Inhibition of CREB Binding and Function with a Dual-Targeting Ligand.

CBP/p300 is a master transcriptional coactivator that regulates gene activation by interacting with multiple transcriptional activators. Dysregulation of protein-protein interactions (PPIs) between the CBP/p300 KIX domain and its activators is implicated in a number of cancers, including breast, leukemia, and colorectal cancer. However, KIX is typically considered "undruggable" because of its shallow binding surfaces lacking both significant topology and promiscuous binding profiles. We previously reported a dual-targeting peptide (MybLL-tide) that inhibits the KIX-Myb interaction with excellent specificity and potency. Here, we demonstrate a branched, second-generation analogue, CREBLL-tide, that inhibits the KIX-CREB PPI with higher potency and selectivity. Additionally, the best of these CREBLL-tide analogues shows excellent and selective antiproliferation activity in breast cancer cells. These results indicate that CREBLL-tide is an effective tool for assessing the role of KIX-activator interactions in breast cancer and expanding the dual-targeting strategy for inhibiting KIX and other coactivators that contain multiple binding surfaces.

A Lipopeptidomimetic of Transcriptional Activation Domains Selectively Disrupts the Coactivator Med25 Protein-Protein Interactions.

Short amphipathic peptides are capable of binding to transcriptional coactivators, often targeting the same binding surfaces as native transcriptional activation domains. However, they do so with modest affinity and generally poor selectivity, limiting their utility as synthetic modulators. Here we show that incorporation of a medium-chain, branched fatty acid to the N-terminus of one such heptameric lipopeptidomimetic (LPPM-8) increases the affinity for the coactivator Med25 >20-fold (Ki >100 µM to 4 µM), rendering it an effective inhibitor of Med25 protein-protein interactions (PPIs). The lipid structure, the peptide sequence, and the C-terminal functionalization of the lipopeptidomimetic each influence the structural propensity of LPPM-8 and its effectiveness as an inhibitor. LPPM-8 engages Med25 through interaction with the H2 face of its activator interaction domain and in doing so stabilizes full-length protein in the cellular proteome. Further, genes regulated by Med25-activator PPIs are inhibited in a cell model of triple-negative breast cancer. Thus, LPPM-8 is a useful tool for studying Med25 and mediator complex biology and the results indicate that lipopeptidomimetics may be a robust source of inhibitors for activator-coactivator complexes.

Garcinolic Acid Distinguishes Between GACKIX Domains and Modulates Interaction Networks.

Natural products are often uniquely suited to modulate protein-protein interactions (PPIs) due to their architectural and functional group complexity relative to synthetic molecules. Here we demonstrate that the natural product garcinolic acid allosterically blocks the CBP/p300 KIX PPI network and displays excellent selectivity over related GACKIX motifs. It does so via a strong interaction (KD 1 µM) with a non-canonical binding site containing a structurally dynamic loop in CBP/p300 KIX. Garcinolic acid engages full-length CBP in the context of the proteome and in doing so effectively inhibits KIX-dependent transcription in a leukemia model. As the most potent small-molecule KIX inhibitor yet reported, garcinolic acid represents an important step forward in the therapeutic targeting of CBP/p300.

Allostery in the dynamic coactivator domain KIX occurs through minor conformational micro-states.

