Assessment of the performance of the Ames MPF™ assay: A multicenter collaborative study with six coded chemicals.

The Ames MPF™ is a miniaturized, microplate fluctuation format of the Ames test. It is a standardized, commercially available product which can be used to assess mutagenicity in Salmonella and E. coli strains in 384-well plates using a color change-based readout. Several peer-reviewed comparisons of the Ames MPF™ to the Ames test in Petri dishes confirmed its suitability to evaluate the mutagenic potential of a variety of test items. An international multicenter study involving seven laboratories tested six coded chemicals with this assay using five bacterial strains, as recommended by the OECD test guideline 471. The data generated by the participating laboratories was in excellent agreement (93%), and the similarity of their dose response curves, as analyzed with sophisticated statistical approaches further confirmed the suitability of the Ames MPF™ assay as an alternative to the Ames test on agar plates, but with advantages with respect to significantly reduced amount of test substance and S9 requirements, speed, hands-on time and, potentially automation.

Identification of a BAZ2A-Bromodomain Hit Compound by Fragment Growing.

BAZ2A is an epigenetic regulator affecting transcription of ribosomal RNA. It is overexpressed in aggressive and recurrent prostate cancer, promoting cellular migration. Its bromodomain is characterized by a shallow and difficult-to-drug pocket. Here, we describe a structure-based fragment-growing campaign for the identification of ligands of the BAZ2A bromodomain. By combining docking, competition binding assays, and protein crystallography, we have extensively explored the interactions of the ligands with the rim of the binding pocket, and in particular ionic interactions with the side chain of Glu1820, which is unique to BAZ2A. We present 23 high-resolution crystal structures of the holo BAZ2A bromodomain and analyze common bromodomain/ligand motifs and favorable intraligand interactions. Binding of some of the compounds is enantiospecific, with affinity in the low micromolar range. The most potent ligand has an equilibrium dissociation constant of 7 µM and a good selectivity over the paralog BAZ2B bromodomain.

Assessment of the miniaturized liquid Ames microplate format (MPF™) for a selection of the test items from the recommended list of genotoxic and non-genotoxic chemicals.

The Ames microplate format (MPF™) is a miniaturized version of the plate agar Ames tests that takes advantage of a liquid microplate approach in 384-well plates with a color change-based readout. This method, already compared to the Ames test in Petri dishes, is used to assess the genotoxic potential of a variety of test items, including (but not limited to) chemicals, environmental samples, and drug candidates. 61 chemicals were selected from the updated recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests and tested in up to five bacterial strains. The agreement with the data from the scientific literature (over 90%) confirms the reliability of the Ames MPF™ as a cost-effective and 3R-compliant alternative to the regulatory Ames test that allows to predict and evaluate chemicals' mutagenicity in a faster, less laborious and, if available, automatable manner.

Hitting a Moving Target: Simulation and Crystallography Study of ATAD2 Bromodomain Blockers.

Small molecule ligand binding to the ATAD2 bromodomain is investigated here through the synergistic combination of molecular dynamics and protein crystallography. A previously unexplored conformation of the binding pocket upon rearrangement of the gatekeeper residue Ile1074 has been found. Further, our investigations reveal how minor structural differences in the ligands result in binding with different plasticity of the ZA loop for this difficult-to-drug bromodomain.

Understanding the mechanism of action of pyrrolo[3,2-b]quinoxaline-derivatives as kinase inhibitors.

The X-ray structure of the catalytic domain of the EphA3 tyrosine kinase in complex with a previously reported type II inhibitor was used to design two novel quinoxaline derivatives, inspired by kinase inhibitors that have reached clinical development. These two new compounds were characterized by an array of cell-based assays and gene expression profiling experiments. A global chemical proteomics approach was used to generate the drug-protein interaction profile, which suggested suitable therapeutic indications. Both inhibitors, studied in the context of angiogenesis and in vivo in a relevant lymphoma model, showed high efficacy in the control of tumor size.

Ligand retargeting by binding site analogy.

