Amphipathic Small Molecules Mimic the Binding Mode and Function of Endogenous Transcription Factors

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 pre-programmed for such multi-modal binding. This is the case for the transcriptional activation domain or TAD of transcriptional activators as these mofifs utilize a poorly characterized multi-partner 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 non-functional 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.Transcriptional activators are essential for high fidelity transcription, responsible for seeking out particular genes and up-regulating them to precise levels in a signal-responsive fashion.(1,2) Indeed, the altered transcription patterns observed in disease states can often be attributed to malfunctioning and/or mis-regulated transcriptional activators.(3-6) Alterations in the function of the tumor suppressor p53, for example, are found in >50% of all human cancers; (7,8) similarly, constitutively active NF-κB, an activator that regulates genes responsible for apoptosis, inflammatory response, and proliferation, is observed in inflammatory disorders and most cancers.(9,10) There is thus tremendous interest in the development of activator artificial transcription factors (activator ATFs), nonnatural molecules programmed to perform the same function as endogenous activators, as both mechanistic tools and as transcription-targeted therapeutic agents. (2,(11)(12)(13)(14) The architecture of activator ATFs is analogous to that of their natural counterparts in that they minimally consist of a DNA binding domain (DBD) that confers gene-targeting specificity and a transcriptional activation domain (TAD) that controls *Corresponding author, amapp@umich.edu.. The challenges associated with small molecule TAD discovery are due in large part to the scarcity of molecular-level details regarding natural TAD function. The largest and most well studied class of activators is the amphipathic class, characterized by interspersed polar a...

Structural basis of CBP/p300 recruitment in leukemia induction by E2A-PBX1

“…It is noteworthy that small molecules based on an isoxazolidine scaffold have been shown to bind KIX selectively at this site. 47,48 The importance of this site for E2A-PBX1-driven oncogenesis, and the accompanying structure of the PCET/KIX complex, opens the exciting possibility of designing novel therapeutics for E2A-PBX1 ϩ acute lymphoblastic leukemia.…”

Dissecting allosteric effects of activator–coactivator complexes using a covalent small molecule ligand

“…HSQC NMR Experiments on 1 H-15 N. Uniformly 15 N-labeled KIX N627C protein was expressed and purified as previously described (47). Aliquots of the purified 15 N-labeled KIX N627C were tethered with the small molecule 1-10 as previously described (30).…”

Thermodynamics of Nucleic Acid Structural Modifications for Biotechnology Applications

“…These experiments demonstrated non-specific binding to the protein targets both in the multiple bands in the gel and in the non-exponential dependence of binding in the competition assay for a generic anionic substrate. The lack of specificity for the target may be due to the amphipathic nature of the modifications introduced, which could lead to several modes of binding similar to the multiple modes of binding of KIX-domain transcription regulatory proteins [80]. These experiments demonstrate the need for a comprehensive examination of the functional groups used from the point of view of the structural folds accessible to DNA.…”

“…The hydrophobic modifications shown in Figure 3A-3D increase the amphipathic nature of DNA aptamers. In order to understand the thermodynamics of amphipathic modes of binding we can compare the possible modes of binding to the class of amphipathic transcription activation domains, which bind to kinase-inducible (KIX) domain of histone acetyl transferase CREB binding protein [80]. The KIX-transcription regulators are amphipathic proteins consisting of alternating regions of negatively-charged aspartates/glutamates and the range of hydrophobic amino acids.…”

“…The KIX-transcription regulators are amphipathic proteins consisting of alternating regions of negatively-charged aspartates/glutamates and the range of hydrophobic amino acids. These amphipathic proteins have multiple binding sites on their target CREB, which underscores the lack of specificity of the binding of hydrophobic groups [80]. Moreover, competitive inhibition by hydrophobic isooxazolidene molecules considerably smaller than the transcription activator peptides strongly suggests that the mode of binding is hydrophobic.…”