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. 2009 May 15;4(5):335-44.
doi: 10.1021/cb900028j.

Amphipathic small molecules mimic the binding mode and function of endogenous transcription factors

Sara J Buhrlage  1 , Caleb A Bates, Steven P Rowe, Aaron R Minter, Brian B Brennan, Chinmay Y Majmudar, David E Wemmer, Hashim Al-Hashimi, Anna K Mapp
Affiliations

Amphipathic small molecules mimic the binding mode and function of endogenous transcription factors

Sara J Buhrlage et al. ACS Chem Biol. .

Abstract

Small molecules that reconstitute the binding mode(s) of a protein and in doing so elicit a programmed functional response offer considerable advantages in the control of complex biological processes. The development challenges of such molecules are significant, however. Many protein-protein interactions require multiple points of contact over relatively large surface areas. More significantly, several binding modes can be superimposed upon a single sequence within a protein, and a true small molecule replacement must be preprogrammed for such multimodal binding. This is the case for the transcriptional activation domain or TAD of transcriptional activators as these motifs utilize a poorly characterized multipartner binding profile in order to stimulate gene expression. Here we describe a unique class of small molecules that exhibit both function and a binding profile analogous to natural transcriptional activation domains. Of particular note, the small molecules are the first reported to bind to the KIX domain within the CREB binding protein (CBP) at a site that is utilized by natural activators. Further, a comparison of functional and nonfunctional small molecules indicates that an interaction with CBP is a key contributor to transcriptional activity. Taken together, the evidence suggests that the small molecule TADs mimic both the function and mechanism of their natural counterparts and thus present a framework for the broader development of small molecule transcriptional switches.

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Figures

Figure 1
Figure 1. Natural and designer transcriptional activation domains (TADs)
a) Key sequences from amphipathic TADs that interact with the coactivator CBP. b) Isoxazolidine TADs (iTADs) that were designed to generically mimic their natural counterparts and up-regulate transcription when localized to a specific promoter (when R = DBD).(42-45)
Figure 2
Figure 2. iTAD 1 interacts with CBP
a) Cross-linking experiments with isoxazolidine 1b and HeLa nuclear extracts. HeLa nuclear extracts were incubated with 1b for 12 hours followed by 30 min irradiation of the mixture with 365 nm light. Following immunoprecipitation with streptavidin, Western blot analysis demonstrates that one interaction partner of 1b is CBP. In the absence of UV irradiation (final lane), no CBP is observed, consistent with a direct interaction between 1 and CBP. Lane 2 (`input' contains nuclear extracts alone. Nuclear extracts are indicated by `NE' and UV indicates irradiation with 365 nm light. b) Fluorescence polarization binding experiments with a fluorescein-labeled variant of 1 (1c) and CBP(KIX domain), TRRAP(Tra1)(3092-3524), Med23(Sur2)(352-625) or Med15(Gal11)(1-357) were used to obtain the indicated dissociation constants. Each experiment was performed in triplicate (R2>0.98) with the error indicated. See Supporting Information for additional details.
Figure 3
Figure 3. iTAD 1 binds the site on the CBP KIX domain bound by endogenous TADs MLL, Jun, Tat, and Tax
a) The 1H, 15N-HSQC spectrum of 15N-His6KIX bound by iTAD 1 (1d, R = N3), red, is overlayed on the spectrum of free 15N-His6KIX. NMR samples were prepared with 400 μM protein in 90% H2O/10% D2O 10 mM phosphate buffer with 150 mM NaCl and 1% CD3OD; the spectrum in the presence of iTAD 1 contained a 5-fold excess of the ligand. b) The amide chemical shifts upon addition of ligand were quantitated (Δδ=[Δδ(1H)2 + 0.1Δδ(15N)2]1/2) and plotted against residue number. The average chemical shift is 0.016 ppm and the largest chemical shift is 0.066 for R623. c) The residues that experience the largest chemical shift perturbation upon binding iTAD 1, MLL and Jun are highlighted in red on the space filling diagrams of the CBP KIX domain.(47, 60) Residues experiencing chemical shifts greater than 2 standard deviations above the average are V608, A618, L620, K621, and R623. Residues experiencing shifts one standard deviation above the average are I611, T614, R624, and E665. Pymol figures were generated from 1kdx.(35)
Figure 4
Figure 4. Additional isoxazolidines evaluated for their ability to interact with the KIX domain of CBP
Figure 5
Figure 5. Synthesis of bis-isoxazolidine 8
Reaction conditions: a) allyl alcohol, NaOCl, CH2Cl2, 0 °C to rt, 5h b) BF3•OEt2, allylmagnesium chloride, tol, -78 °C, 4h c) TBSOTf, Et3N, DMAP, THF, 0 °C, 2h d) BnBr, iPr2NEt, DMF, μwave e) TBAF, THF, 0 °C, 3h f) MsCl, Et3N, CH2Cl2, 0 °C, 30 min g) NaN3, DMSO, 100 °C, 12h h) OsO4, NMO, tBuOH/THF/H2O, rt, 5h.
Figure 6
Figure 6. iTAD 8 binds the site on the CBP KIX domain bound by endogenous TADs MLL, Jun, Tat, and Tax
iTAD 8 binds the site on the CBP KIX domain bound by endogenous TADs MLL, Tat, Tax, Jun, and others. a) A 1H, 15N-HSQC of His6KIXwas collected in the presence of excess iTAD 8 and the amide chemical shifts were quantitated (Δδ=[Δδ(1H)2 + 0.1 Δδ(15N)2]1/2) and plotted against residue number.(69) b) The average chemical shift is 0.04 ppm and the largest chemical shift is 0.25 ppm (L620). The black bar indicated the average chemical shift. c) The residues that experience the largest chemical shift upon binding iTAD 8 and MLL(47) are highlighted in red on the space filling diagrams of the CBP KIX domain. Pymol figures were generated from 1kdx.(35)
Figure 7
Figure 7. MLL•KIX solution structure
The solution structure of MLL•KIX shows that one polar (T2857) and four hydrophobic (I2849, F2852, V2853, and L2854) residues make extensive contacts with KIX.(32) MLL residues F2852, L2854, and T2857 are predicted to be mimicked by iTAD 1 functional groups benzyl, isobutyl, and hydroxyl, respectively. MLL amino acid I2849 is predicted to be mimicked by a ring B substituent of 8 with ring A binding in a similar orientation to iTAD 1.
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