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DNA damage in oocytes induces a switch of the quality control factor TAp63α from dimer to tetramer
(2011)
TAp63a, a homolog of the p53 tumor suppressor, is a quality control factor in the female germline. Remarkably, already undamaged oocytes express high levels of the protein, suggesting that TAp63a’s activity is under tight control of an inhibitory mechanism. Biochemical studies have proposed that inhibition requires the C-terminal transactivation inhibitory domain. However, the structural mechanism of TAp63a inhibition remains unknown. Here, we show that TAp63a is kept in an inactive dimeric state. We reveal that relief of inhibition leads to tetramer formation with ~20-fold higher DNA affinity. In vivo, phosphorylation-triggered tetramerization of TAp63a is not reversible by dephosphorylation. Furthermore, we show that a helix in the oligomerization domain of p63 is crucial for tetramer stabilization and competes with the transactivation domain for the same binding site. Our results demonstrate how TAp63a is inhibited by complex domain-domain interactions that provide the basis for regulating quality control in oocytes.
Binding free energy calculations that make use of alchemical pathways are becoming increasingly feasible thanks to advances in hardware and algorithms. Although relative binding free energy (RBFE) calculations are starting to find widespread use, absolute binding free energy (ABFE) calculations are still being explored mainly in academic settings due to the high computational requirements and still uncertain predictive value. However, in some drug design scenarios, RBFE calculations are not applicable and ABFE calculations could provide an alternative. Computationally cheaper end-point calculations in implicit solvent, such as molecular mechanics Poisson–Boltzmann surface area (MMPBSA) calculations, could too be used if one is primarily interested in a relative ranking of affinities. Here, we compare MMPBSA calculations to previously performed absolute alchemical free energy calculations in their ability to correlate with experimental binding free energies for three sets of bromodomain–inhibitor pairs. Different MMPBSA approaches have been considered, including a standard single-trajectory protocol, a protocol that includes a binding entropy estimate, and protocols that take into account the ligand hydration shell. Despite the improvements observed with the latter two MMPBSA approaches, ABFE calculations were found to be overall superior in obtaining correlation with experimental affinities for the test cases considered. A difference in weighted average Pearson () and Spearman () correlations of 0.25 and 0.31 was observed when using a standard single-trajectory MMPBSA setup ( = 0.64 and = 0.66 for ABFE; = 0.39 and = 0.35 for MMPBSA). The best performing MMPBSA protocols returned weighted average Pearson and Spearman correlations that were about 0.1 inferior to ABFE calculations: = 0.55 and = 0.56 when including an entropy estimate, and = 0.53 and = 0.55 when including explicit water molecules. Overall, the study suggests that ABFE calculations are indeed the more accurate approach, yet there is also value in MMPBSA calculations considering the lower compute requirements, and if agreement to experimental affinities in absolute terms is not of interest. Moreover, for the specific protein–ligand systems considered in this study, we find that including an explicit ligand hydration shell or a binding entropy estimate in the MMPBSA calculations resulted in significant performance improvements at a negligible computational cost.
The chemopreventive and anticancer effects of resveratrol (RSV) are widely reported in the literature. Specifically, mechanisms involving epigenetic regulation are promising targets to regulate tumor development. Bromodomains act as epigenetic readers by recognizing lysine acetylation on histone tails and boosting gene expression in order to regulate tissue-specific transcription. In this work, we showed that RSV is a pan-BET inhibitor. Using Differential Scanning Fluorimetry (DSF), we showed that RSV at 100 µM increased the melting temperature (∆Tm) of BET bromodomains by around 2.0 °C. The micromolar dissociation constant (Kd) range was characterized using Isothermal Titration Calorimetry (ITC). The RSV Kd value accounted to 6.6 µM in case of BRD4(1). Molecular docking proposed the binding mode of RSV against BRD4(1) mimicking the acetyl-lysine interactions. All these results suggest that RSV can also recognize epigenetic readers domains by interacting with BET bromodomains.
The β-carboline alkaloid harmine is a potent DYRK1A inhibitor, but suffers from undesired potent inhibition of MAO-A, which strongly limits its application. We synthesized more than 60 analogues of harmine, either by direct modification of the alkaloid or by de novo synthesis of β-carboline and related scaffolds aimed at learning about structure-activity relationships for inhibition of both DYRK1A and MAO-A, with the ultimate goal of separating desired DYRK1A inhibition from undesired MAO-A inhibition. Based on evidence from published crystal structures of harmine bound to each of these enzymes, we performed systematic structure modifications of harmine yielding DYRK1A-selective inhibitors characterized by small polar substituents at N-9 (which preserve DYRK1A inhibition and eliminate MAO-A inhibition) and beneficial residues at C-1 (methyl or chlorine). The top compound AnnH75 remains a potent DYRK1A inhibitor, and it is devoid of MAO-A inhibition. Its binding mode to DYRK1A was elucidated by crystal structure analysis, and docking experiments provided additional insights for this attractive series of DYRK1A and MAO-A inhibitors.
The P300/CBP-associated factor plays a central role in retroviral infection and cancer development, and the C-terminal bromodomain provides an opportunity for selective targeting. Here, we report several new classes of acetyl-lysine mimetic ligands ranging from mM to low micromolar affinity that were identified using fragment screening approaches. The binding modes of the most attractive fragments were determined using high resolution crystal structures providing chemical starting points and structural models for the development of potent and selective PCAF inhibitors.
Characterization of a dual BET/HDAC inhibitor for treatment of pancreatic ductal adenocarcinoma
(2020)
Pancreatic ductal adenocarcinoma (PDAC) is resistant to virtually all chemo‐ and targeted therapeutic approaches. Epigenetic regulators represent a novel class of drug targets. Among them, BET and HDAC proteins are central regulators of chromatin structure and transcription, and preclinical evidence suggests effectiveness of combined BET and HDAC inhibition in PDAC. Here, we describe that TW9, a newly generated adduct of the BET inhibitor (+)‐JQ1 and class I HDAC inhibitor CI994, is a potent dual inhibitor simultaneously targeting BET and HDAC proteins. TW9 has a similar affinity to BRD4 bromodomains as (+)‐JQ1 and shares a conserved binding mode, but is significantly more active in inhibiting HDAC1 compared to the parental HDAC inhibitor CI994. TW9 was more potent in inhibiting tumor cell proliferation compared to (+)‐JQ1, CI994 alone or combined treatment of both inhibitors. Sequential administration of gemcitabine and TW9 showed additional synergistic antitumor effects. Microarray analysis revealed that dysregulation of a FOSL1‐directed transcriptional program contributed to the antitumor effects of TW9. Our results demonstrate the potential of a dual chromatin‐targeting strategy in the treatment of PDAC and provide a rationale for further development of multitarget inhibitors.