The DNA-repair enzyme MutT homolog 1 (MTH1) is a potential target for a broad range of tumors. Its substrate binding site features a non-catalytical pair of aspartic acids which resembles the catalytic dyad of aspartic proteases. We hypothesized that inhibitors of the latter might be re-targeted for MTH1 despite the two enzyme classes having different substrates and catalyze different reactions. We selected from the crystal structures of holo aspartic proteases a library of nearly 350 inhibitors for in silico screening. Three fragment hits were identified by docking and scoring according to a force field-based energy with continuum dielectric solvation. These fragments showed good ligand efficiency in a colorimetric assay (MW <300 Da and IC50<50µM). Molecular dynamics simulations were carried out for determining the most favorable interaction patterns. On the basis of the simulation results we evaluated in vitro seven commercially available compounds, two of which showed submicromolar potency for MTH1. To obtain definitive evidence of the predicted binding modes we solved the crystal structures of five of the 10 inhibitors predicted in silico. The final step of hit optimization was guided by protein crystallography and involved the synthesis of a single compound, the lead 11, which shows nanomolar affinity for MTH1 in two orthogonal binding assays, and selectivity higher than 2000-fold against its original target (BACE1). The high rate of fragment-hit identification and the fast optimization suggest that ligand retargeting by binding site analogy is an efficient strategy for drug design.

Structural Analysis of Small-Molecule Binding to the BAZ2A and BAZ2B Bromodomains.

The bromodomain-containing protein BAZ2A is a validated target in prostate cancer research, whereas the function of its paralogue BAZ2B is still undefined. The bromodomains of BAZ2A and BAZ2B have a similar binding site for their natural ligand, the acetylated lysine side chain. Here, we present an analysis of the binding modes of eight compounds belonging to three distinct chemical classes. For all compounds, the moiety mimicking the natural ligand engages in essentially identical interactions in the BAZ2A and BAZ2B bromodomains. In contrast, the rest of the molecule is partially solvent-exposed and adopts different orientations with different interactions in the two bromodomains. Some of these differences could be exploited for designing inhibitors with selectivity within the BAZ2 bromodomain subfamily.

Chemical Space Expansion of Bromodomain Ligands Guided by in Silico Virtual Couplings (AutoCouple).

Expanding the chemical space and simultaneously ensuring synthetic accessibility is of upmost importance, not only for the discovery of effective binders for novel protein classes but, more importantly, for the development of compounds against hard-to-drug proteins. Here, we present AutoCouple, a de novo approach to computational ligand design focused on the diversity-oriented generation of chemical entities via virtual couplings. In a benchmark application, chemically diverse compounds with low-nanomolar potency for the CBP bromodomain and high selectivity against the BRD4(1) bromodomain were achieved by the synthesis of about 50 derivatives of the original fragment. The binding mode was confirmed by X-ray crystallography, target engagement in cells was demonstrated, and antiproliferative activity was showcased in three cancer cell lines. These results reveal AutoCouple as a useful in silico coupling method to expand the chemical space in hit optimization campaigns resulting in potent, selective, and cell permeable bromodomain ligands.

Discovery of BAZ2A bromodomain ligands.

The bromodomain adjacent to zinc finger domain protein 2A (BAZ2A) is implicated in aggressive prostate cancer. The BAZ2A bromodomain is a challenging target because of the shallow pocket of its natural ligand, the acetylated side chain of lysine. Here, we report the successful screening of a library of nearly 1500 small molecules by high-throughput docking and force field-based binding-energy evaluation. For seven of the 20 molecules selected in silico, evidence of binding to the BAZ2A bromodomain is provided by ligand-observed NMR spectroscopy. Two of these compounds show a favorable ligand efficiency of 0.42 kcal/mol per non-hydrogen atom in a competition-binding assay. The crystal structures of the BAZ2A bromodomain in complex with four fragment hits validate the predicted binding modes. The binding modes of compounds 1 and 3 are compatible with ligand growing for optimization of affinity for BAZ2A and selectivity against the close homologue BAZ2B.

Virtual screen to NMR (VS2NMR): Discovery of fragment hits for the CBP bromodomain.

