A systematic method for identifying small-molecule modulators of protein–protein interactions

Horswill et al. 10.1073/pnas.0406999101.

Supporting Information

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Supporting Materials and Methods
Supporting Table 1
Supporting Figure 5
Supporting Figure 6
Supporting Results
Supporting Figure 7
Supporting Figure 8
Supporting Figure 9
Supporting Figure 10
Supporting Figure 11

Fig. 5. Graphic map of the reverse two-hybrid system (RTHS) plasmids for making repressor fusions and promoter sequences. (A) Plasmid pTHCP14 for constructing heterodimeric fusions for strains containing the chimeric phage 434·P22 promoter (strain SNS126 derivatives). (B and C) Plasmid pTHCP16 (B) and plasmid pTHCP17 (C) for constructing fusions for strains containing the phage 434 wild-type promoter (strain SNS118 derivatives). (D) Chimeric 434× P22 promoter. (E) Wild-type 434 promoter.

Fig. 6. Results of β-galactosidase assays for testing reporter strain selectivity with the FKBP12–FRAP pairing. Fusions with either 434 wild-type or 434·P22 chimeric DNA-binding domains were transformed into strains containing either 434 wild-type (SNS118) or 434·P22 chimeric (SNS126) promoters. β-Galactosidase assays were performed in the absence (white bars) and presence of 10 µM rapamycin (black bars) for each strain type.

Fig. 7. Results from β-galactosidase activity assays documenting levels of protein–protein interactions. (A) Reporter strain SNS118 expressing DNA-binding domain (negative control), 434 repressor (positive control), GCN4 transcription factor, and HIV-1 protease. Isopropyl β-D-thiogalactoside (IPTG) concentrations: 0 (white bars), 10 (gray bars), and 50 µM (black bars). (B) Reporter strain SNS126 with integrated null (negative control), ribonucleotide reductase (RR), and FKBP12–FRAP fusions (with and without 1 µM rapamycin). IPTG concentrations: 0 (white bars), 20 (gray bars), and 650 µM (black bars).

Fig. 8. Results from β-galactosidase assays demonstrating the effects of linear peptide inhibitors on the oligomeric state of HIV-1 protease and ribonucleotide reductase (RR). (A) Comparison of the activity of the known inhibitor (pHIV16, encoding for MTVSYEL) versus a control (pHIV17, encoding for MDSATYV) induced with arabinose concentrations of 0, 33, and 66 µM, using a strain expressing an integrated HIV-1 protease fusion at 100 µM isopropyl β-D-thiogalactoside (IPTG). (B) Comparison of the activity of the known inhibitor (pTHCP35, encoding for MSFTLDADF) versus a scrambled control (pTHCP37, encoding for MDTAFSFLD) induced with 13 µM arabinose, using a strain expressing an integrated RR fusion at IPTG concentrations of 0, 10, and 30 µM.

Fig. 9. Identification of false positives. Restreaks of six representative candidates on minimal media without arabinose and with isopropyl β-D-thiogalactoside (IPTG) drops (indicated by circles, 200 mM solution) (A) and minimal media with arabinose (13 µM) and IPTG (1 mM) (B). A positive control (unrepressed strain) and a negative control (SICLOPPS control plasmid) are indicated on all plates as reference points. (A) Candidates numbered 4 and 5 were identified as false positives by growth in the presence of IPTG, characterized by lack of response to the localized expression of ribonucleotide reductase (RR) fusions . (B) All six candidates show enhanced growth over the negative control in the presence of arabinose.

Fig. 10. Representative plates demonstrating the iterative processing of ribonucleotide reductase (RR) inhibitor candidates. Positive control (P; unrepressed strain) and the negative control (N; SICLOPPS control plasmid) are indicated on all plates as reference points. Ranking of six representative candidates by serial dilutions (10^{6 -} 10 cells per drop) on minimal media without (A) and with (B) arabinose. Those with strong growth enhancement and arabinose-dependent growth response were characterized further.

Fig. 11. Target selectivity test. The eight best candidates were transformed into the ribonucleotide reductase (RR) selection strain (red labels) and a testing strain, which expresses the FKBP12 and FRAP fusions (yellow labels). Candidates in both strains are shown in A (RR84, RR93, RR112, and RR120) and B (RR127, RR130, RR131, and RR133). The transformants were serially diluted (10–10⁶ cells) on selective media (50 µg/ml kanamycin; 10 mM 3-amino-1,2,4-triazole) with inducer (13 µM arabinose). To allow direct target specificity comparisons between the native and the FKBP12–FRAP systems, the reverse two-hybrid system (RTHS) repression level was equalized with isopropyl β-D-thiogalactoside (IPTG; 1 mM) and rapamycin (35 nM), whose combined effect induced fusion expression and allowed the association of the FKPB12 and FRAP proteins. The relative growth advantage of the selectants was demonstrated by including a functionally inert SICLOPPS plasmid, transformed into both strains (shown as RR and FF, FKBP12–FRAP).

Table 1. Mass spectrometry results and synthesis yields of cyclic peptides