Discovery of Novel PROTAC Degraders of p300/CBP as Potential Therapeutics for Hepatocellular Carcinoma.

Adenoviral E1A binding protein 300 kDa (p300) and its closely related paralog CREB binding protein (CBP) are promising therapeutic targets for human cancer. Here, we report the first discovery of novel potent small-molecule PROTAC degraders of p300/CBP against hepatocellular carcinoma (HCC), one of the most common solid tumors. Based upon the clinical p300/CBP bromodomain inhibitor CCS1477, a conformational restriction strategy was used to optimize the linker to generate a series of PROTACs, culminating in the identification of QC-182. This compound effectively induces p300/CBP degradation in the SK-HEP-1 HCC cells in a dose-, time-, and ubiquitin-proteasome system-dependent manner. QC-182 significantly downregulates p300/CBP-associated transcriptome in HCC cells, leading to more potent cell growth inhibition compared to the parental inhibitors and the reported degrader dCBP-1. Notably, QC-182 potently depletes p300/CBP proteins in mouse SK-HEP-1 xenograft tumor tissue. QC-182 is a promising lead compound toward the development of p300/CBP-targeted HCC therapy.

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.

Molecular insight into CREBBP and TANGO2 variants causing intellectual disability.

BACKGROUND: Intellectual disability (ID) can be associated with different syndromes such as Rubinstein-Taybi syndrome (RSTS) and can also be related to conditions such as metabolic encephalomyopathic crises, recurrent,with rhabdomyolysis, cardiac arrhythmias and neurodegeneration. Rare congenital RSTS1 (OMIM 180849) is characterized by mental and growth retardation, significant and duplicated distal phalanges of thumbs and halluces, facial dysmorphisms, and an elevated risk of malignancies. Microdeletions and point mutations in the CREB-binding protein (CREBBP) gene, located at 16p13.3, have been reported to cause RSTS. By contrast, TANGO2-related metabolic encephalopathy and arrhythmia (TRMEA) is a rare metabolic condition that causes repeated metabolic crises, hypoglycemia, lactic acidosis, rhabdomyolysis, arrhythmias and encephalopathy with cognitive decline. Clinicians need more clinical and genetic evidence to detect and comprehend the phenotypic spectrum of this disorder. METHODS: Exome sequencing was used to identify the disease-causing variants in two affected families A and B from District Kohat and District Karak, Khyber Pakhtunkhwa. Affected individuals from both families presented symptoms of ID, developmental delay and behavioral abnormalities. The validation and co-segregation analysis of the filtered variant was carried out using Sanger sequencing. RESULTS: In the present study, two families (A and B) exhibiting various forms of IDs were enrolled. In Family A, exome sequencing revealed a novel missense variant (NM 004380.3: c.4571A>G; NP_004371.2: p.Lys1524Arg) in the CREBBP gene, whereas, in Family B, a splice site variant (NM 152906.7: c.605 + 1G>A) in the TANGO2 gene was identified. Sanger sequencing of both variants confirmed their segregation with ID in both families. The in silico tools verified the aberrant changes in the CREBBP protein structure. Wild-type and mutant CREBBP protein structures were superimposed and conformational changes were observed likely altering the protein function. CONCLUSIONS: RSTS and TRMEA are exceedingly rare disorders for which specific clinical characteristics have been clearly established, but more investigations are underway and required. Multicenter studies are needed to increase our understanding of the clinical phenotypes, mainly showing the genotype-phenotype associations.

Comparative Analysis of Drug-like EP300/CREBBP Acetyltransferase Inhibitors.

The human acetyltransferase paralogues EP300 and CREBBP are master regulators of lysine acetylation whose activity has been implicated in various cancers. In the half-decade since the first drug-like inhibitors of these proteins were reported, three unique molecular scaffolds have taken precedent: an indane spiro-oxazolidinedione (A-485), a spiro-hydantoin (iP300w), and an aminopyridine (CPI-1612). Despite increasing use of these molecules to study lysine acetylation, the dearth of data regarding their relative biochemical and biological potencies makes their application as chemical probes a challenge. To address this gap, here we present a comparative study of drug-like EP300/CREBBP acetyltransferase inhibitors. First, we determine the biochemical and biological potencies of A-485, iP300w, and CPI-1612, highlighting the increased potencies of the latter two compounds at physiological acetyl-CoA concentrations. Cellular evaluation shows that inhibition of histone acetylation and cell growth closely aligns with the biochemical potencies of these molecules, consistent with an on-target mechanism. Finally, we demonstrate the utility of comparative pharmacology by using it to investigate the hypothesis that increased CoA synthesis caused by knockout of PANK4 can competitively antagonize the binding of EP300/CREBBP inhibitors and demonstrate proof-of-concept photorelease of a potent inhibitor molecule. Overall, our study demonstrates how knowledge of the relative inhibitor potency can guide the study of EP300/CREBBP-dependent mechanisms and suggests new approaches to target delivery, thus broadening the therapeutic window of these preclinical epigenetic drug candidates.