The coactivator KIX of CBP uses two binding surfaces to recognize multiple activators and exhibits allostery in ternary complex formation. Activator•coactivator interactions are central to transcriptional regulation, yet the microscopic origins of allostery in dynamic proteins like KIX are largely unknown. Here, we investigate the molecular recognition and allosteric manifestations involved in two KIX ternary systems c-Myb•KIX•MLL and pKID•KIX•MLL. Exploring the hypothesis that binary complex formation prepays an entropic cost for positive cooperativity, we utilize molecular dynamics simulations, side chain methyl order parameters, and differential scanning fluorimetry (DSF) to explore conformational entropy changes in KIX. The protein's configurational micro-states from structural clustering highlight the utility of protein plasticity in molecular recognition and allostery. We find that apo KIX occupies a wide distribution of lowly-populated configurational states. Each binding partner has its own suite of KIX states that it selects, building a model of molecular recognition fingerprints. Allostery is maximized with MLL pre-binding, which corresponds to the observation of a significant reduction in KIX micro-states observed when MLL binds. With all binding partners, the changes in KIX conformational entropy arise predominantly from changes in the most flexible loop. Likewise, we find that a small molecule and mutations allosterically inhibit/enhance activator binding by tuning loop dynamics, suggesting that loop-targeting chemical probes could be developed to alter KIX•activator interactions. Experimentally capturing KIX stabilization is challenging, particularly because of the disordered nature of particular activators. However, DSF melting curves allow for inference of relative entropic changes that occur across complexes, which we compare to our computed entropy changes using simulation methyl order parameters.

Unexpected specificity within dynamic transcriptional protein-protein complexes.

A key functional event in eukaryotic gene activation is the formation of dynamic protein-protein interaction networks between transcriptional activators and transcriptional coactivators. Seemingly incongruent with the tight regulation of transcription, many biochemical and biophysical studies suggest that activators use nonspecific hydrophobic and/or electrostatic interactions to bind to coactivators, with few if any specific contacts. Here a mechanistic dissection of a set of representative dynamic activator•coactivator complexes, comprised of the ETV/PEA3 family of activators and the coactivator Med25, reveals a different molecular recognition model. The data demonstrate that small sequence variations within an activator family significantly redistribute the conformational ensemble of the complex while not affecting overall affinity, and distal residues within the activator-not often considered as contributing to binding-play a key role in mediating conformational redistribution. The ETV/PEA3•Med25 ensembles are directed by specific contacts between the disordered activator and the Med25 interface, which is facilitated by structural shifts of the coactivator binding surface. Taken together, these data highlight the critical role coactivator plasticity plays in recognition of disordered activators and indicate that molecular recognition models of disordered proteins must consider the ability of the binding partners to mediate specificity.

Copper-Mediated Radiocyanation of Unprotected Amino Acids and Peptides.

This report describes a copper-mediated radiocyanation of aryl halides that is applicable to complex molecules. This transformation tolerates an exceptionally wide range of functional groups, including unprotected amino acids. As such, it enables the site-specific introduction of [11C]CN into peptides at an iodophenylalanine residue. The use of a diamine-ligated copper(I) mediator is crucial for achieving high radiochemical yield under relatively mild conditions, thus limiting racemization and competing side reactions of other amino acid side chains. The reaction has been scaled and automated to deliver radiolabeled peptides, including analogues of adrenocorticotropic hormone 1-27 (ACTH) and nociceptin (NOP). For instance, this Cu-mediated radiocyanation was leveraged to prepare >40 mCi of [11C]cyano-NOP to evaluate biodistribution in a primate using positron emission tomography. This investigation provides preliminary evidence that nociceptin crosses the blood-brain barrier and shows uptake across all brain regions (SUV > 1 at 60 min post injection), consistent with the known distribution of NOP receptors in the rhesus brain.

A Dual-Site Inhibitor of CBP/p300 KIX is a Selective and Effective Modulator of Myb.

The protein-protein interaction between the KIX motif of the transcriptional coactivator CBP/p300 and the transcriptional activator Myb is a high-value target due to its established role in certain acute myeloid leukemias (AML) and potential contributions to other cancers. However, the CBP/p300 KIX domain has multiple binding sites, several structural homologues, many binding partners, and substantial conformational plasticity, making it challenging to specifically target using small-molecule inhibitors. Here, we report a picomolar dual-site inhibitor (MybLL-tide) of the Myb-CBP/p300 KIX interaction. MybLL-tide has higher affinity for CBP/p300 KIX than any previously reported compounds while also possessing 5600-fold selectivity for the CBP/p300 KIX domain over other coactivator domains. MybLL-tide blocks the association of CBP and p300 with Myb in the context of the proteome, leading to inhibition of key Myb·KIX-dependent genes in AML cells. These results show that MybLL-tide is an effective, modifiable tool to selectively target the KIX domain and assess transcriptional effects in AML cells and potentially other cancers featuring aberrant Myb behavior. Additionally, the dual-site design has applicability to the other challenging coactivators that bear multiple binding surfaces.

