114 ], antigens [ 115 , 116 , 117 , 118 ], polypeptides with special binding ability [ 119 , 120 , 121 , 122 ], GFP and its color variants [ 123 , 124 , 125 , 126 , 127 , 128 , 129 ], etc. Based on the phenomenon of assembling split luciferase derivatives, a whole spectrum of homogeneous methods has been developed [ 125 , 126 , 127 , 128 , 129 ] and applied for studying protein–protein/ligand interaction [ 130 , 131 , 132 , 133 ], protein activity [ 134 , 135 ], and protein folding [ 136 , 137 ].…”