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.

Self-Effected Allosteric Coupling and Cooperativity in Hypoxic Response Regulation with Disordered Proteins.

Hypersensitive regulation of cellular hypoxic response relies on cooperative displacement of one disordered protein (HIF-1α) by another disordered protein (CITED2) from the target in a negative feedback loop. Considering the weak intramolecule coupling in disordered proteins, the molecular mechanism of high cooperativity in the molecular displacement event remains elusive. Herein, we show that disordered proteins utilize a "self-effected allostery" mechanism to achieve high binding cooperativity. Different from the conventional allostery mechanisms shown by many structured or disordered proteins, this mechanism utilizes one part of the disordered protein as the effector to trigger the allosteric coupling and enhance the binding of the remaining part of the same disordered protein, contributing to high cooperativity of the displacement event. The conserved charge motif of CITED2 is the key determinant of the molecular displacement event by serving as the effector of allosteric coupling. Such self-effected allostery provides an efficient strategy to achieve high cooperativity in the molecular events involving disordered proteins.

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.

Dynamics of the Histone Acetyltransferase Lysine-Rich Loop in the Catalytic Core of the CREB-Binding Protein.

The tight control of transcriptional coactivators is a fundamental aspect of gene expression in cells. The regulation of the CREB-binding protein (CBP) and p300 coactivators, two paralog multidomain proteins, involves an autoinhibitory loop (AIL) of the histone acetyltransferase (HAT) domain. There is experimental evidence for the AIL engaging with the HAT binding site, thus interrupting the acetylation of histone tails or other proteins. Both CBP and p300 contain a domain of about 110 residues (called the bromodomain) that recognizes histone tails with one or more acetylated lysine side chains. Here, we investigate by molecular dynamics simulations whether the AIL of CBP (residues 1556-1618) acetylated at the side chain of Lys1595 can bind to the bromodomain. The structural instability and fast unbinding kinetics of the AIL from the bromodomain pocket suggest that the AIL is not a ligand of the bromodomain on the same protein chain. This is further supported by the absence of strong and persistent contacts at the binding interface. Furthermore, the simulations of unbinding show an initial fast detachment of the acetylated lysine and a slower phase necessary for complete AIL dissociation. We provide further evidence for the instability of the AIL intramolecular binding by comparison with a natural ligand, the histone peptide H3K56ac, which shows higher stability in the pocket.

Rational design of a helical peptide inhibitor targeting c-Myb-KIX interaction.

The transcription factor c-Myb promotes the proliferation of hematopoietic cells by interacting with the KIX domain of CREB-binding protein; however, its aberrant expression causes leukemia. Therefore, inhibitors of the c-Myb-KIX interaction are potentially useful as antitumor drugs. Since the intrinsically disordered transactivation domain (TAD) of c-Myb binds KIX via a conformational selection mechanism where helix formation precedes binding, stabilizing the helical structure of c-Myb TAD is expected to increase the KIX-binding affinity. Here, to develop an inhibitor of the c-Myb-KIX interaction, we designed mutants of the c-Myb TAD peptide fragment where the helical structure is stabilized, based on theoretical predictions using AGADIR. Three of the four initially designed peptides each had a different Lys-to-Arg substitution on the helix surface opposite the KIX-binding interface. Furthermore, the triple mutant with three Lys-to-Arg substitutions, named RRR, showed a high helical propensity and achieved three-fold higher affinity to KIX than the wild-type TAD with a dissociation constant of 80 nM. Moreover, the RRR inhibitor efficiently competed out the c-Myb-KIX interaction. These results suggest that stabilizing the helical structure based on theoretical predictions, especially by conservative Lys-to-Arg substitutions, is a simple and useful strategy for designing helical peptide inhibitors of protein-protein interactions.

Characterization of the High-Affinity Fuzzy Complex between the Disordered Domain of the E7 Oncoprotein from High-Risk HPV and the TAZ2 Domain of CBP.