Norstictic Acid Is a Selective Allosteric Transcriptional Regulator.

Inhibitors of transcriptional protein-protein interactions (PPIs) have high value both as tools and for therapeutic applications. The PPI network mediated by the transcriptional coactivator Med25, for example, regulates stress-response and motility pathways, and dysregulation of the PPI networks contributes to oncogenesis and metastasis. The canonical transcription factor binding sites within Med25 are large (∼900 Å2) and have little topology, and thus, they do not present an array of attractive small-molecule binding sites for inhibitor discovery. Here we demonstrate that the depsidone natural product norstictic acid functions through an alternative binding site to block Med25-transcriptional activator PPIs in vitro and in cell culture. Norstictic acid targets a binding site comprising a highly dynamic loop flanking one canonical binding surface, and in doing so, it both orthosterically and allosterically alters Med25-driven transcription in a patient-derived model of triple-negative breast cancer. These results highlight the potential of Med25 as a therapeutic target as well as the inhibitor discovery opportunities presented by structurally dynamic loops within otherwise challenging proteins.

Conservation of coactivator engagement mechanism enables small-molecule allosteric modulators.

Transcriptional coactivators are a molecular recognition marvel because a single domain within these proteins, the activator binding domain or ABD, interacts with multiple compositionally diverse transcriptional activators. Also remarkable is the structural diversity among ABDs, which range from conformationally dynamic helical motifs to those with a stable core such as a ß-barrel. A significant objective is to define conserved properties of ABDs that allow them to interact with disparate activator sequences. The ABD of the coactivator Med25 (activator interaction domain or AcID) is unique in that it contains secondary structural elements that are on both ends of the spectrum: helices and loops that display significant conformational mobility and a seven-stranded ß-barrel core that is structurally rigid. Using biophysical approaches, we build a mechanistic model of how AcID forms binary and ternary complexes with three distinct activators; despite its static core, Med25 forms short-lived, conformationally mobile, and structurally distinct complexes with each of the cognate partners. Further, ternary complex formation is facilitated by allosteric communication between binding surfaces on opposing faces of the ß-barrel. The model emerging suggests that the conformational shifts and cooperative binding is mediated by a flexible substructure comprised of two dynamic helices and flanking loops, indicating a conserved mechanistic model of activator engagement across ABDs. Targeting a region of this substructure with a small-molecule covalent cochaperone modulates ternary complex formation. Our data support a general strategy for the identification of allosteric small-molecule modulators of ABDs, which are key targets for mechanistic studies as well as therapeutic applications.

X-linked inhibitor of apoptosis protein (XIAP) is a client of heat shock protein 70 (Hsp70) and a biomarker of its inhibition.

Heat shock protein 70 (Hsp70) and Hsp90 are molecular chaperones that play essential roles in tumor growth by stabilizing pro-survival client proteins. However, although the development of Hsp90 inhibitors has benefited from the identification of clients, such as Raf-1 proto-oncogene, Ser/Thr kinase (RAF1), that are particularly dependent on this chaperone, no equivalent clients for Hsp70 have been reported. Using chemical probes and MDA-MB-231 breast cancer cells, we found here that the inhibitors of apoptosis proteins, including c-IAP1 and X-linked inhibitor of apoptosis protein (XIAP), are obligate Hsp70 clients that are rapidly (within ∼3-12 h) lost after inhibition of Hsp70 but not of Hsp90. Mutagenesis and pulldown experiments revealed multiple Hsp70-binding sites on XIAP, suggesting that it is a direct, physical Hsp70 client. Interestingly, this interaction was unusually tight (∼260 nm) for an Hsp70-client interaction and involved non-canonical regions of the chaperone. Finally, we also found that Hsp70 inhibitor treatments caused loss of c-IAP1 and XIAP in multiple cancer cell lines and in tumor xenografts, but not in healthy cells. These results are expected to significantly accelerate Hsp70 drug discovery by providing XIAP as a pharmacodynamic biomarker. More broadly, our findings further suggest that Hsp70 and Hsp90 have partially non-overlapping sets of obligate protein clients in cancer cells.