Overexpression of the CREB-binding protein (CBP), a bromodomain-containing transcription coactivator involved in a variety of cellular processes, has been observed in several types of cancer with a correlation to aggressiveness. We have screened a library of nearly 1500 fragments by high-throughput docking into the CBP bromodomain followed by binding energy evaluation using a force field with electrostatic solvation. Twenty of the 39 fragments selected by virtual screening are positive in one or more ligand-observed nuclear magnetic resonance (NMR) experiments. Four crystal structures of the CBP bromodomain in complex with in silico screening hits validate the pose predicted by docking. Thus, the success ratio of the high-throughput docking procedure is 50% or 10% if one considers the validation by ligand-observed NMR spectroscopy or X-ray crystallography, respectively. Compounds 1 and 3 show favorable ligand efficiency in two different in vitro binding assays. The structure of the CBP bromodomain in the complex with the brominated pyrrole 1 suggests fragment growing by Suzuki coupling.

Fragment-based in silico screening of bromodomain ligands.

We review the results of fragment-based high-throughput docking to the N-terminal bromodomain of BRD4 and the CREBBP bromodomain. In both docking campaigns the ALTA (anchor-based library tailoring) procedure was used to reduce the size of the initial library by selecting for flexible docking only the molecules that contain a fragment with favorable predicted binding energy. Ranking by a force field-based energy with solvation has resulted in small-molecule hits with low-micromolar affinity and favorable ligand efficiency. Importantly, the binding modes predicted by docking have been validated by X-ray crystallography. One of the hits for the CREBBP bromodomain has been optimized by medicinal chemistry into a series of potent and selective ligands.

dMM-PBSA: A New HADDOCK Scoring Function for Protein-Peptide Docking.

Molecular-docking programs coupled with suitable scoring functions are now established and very useful tools enabling computational chemists to rapidly screen large chemical databases and thereby to identify promising candidate compounds for further experimental processing. In a broader scenario, predicting binding affinity is one of the most critical and challenging components of computer-aided structure-based drug design. The development of a molecular docking scoring function which in principle could combine both features, namely ranking putative poses and predicting complex affinity, would be of paramount importance. Here, we systematically investigated the performance of the MM-PBSA approach, using two different Poisson-Boltzmann solvers (APBS and DelPhi), in the currently rising field of protein-peptide interactions (PPIs), identifying the correct binding conformations of 19 different protein-peptide complexes and predicting their binding free energies. First, we scored the decoy structures from HADDOCK calculation via the MM-PBSA approach in order to assess the capability of retrieving near-native poses in the best-scoring clusters and of evaluating the corresponding free energies of binding. MM-PBSA behaves well in finding the poses corresponding to the lowest binding free energy, however the built-in HADDOCK score shows a better performance. In order to improve the MM-PBSA-based scoring function, we dampened the MM-PBSA solvation and coulombic terms by 0.2, as proposed in the HADDOCK score and LIE approaches. The new dampened MM-PBSA (dMM-PBSA) outperforms the original MM-PBSA and ranks the decoys structures as the HADDOCK score does. Second, we found a good correlation between the dMM-PBSA and HADDOCK scores for the near-native clusters of each system and the experimental binding energies, respectively. Therefore, we propose a new scoring function, dMM-PBSA, to be used together with the built-in HADDOCK score in the context of protein-peptide docking simulations.

Structural basis for PHDVC5HCHNSD1-C2HRNizp1 interaction: implications for Sotos syndrome.

Sotos syndrome is an overgrowth syndrome caused by mutations within the functional domains ofNSD1 gene coding for NSD1, a multidomain protein regulating chromatin structure and gene expression. In particular, PHDVC5HCHNSD1 tandem domain, composed by a classical (PHDV) and an atypical (C5HCH) plant homeo-domain (PHD) finger, is target of several pathological missense-mutations. PHDVC5HCHNSD1 is also crucial for NSD1-dependent transcriptional regulation and interacts with the C2HR domain of transcriptional repressor Nizp1 (C2HRNizp1)in vitro To get molecular insights into the mechanisms dictating the patho-physiological relevance of the PHD finger tandem domain, we solved its solution structure and provided a structural rationale for the effects of seven Sotos syndrome point-mutations. To investigate PHDVC5HCHNSD1 role as structural platform for multiple interactions, we characterized its binding to histone H3 peptides and to C2HRNizp1 by ITC and NMR. We observed only very weak electrostatic interactions with histone H3 N-terminal tails, conversely we proved specific binding to C2HRNizp1 We solved C2HRNizp1 solution structure and generated a 3D model of the complex, corroborated by site-directed mutagenesis. We suggest a mechanistic scenario where NSD1 interactions with cofactors such as Nizp1 are impaired by PHDVC5HCHNSD1 pathological mutations, thus impacting on the repression of growth-promoting genes, leading to overgrowth conditions.