The intrinsically disordered N-terminal region of the E7 protein from high-risk human papillomavirus (HPV) strains is responsible for oncogenic transformation of host cells through its interaction with a number of cellular factors, including the TAZ2 domain of the transcriptional coactivator CREB-binding protein. Using a variety of spectroscopic and biochemical tools, we find that despite its nanomolar affinity, the HPV16 E7 complex with TAZ2 is disordered and highly dynamic. The disordered domain of HPV16 E7 protein does not adopt a single conformation on the surface of TAZ2 but engages promiscuously with its target through multiple interactions involving two conserved motifs, termed CR1 and CR2, that occupy an extensive binding surface on TAZ2. The fuzzy nature of the complex is a reflection of the promiscuous binding repertoire of viral proteins, which must efficiently dysregulate host cell processes by binding to a variety of host factors in the cellular environment.

Universal two-point interaction of mediator KIX with 9aaTAD activation domains.

The nine-amino-acid activation domain (9aaTAD) is defined by a short amino acid pattern including two hydrophobic regions (positions p3-4 and p6-7). The KIX domain of mediator transcription CBP interacts with the 9aaTAD domains of transcription factors MLL, E2A, NF-kB, and p53. In this study, we analyzed the 9aaTADs-KIX interactions by nuclear magnetic resonance. The positions of three KIX helixes α1-α2-α3 are influenced by sterically-associated hydrophobic I611, L628, and I660 residues that are exposed to solvent. The positions of two rigid KIX helixes α1 and α2 generate conditions for structural folding in the flexible KIX-L12-G2 regions localized between them. The three KIX I611, L628, and I660 residues interact with two 9aaTAD hydrophobic residues in positions p3 and p4 and together build a hydrophobic core of five residues (5R). Numerous residues in 9aaTAD position p3 and p4 could provide this interaction. Following binding of the 9aaTAD to KIX, the hydrophobic I611, L628, and I660 residues are no longer exposed to solvent and their position changes inside the hydrophobic core together with position of KIX α1-α2-α3 helixes. The new positions of the KIX helixes α1 and α2 allow the KIX-L12-G2 enhanced formation. The second hydrophobic region of the 9aaTAD (positions p6 and p7) provides strong binding with the KIX-L12-G2 region. Similarly, multiple residues in 9aaTAD position p6 and p7 could provide this interaction. In conclusion, both 9aaTAD regions p3, p4 and p6, p7 provide co-operative and highly universal binding to mediator KIX. The hydrophobic core 5R formation allows new positions of the rigid KIX α-helixes and enables the enhanced formation of the KIX-L12-G2 region. This contributes to free energy and is the key for the KIX-9aaTAD binding. Therefore, the 9aaTAD-KIX interactions do not operate under the rigid key-and-lock mechanism what explains the 9aaTAD natural variability.

Insights into interaction mechanism of inhibitors E3T, E3H and E3B with CREB binding protein by using molecular dynamics simulations and MM-GBSA calculations.

CREB binding protein (CBP) and its paralog E1A binding protein (p300) are related to the development of inflammatory diseases, cancers and other diseases, and have been potential targets for the treatment of human diseases. In this work, interaction mechanism of three small molecules E3T, E3H, and E3B with CBP was investigated by employing molecular dynamics (MD) simulations, principal component analysis (PCA), and molecular mechanics/generalized born surface area (MM-GBSA) method. The results indicate that inhibitor bindings cause the changes of movement modes and structural flexibility of CBP, and van der Waals interactions mostly drive associations of inhibitors with CBP. In the meantime, the results based on inhibitor-residue interactions not only show that eight residues of CBP can strongly interact with E3T, E3H and E3B but also verify that the CH-CH, CH-π, and π-π interactions are responsible for vital contributions in associations of E3T, E3H and E3B with CBP. In addition, the H-O radial distribution functions (RDFs) were computed to assess the stability of hydrogen bonding interactions between inhibitors and CBP, and the obtained information identifies several key hydrogen bonds playing key roles in bindings of E3T, E3H and E3B to CBP. Potential new inhibitors have been proposed.

Update and Potential Opportunities in CBP [Cyclic Adenosine Monophosphate (cAMP) Response Element-Binding Protein (CREB)-Binding Protein] Research Using Computational Techniques.