Covalent Chemical Cochaperones of the p300/CBP GACKIX Domain.

The GACKIX activator binding domain has been a compelling target for small-molecule probe discovery because of the central role of activator-GACKIX complexes in diseases ranging from leukemia to memory disorders. Additionally, GACKIX is an ideal model to dissect the context-dependent function of activator-coactivator complexes. However, the dynamic and transient protein-protein interactions (PPIs) formed by GACKIX are difficult targets for small molecules. An additional complication is that activator-binding motifs, such as GACKIX, are found in multiple coactivators, making specificity difficult to attain. In this study, we demonstrate that the strategy of tethering can be used to rapidly discover highly specific covalent modulators of the dynamic PPIs between activators and coactivators. These serve as both ortho- and allosteric modulators, enabling the tunable assembly or disassembly of the activator-coactivator complexes formed between the KIX domain and its cognate activator binding partners MLL and CREB. The molecules maintain their function and selectivity, even in human cell lysates and in bacterial cells, and thus, will ultimately be highly useful probes for cellular studies.

From Fuzzy to Function: The New Frontier of Protein-Protein Interactions.

Conformationally heterogenous or "fuzzy" proteins have often been described as lacking specificity in binding and in function. The activation domains, for example, of transcriptional activators were labeled as negative noodles, with little structure or specificity. However, emerging data illustrates that the opposite is true: conformational heterogeneity enables context-specific function to emerge in response to changing cellular conditions and, furthermore, allows a single structural motif to be used in multiple settings. A further benefit is that conformational heterogeneity can be harnessed for the discovery of allosteric drug-like modulators, targeting critical pathways in protein homeostasis and transcription.

Protein cross-linking capillary electrophoresis at increased throughput for a range of protein-protein interactions.

Tools for measuring affinities and stoichiometries of protein-protein complexes are valuable for elucidating the role of protein-protein interactions (PPIs) in governing cell functions and screening for PPI modulators. Such measurements can be challenging because PPIs can span a wide range of affinities and include stoichiometries from dimers to high order oligomers. Also, most techniques require large amounts of protein which can hamper research for difficult to obtain proteins. Protein cross-linking capillary electrophoresis (PXCE) has the potential to directly measure PPIs and even resolve multiple PPIs while consuming attomole quantities. Previously PXCE has only been used for high affinity, 1 : 1 complexes; here we expand the utility of PXCE to access a wide range of PPIs including weak and multimeric oligomers. Use of glutaraldehyde as the cross-linking agent was key to advancing the method because of its rapid reaction kinetics. A 10 s reaction time was found to be sufficient for cross-linking and quantification of seven different PPIs with Kd values ranging from low µM to low nM including heat shock protein 70 (Hsp70) interacting with heat shock organizing protein (3.8 ± 0.7 µM) and bcl2 associated anthanogene (26 ± 6 nM). Non-specific cross-linking of protein aggregates was found to be minimal at protein concentrations <20 µM as assessed by size exclusion chromatography. PXCE was sensitive enough to measure changes in PPI affinity induced by the protein nucleotide state or point mutations in the protein-binding site. Further, several interactions could be resolved in a single run, including Hsp70 monomer, homodimer and Hsp70 complexed the with c-terminus of Hsp70 interacting protein (CHIP). Finally, the throughput of PXCE was increased to 1 min per sample suggesting potential for utility in screening.

BET bromodomain ligands: Probing the WPF shelf to improve BRD4 bromodomain affinity and metabolic stability.