Discovery of CREBBP Bromodomain Inhibitors by High-Throughput Docking and Hit Optimization Guided by Molecular Dynamics.

We have identified two chemotypes of CREBBP bromodomain ligands by fragment-based high-throughput docking. Only 17 molecules from the original library of two-million compounds were tested in vitro. Optimization of the two low-micromolar hits, the 4-acylpyrrole 1 and acylbenzene 9, was driven by molecular dynamics results which suggested improvement of the polar interactions with the Arg1173 side chain at the rim of the binding site. The synthesis of only two derivatives of 1 yielded the 4-acylpyrrole 6 which shows a single-digit micromolar affinity for the CREBBP bromodomain and a ligand efficiency of 0.34 kcal/mol per non-hydrogen atom. Optimization of the acylbenzene hit 9 resulted in a series of derivatives with nanomolar potencies, good ligand efficiency and selectivity (see Unzue, A.; Xu, M.; Dong, J.; Wiedmer, L.; Spiliotopoulos, D.; Caflisch, A.; Nevado, C.Fragment-Based Design of Selective Nanomolar Ligands of the CREBBP Bromodomain. J. Med. Chem. 2015, DOI: 10.1021/acs.jmedchem.5b00172). The in silico predicted binding mode of the acylbenzene derivative 10 was validated by solving the structure of the complex with the CREBBP bromodomain.

Fragment-Based Design of Selective Nanomolar Ligands of the CREBBP Bromodomain.

Novel ligands of the CREBBP bromodomain were identified by fragment-based docking. The in silico discovered hits have been optimized by chemical synthesis into selective nanomolar compounds, thereby preserving the ligand efficiency. The selectivity for the CREBBP bromodomain over other human bromodomain subfamilies has achieved by a benzoate moiety which was predicted by docking to be involved in favorable electrostatic interactions with the Arg1173 side chain, a prediction that could be verified a posteriori by the high-resolution crystal structure of the CREBBP bromodomain in complex with ligand 6 and also by MD simulations (see Xu, M.; Unzue, A.; Dong, J.; Spiliotopoulos, D.; Nevado, C.; Caflisch, A. Discovery of CREBBP bromodomain inhibitors by high-throughput docking and hit optimization guided by molecular dynamics. J. Med. Chem. 2015, DOI: 10.1021/acs.jmedchem.5b00171).

Structural Analysis of the Binding of Type I, I1/2, and II Inhibitors to Eph Tyrosine Kinases.

We have solved the crystal structures of the EphA3 tyrosine kinase in complex with nine small-molecule inhibitors, which represent five different chemotypes and three main binding modes, i.e., types I and I1/2 (DFG in) and type II (DFG out). The three structures with type I1/2 inhibitors show that the higher affinity with respect to type I is due to an additional polar group (hydroxyl or pyrazole ring of indazole) which is fully buried and is involved in the same hydrogen bonds as the (urea or amide) linker of the type II inhibitors. Overall, the type I and type II binding modes belong to the lock-and-key and induced fit mechanism, respectively. In the type II binding, the scaffold in contact with the hinge region influences the position of the Phe765 side chain of the DFG motif and the orientation of the Gly-rich loop. The binding mode of Birb796 in the EphA3 kinase does not involve any hydrogen bond with the hinge region, which is different from the Birb796/p38 MAP kinase complex. Our structural analysis emphasizes the importance of accounting for structural plasticity of the ATP binding site in the design of type II inhibitors of tyrosine kinases.

Identification of a novel de novo deletion in RAF1 associated with biventricular hypertrophy in Noonan syndrome.