CBP [cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB)-binding protein] is one of the most researched proteins for its therapeutic function. Several studies have identified its vast functions and interactions with other transcription factors to initiate cellular signals of survival. In cancer and other diseases such as Alzheimer's, Rubinstein-taybi syndrome, and inflammatory diseases, CBP has been implicated and hence an attractive target in drug design and development. In this review, we explore the various computational techniques that have been used in CBP research, furthermore we identified computational gaps that could be explored to facilitate the development of highly therapeutic CBP inhibitors.

The interaction strength of an intrinsically disordered protein domain with its binding partner is little affected by very different cosolutes.

A common feature of intrinsically disordered proteins (IDPs) is a disorder-to-order transition upon binding to other proteins, which has been tied to multiple benefits, including accelerated association rates or binding with low affinity, yet high specificity. Given the balanced equilibrium concentrations of folded and unfolded state of an IDP we asked the question if changes in the chemical environment, such as the presence of osmolytes or crowding agents, have a strong influence on the interaction of an IDP. Here, we demonstrate the impact of cosolutes on the interaction of the intrinsically disordered transcription factor c-Myb and its binding partner, the kinase-inducible interaction domain (KIX) of the CREB-binding protein. Temperature jump relaxation kinetics and microscale thermophoresis were employed in order to quantify the rate constants and the binding affinity of the c-Myb/KIX complex, respectively, in the presence of various cosolutes. We find the binding free energy of the c-Myb/KIX complex only marginally modulated by cosolutes, whereas the enthalpy and entropy of the interaction are very sensitive to the respective solvent conditions. For different cosolutes we observe substantial changes in enthalpy, both favorable and unfavorable, which are going with entropy changes largely compensating the enthalpy effects in each case. These characteristics might reflect a potential mechanism by which c-Myb offsets changes in the physico-chemical environment to maintain a roughly unaltered binding affinity.

The CBP KIX domain regulates long-term memory and circadian activity.

BACKGROUND: CREB-dependent transcription necessary for long-term memory is driven by interactions with CREB-binding protein (CBP), a multi-domain protein that binds numerous transcription factors potentially affecting expression of thousands of genes. Identifying specific domain functions for multi-domain proteins is essential to understand processes such as cognitive function and circadian clocks. We investigated the function of the CBP KIX domain in hippocampal memory and gene expression using CBPKIX/KIX mice with mutations that prevent phospho-CREB (Ser133) binding. RESULTS: We found that CBPKIX/KIX mice were impaired in long-term memory, but not learning acquisition or short-term memory for the Morris water maze. Using an unbiased analysis of gene expression in the dorsal hippocampus after training in the Morris water maze or contextual fear conditioning, we discovered dysregulation of CREB, CLOCK, and BMAL1 target genes and downregulation of circadian genes in CBPKIX/KIX mice. Given our finding that the CBP KIX domain was important for transcription of circadian genes, we profiled circadian activity and phase resetting in CBPKIX/KIX mice. CBPKIX/KIX mice exhibited delayed activity peaks after light offset and longer free-running periods in constant dark. Interestingly, CBPKIX/KIX mice displayed phase delays and advances in response to photic stimulation comparable to wildtype littermates. Thus, this work delineates site-specific regulation of the circadian clock by a multi-domain protein. CONCLUSIONS: These studies provide insight into the significance of the CBP KIX domain by defining targets of CBP transcriptional co-activation in memory and the role of the CBP KIX domain in vivo on circadian rhythms.

Screening of a large Rubinstein-Taybi cohort identified many novel variants and emphasizes the importance of the CREBBP histone acetyltransferase domain.

Pathogenic variants within the CREBBP and EP300 genes account for the majority of individuals with Rubinstein-Taybi syndrome (RSTS). Data are presented from a large cohort of 395 individuals referred for diagnostic testing of CREBBP, and of the 19 CREBBP missense variants classified as likely pathogenic in this study, 17 were within the histone acetyltransferase (HAT) domain, providing evidence that this domain is critical to the normal function of the CREBBP protein (CBP). The data presented here, combined with other published results, suggest that the presence of a missense variant within the CBP HAT domain can be considered as moderate evidence of pathogenicity in the context of official variant interpretation guidelines. Within our study cohort, 129 had a pathogenic or likely pathogenic CREBBP variant and 5 had a variant of uncertain significance (VUS) which warranted familial studies. 147 of the remaining probands were also screened for EP300 and a further 16 pathogenic or likely pathogenic variants were identified, plus one VUS. Therefore, this analysis has provided a molecular diagnosis in at least 145 individuals with RSTS (37%) and identified a wide range of variants (n = 133) of which 103 were novel.