Ligands for the bromodomain and extra-terminal domain (BET) family of bromodomains have shown promise as useful therapeutic agents for treating a range of cancers and inflammation. Here we report that our previously developed 3,5-dimethylisoxazole-based BET bromodomain ligand (OXFBD02) inhibits interactions of BRD4(1) with the RelA subunit of NF-κB, in addition to histone H4. This ligand shows a promising profile in a screen of the NCI-60 panel but was rapidly metabolised (t½â€¯= 39.8 min). Structure-guided optimisation of compound properties led to the development of the 3-pyridyl-derived OXFBD04. Molecular dynamics simulations assisted our understanding of the role played by an internal hydrogen bond in altering the affinity of this series of molecules for BRD4(1). OXFBD04 shows improved BRD4(1) affinity (IC50 = 166 nM), optimised physicochemical properties (LE = 0.43; LLE = 5.74; SFI = 5.96), and greater metabolic stability (t½â€¯= 388 min).

A Bifunctional Amino Acid Enables Both Covalent Chemical Capture and Isolation of in Vivo Protein-Protein Interactions.

In vivo covalent chemical capture by using photoactivatable unnatural amino acids (UAAs) is a powerful tool for the identification of transient protein-protein interactions (PPIs) in their native environment. However, the isolation and characterization of the crosslinked complexes can be challenging. Here, we report the first in vivo incorporation of the bifunctional UAA BPKyne for the capture and direct labeling of crosslinked protein complexes through post-crosslinking functionalization of a bioorthogonal alkyne handle. Using the prototypical yeast transcriptional activator Gal4, we demonstrate that BPKyne is incorporated at the same level as the commonly used photoactivatable UAA pBpa and effectively captures the Gal4-Gal80 transcriptional complex. Post-crosslinking, the Gal4-Gal80 adduct was directly labeled by treatment of the alkyne handle with a biotin-azide probe; this enabled facile isolation and visualization of the crosslinked adduct from whole-cell lysate. This bifunctional amino acid extends the utility of the benzophenone crosslinker and expands our toolbox of chemical probes for mapping PPIs in their native cellular environment.

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

Allosteric binding events play a critical role in the formation and stability of transcriptional activator-coactivator complexes, perhaps in part due to the often intrinsically disordered nature of one or more of the constituent partners. The kinase-inducible domain interacting (KIX) domain of the master coactivator CREB binding protein/p300 is a conformationally dynamic domain that complexes with transcriptional activators at two discrete binding sites in allosteric communication. The complexation of KIX with the transcriptional activation domain of mixed-lineage leukemia protein leads to an enhancement of binding by the activation domain of CREB (phosphorylated kinase-inducible domain of CREB) to the second site. A transient kinetic analysis of the ternary complex formation aided by small molecule ligands that induce positive or negative cooperative binding reveals that positive cooperativity is largely governed by stabilization of the bound complex as indicated by a decrease in koff. Thus, this suggests the increased binding affinity for the second ligand is not due to an allosteric creation of a more favorable binding interface by the first ligand. This is consistent with data from us and from others indicating that the on rates of conformationally dynamic proteins approach the limits of diffusion. In contrast, negative cooperativity is manifested by alterations in both kon and koff, suggesting stabilization of the binary complex.

Prepaying the entropic cost for allosteric regulation in KIX.

The kinase-inducible domain interacting (KIX) domain of the CREB binding protein (CBP) is capable of simultaneously binding two intrinsically disordered transcription factors, such as the mixed-lineage leukemia (MLL) and c-Myb peptides, at isolated interaction sites. In vitro, the affinity for binding c-Myb is approximately doubled when KIX is in complex with MLL, which suggests a positive cooperative binding mechanism, and the affinity for MLL is also slightly increased when KIX is first bound by c-Myb. Expanding the scope of recent NMR and computational studies, we explore the allosteric mechanism at a detailed molecular level that directly connects the microscopic structural dynamics to the macroscopic shift in binding affinities. To this end, we have performed molecular dynamics simulations of free KIX, KIX-c-Myb, MLL-KIX, and MLL-KIX-c-Myb using a topology-based Go-like model. Our results capture an increase in affinity for the peptide in the allosteric site when KIX is prebound by a complementary effector and both peptides follow an effector-independent folding-and-binding mechanism. More importantly, we discover that MLL binding lowers the entropic cost for c-Myb binding, and vice versa, by stabilizing the L12-G2 loop and the C-terminal region of the α3 helix on KIX. This work demonstrates the importance of entropy in allosteric signaling between promiscuous molecular recognition sites and can inform the rational design of small molecule stabilizers to target important regions of conformationally dynamic proteins.