Biventricular hypertrophy (BVH) is a disease state characterized by the thickening of the ventricle walls. The differential diagnosis of BVH with other congenital and familial diseases in which increased ventricle wall thickness is a prominent clinical feature is fundamental due to its therapeutic and prognostic value, mainly during infancy. We describe a 2-month-old infant presenting BVH. Using exome sequencing, we identified a novel de novo 3-bp deletion in the RAF1 gene that is located in the binding active site for the 14-3-3 peptide. Based on docking calculations, we demonstrate that this novel mutation impairs protein/target binding, thus constitutively activating Ras signaling, which is a dysregulation associated with Noonan syndrome. Finally, our study underlines the importance of molecular modeling to understand the roles of novel mutations in pathogenesis.

Discovery of BRD4 bromodomain inhibitors by fragment-based high-throughput docking.

Bromodomains (BRDs) recognize acetyl-lysine modified histone tails mediating epigenetic processes. BRD4, a protein containing two bromodomains, has emerged as an attractive therapeutic target for several types of cancer as well as inflammatory diseases. Using a fragment-based in silico screening approach, we identified two small molecules that bind to the first bromodomain of BRD4 with low-micromolar affinity and favorable ligand efficiency (0.37 kcal/mol per non-hydrogen atom), selectively over other families of bromodomains. Notably, the hit rate of the fragment-based in silico approach is about 10% as only 24 putative inhibitors, from an initial library of about 9 million molecules, were tested in vitro.

AIRE-PHD fingers are structural hubs to maintain the integrity of chromatin-associated interactome.

Mutations in autoimmune regulator (AIRE) gene cause autoimmune polyendocrinopathy candidiasis ectodermal dystrophy. AIRE is expressed in thymic medullary epithelial cells, where it promotes the expression of peripheral-tissue antigens to mediate deletional tolerance, thereby preventing self-reactivity. AIRE contains two plant homeodomains (PHDs) which are sites of pathological mutations. AIRE-PHD fingers are important for AIRE transcriptional activity and presumably play a crucial role in the formation of multimeric protein complexes at chromatin level which ultimately control immunological tolerance. As a step forward the understanding of AIRE-PHD fingers in normal and pathological conditions, we investigated their structure and used a proteomic SILAC approach to assess the impact of patient mutations targeting AIRE-PHD fingers. Importantly, both AIRE-PHD fingers are structurally independent and mutually non-interacting domains. In contrast to D297A and V301M on AIRE-PHD1, the C446G mutation on AIRE-PHD2 destroys the structural fold, thus causing aberrant AIRE localization and reduction of AIRE target genes activation. Moreover, mutations targeting AIRE-PHD1 affect the formation of a multimeric protein complex at chromatin level. Overall our results reveal the importance of AIRE-PHD domains in the interaction with chromatin-associated nuclear partners and gene regulation confirming the role of PHD fingers as versatile protein interaction hubs for multiple binding events.

Exploring PHD fingers and H3K4me0 interactions with molecular dynamics simulations and binding free energy calculations: AIRE-PHD1, a comparative study.

PHD fingers represent one of the largest families of epigenetic readers capable of decoding post-translationally modified or unmodified histone H3 tails. Because of their direct involvement in human pathologies they are increasingly considered as a potential therapeutic target. Several PHD/histone-peptide structures have been determined, however relatively little information is available on their dynamics. Studies aiming to characterize the dynamic and energetic determinants driving histone peptide recognition by epigenetic readers would strongly benefit from computational studies. Herein we focus on the dynamic and energetic characterization of the PHD finger subclass specialized in the recognition of histone H3 peptides unmodified in position K4 (H3K4me0). As a case study we focused on the first PHD finger of autoimmune regulator protein (AIRE-PHD1) in complex with H3K4me0. PCA analysis of the covariance matrix of free AIRE-PHD1 highlights the presence of a "flapping" movement, which is blocked in an open conformation upon binding to H3K4me0. Moreover, binding free energy calculations obtained through Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) methodology are in good qualitative agreement with experiments and allow dissection of the energetic terms associated with native and alanine mutants of AIRE-PHD1/H3K4me0 complexes. MM/PBSA calculations have also been applied to the energetic analysis of other PHD fingers recognizing H3K4me0. In this case we observe excellent correlation between computed and experimental binding free energies. Overall calculations show that H3K4me0 recognition by PHD fingers relies on compensation of the electrostatic and polar solvation energy terms and is stabilized by non-polar interactions.