Exploiting binding-site arginines in drug design: Recent examples.

Active or allosteric site arginines can form diverse interactions with ligands including different types of cation-π interactions, H-bond interactions and non-bond, non-canonical interactions. This provides many opportunities for creative structure-based drug design to improve potency, introduce novelty, and modulate MoA (mode of action), and even to achieve selectivity. This digest will use some recent drug targets of interest as examples to illustrate different types of interactions and how these interactions impact on potency, MoA, and selectivity.

Design, synthesis, and biological evaluation of tetrahydroquinolin derivatives as potent inhibitors of CBP bromodomain.

CREB-binding protein (CBP) is a large multi-domain protein containing a HAT domain catalyzing transacetylation and a bromodomain responsible for acetylated lysine recognition. CBPs could act as transcription co-activators to regulate gene expression and have been shown to play a significant role in the development and progression of many cancers. Herein, through in silico screening two hit compounds with tetrahydroquinolin methyl carbamate scaffold were discovered, among which DC-CPin7 showed an in vitro inhibitory activity with the TR-FRET IC50 value of 2.5 ± 0.3 µM. We obtained a high-resolution co-crystal structure of the CBP bromodomain in complex with DC-CPin7 to guide following structure-based rational drug design, which yielded over ten DC-CPin7 derivatives with much higher potency, among which DC-CPin711 showed approximately 40-fold potency compared with hit compound DC-CPin7 with an in vitro TR-FRET IC50 value of 63.3 ± 4.0 nM. Notably, DC-CPin711 showed over 150-fold selectivity against BRD4 bromodomains. Moreover, DC-CPin711 showed micromolar level of anti-leukemia proliferation through G1 phase cell cycle arrest and cell apoptosis. In summary, through a combination of computational and crystal-based structure optimization, DC-CPin711 showed potent in vitro inhibitory activities to CBP bromodomain with a decent selectivity towards BRD4 bromodomains and good cellular activity to leukemia cells, which could further be applied to related biological and translational studies as well as serve as a lead compound for future development of potent and selective CBP bromodomain inhibitors.

Revisiting allostery in CREB-binding protein (CBP) using residue-based interaction energy.

CREB-binding protein (CBP) is a multi-subunit scaffold protein complex in transcription regulation process, binding and interacting with ligands such as mixed-lineage leukemia (MLL) and c-Myb allosterically. Here in this study, we have revisited the concept of allostery in CBP via residue-based interaction energy calculation based on molecular dynamics (MD) simulations. To this end, we conducted MD simulations of KIX:MLL:c-Myb ternary complex, its binary components and kinase-inducible domain (KID) interacting domain (KIX) backbone. Interaction energy profiles and cross correlation analysis were performed and the results indicated that KIX:MLL and KIX:c-Myb:MLL complexes demonstrate significant similarities according to both analysis methods. Two regions in the KIX backbone were apparent from the interaction energy and cross correlation maps that hold a key to allostery phenomena observed in CBP. While one of these regions are related to the ligand binding residues, the other comprises of L12-G2 loop and α3 helix regions that have been found to have a significant role in allosteric signal propagation. All in all, residue-based interaction energy calculation method is demonstrated to be a valuable calculation technique for the detection of allosteric signal propagation and ligand interaction regions.

Aggregation and Amyloidogenicity of the Nuclear Coactivator Binding Domain of CREB-Binding Protein.

The nuclear coactivator binding domain (NCBD) of transcriptional co-regulator CREB-binding protein (CBP) is an example of conformationally malleable proteins that can bind to structurally unrelated protein targets and adopt distinct folds in the respective protein complexes. Here, we show that the folding landscape of NCBD contains an alternative pathway that results in protein aggregation and self-assembly into amyloid fibers. The initial steps of such protein misfolding are driven by intermolecular interactions of its N-terminal α-helix bringing multiple NCBD molecules into contact. These oligomers then undergo slow but progressive interconversion into ß-sheet-containing aggregates. To reveal the concealed aggregation potential of NCBD we used a chemically synthesized mirror-image d-NCBD form. The addition of d-NCBD promoted self-assembly into amyloid precipitates presumably due to formation of thermodynamically more stable racemic ß-sheet structures. The unexpected aggregation of NCBD needs to be taken into consideration given the multitude of protein-protein interactions and resulting biological functions mediated by CBP.