Discovery of Enzymatic Targets of Transcriptional Activators via in Vivo Covalent Chemical Capture.

The network of activator protein-protein interactions (PPIs) that underpin transcription initiation is poorly defined, particularly in the cellular context. The transient nature of these contacts and the often low abundance of the participants present significant experimental hurdles. Through the coupling of in vivo covalent chemical capture and shotgun LC-MS/MS (MuDPIT) analysis, we can trap the PPIs of transcriptional activators in a cellular setting and identify the binding partners in an unbiased fashion. Using this approach, we discover that the prototypical activators Gal4 and VP16 target the Snf1 (AMPK) kinase complex via direct interactions with both the core enzymatic subunit Snf1 and the exchangeable subunit Gal83. Further, we use a tandem reversible formaldehyde and irreversible covalent chemical capture approach (TRIC) to capture the Gal4-Snf1 interaction at the Gal1 promoter in live yeast. Together, these data support a critical role for activator PPIs in both the recruitment and positioning of important enzymatic complexes at a gene promoter and represent a technical advancement in the discovery of new cellular binding targets of transcriptional activators.

A Synthetic Loop Replacement Peptide That Blocks Canonical NF-κB Signaling.

Aberrant canonical NF-κB signaling is implicated in diseases from autoimmune disorders to cancer. A major therapeutic challenge is the need for selective inhibition of the canonical pathway without impacting the many non-canonical NF-κB functions. Here we show that a selective peptide-based inhibitor of canonical NF-κB signaling, in which a hydrogen bond in the NBD peptide is synthetically replaced by a non-labile bond, shows an about 10-fold increased potency relative to the original inhibitor. Not only is this molecule, NBD2, a powerful tool for dissection of canonical NF-κB signaling in disease models and healthy tissues, the success of the synthetic loop replacement suggests that the general strategy could be useful for discovering modulators of the many protein-protein interactions mediated by such structures.

Direct measurements of oscillatory glycolysis in pancreatic islet ß-cells using novel fluorescence resonance energy transfer (FRET) biosensors for pyruvate kinase M2 activity.

Pulses of insulin released from pancreatic ß-cells maintain blood glucose in a narrow range, although the source of these pulses is unclear. We and others have proposed that positive feedback mediated by the glycolytic enzyme phosphofructokinase-1 (PFK1) enables ß-cells to generate metabolic oscillations via autocatalytic activation by its product fructose 1,6-bisphosphate (FBP). Although much indirect evidence has accumulated in favor of this hypothesis, a direct measurement of oscillating glycolytic intermediates has been lacking. To probe glycolysis directly, we engineered a family of inter- and intramolecular FRET biosensors based on the glycolytic enzyme pyruvate kinase M2 (PKAR; pyruvate kinase activity reporter), which multimerizes and is activated upon binding FBP. When introduced into Min6 ß-cells, PKAR FRET efficiency increased rapidly in response to glucose. Importantly, however, metabolites entering downstream of PFK1 (glyceraldehyde, pyruvate, and ketoisocaproate) failed to activate PKAR, consistent with sensor activation by FBP; the dependence of PKAR on FBP was further confirmed using purified sensor in vitro. Using a novel imaging modality for monitoring mitochondrial flavin fluorescence in mouse islets, we show that slow oscillations in mitochondrial redox potential stimulated by 10 mm glucose are in phase with glycolytic efflux through PKM2, measured simultaneously from neighboring islet ß-cells expressing PKAR. These results indicate that PKM2 activity in ß-cells is oscillatory and are consistent with pulsatile PFK1 being the mediator of slow glycolytic oscillations.