4-Acyl Pyrrole Derivatives Yield Novel Vectors for Designing Inhibitors

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4-acyl pyrrole derivatives yield novel vectors for designing inhibitors of the acetyl-lysine recognition site of BRD4(1) Martin Hügle, Xavier Lucas, Gerhard Weitzel, Dmytro Ostrovskyi, Bernhard Breit, Stefan Gerhardt, Oliver Einsle, Stefan Günther, and Daniel Wohlwend J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01267 • Publication Date (Web): 05 Jan 2016 Downloaded from http://pubs.acs.org on January 12, 2016

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Journal of Medicinal Chemistry

4-acyl pyrrole derivatives yield novel vectors for designing inhibitors of the acetyl-lysine recognition site of BRD4(1) Martin Hügle1, Xavier Lucas2, Gerhard Weitzel3†, Dmytro Ostrovskyi3, Bernhard Breit3, Stefan Gerhardt1, Oliver Einsle1, Stefan Günther4 & Daniel Wohlwend1* 1

Albert-Ludwigs-Universität Freiburg, Institut für Biochemie, Albertstr. 21, D-79104 Freiburg College of Life Sciences, Division of Biological Chemistry and Drug Discovery, University of Dundee, James Black Centre, Dow Street, Dundee, DD1 5EH, United Kingdom 3 Albert-Ludwigs-Universität Freiburg, Institut für Organische Chemie, Albertstr. 21, D-79104 Freiburg 4 Albert-Ludwigs-Universität Freiburg, Institut für Pharmazeutische Wissenschaften, Hermann-HerderStr. 8, D-79104 Freiburg 2

† Present address: Bachem AG, Hauptstrasse 144, CH-4416 Bubendorf * Corresponding author

ABSTRACT: Several human diseases, including cancer, show altered signaling pathways resulting from changes in the activity levels of epigenetic modulators. In the last few years, small-molecule inhibitors against specific modulators, including the bromodomain and extra-terminal (BET) bromodomain family of acetylation readers, have shown early promise in the treatment of the genetically defined midline carcinoma and hematopoietic malignancies. We have recently developed a novel potent inhibitor of BET proteins, 1 (XD141), which exerts a strong inhibitory potential on the proliferation of specific leukemia cell lines. In the study presented here, we designed analogs of 1 to study the potential of substitutions on the 4-acyl pyrrole backbone to occupy additional sites within the substrate recognition site of BRD4(1). The compounds were profiled using ITC, DSF, and X-ray crystallography. We could introduce several substitutions that address previously untargeted areas of the substrate recognition site. This work may substantially contribute to the development of therapeutics with increased target specificity against BRD4-related malignancies.

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Introduction Epigenetic mechanisms are non-genetic pathways for the inheritance of biological information. They are alternatively described as heritable changes in gene activity, that occur without alteration in DNA sequence. The best studied epigenetic mechanisms comprise the covalent modification of DNA bases and histones2. They have the potential to regulate DNA packing and repair. Further, they exert a direct influence on the expression of numerous genes. Thus, gene expression at cell and tissue level is extremely heterogeneous and is controlled by a complex network of regulators. While some genes are constitutively expressed in a specific tissue, others remain permanently repressed. These non-genetic alterations are tightly linked to the binding of histone proteins to DNA. Chemical modifications of a histone can affect its interaction with DNA and other biomolecules. Such modifications mainly include methylation, acetylation, phosphorylation, and ubiquitination3-6, and may occur on any accessible amino acid side chain of the protein, including the histone tails and the central, globular domain. Hence, diverse and complex modification patterns exist, which have become generally known as the histone code6,7. Functionally, the histone code is translated into modulated expression levels and affects gene expression and chromatin organization during the cell cycle7,8. Should the corresponding regulative mechanisms fail, inflammatory diseases and cancer could emerge. Hence, it is not surprising that within the last decade numerous research projects unveiled fundamental contributions of epigenetic mechanisms to both inflammatory diseases and malignancies9,10. Specific proteins were identified that modify certain residues of the histone tails6. Acetylation involves mainly two enzyme classes, histone acetyl transferases (HATs) and histone deacetylases (HDACs). HATs catalyze the acetylation of the terminal ε-amino group of lysine side chains, and together with other transferases have been termed writers7. HDACs catalyze the removal of such acetyl substituents and, due to their



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function as antagonists of the writer proteins, they are commonly known as erasers7. The presence or absence of acetylation marks has a major impact on the electrostatic surface potential of histones and concomitantly influences binding to DNA and the recruitment of co-factors and other effector proteins for gene expression7,8. Notably, some proteins specifically recognize distinct histone acetylation patterns and can therefore directly act on the expression rate of the underlying genes8. In full analogy to the writers and erasers these proteins are dubbed histone readers7. Histone tail reader proteins including the family of the bromodomain containing proteins (BDCPs)11,12 are particularly frequently involved in signaling pathways that - once misregulated are linked to cancer progression9,10. Indeed, mutations of BDCPs have often been related to such misregulations9,13--15. Among the various BDCPs, the members of the bromodomain and extra terminal (BET) family of proteins, namely BRD2, BRD3, BRD4, and BRDT16, have recently entered the spotlight as therapeutic targets due to their implication in many epigenetically related diseases. The wealth of available sequence and structure information on BDCPs shows that bromodomains are structurally conserved and comprise roughly 120 amino acids forming an antiparallel four helix bundle (helices αZ, αA, αB, and αC), although the interconnecting loops ZA and ZB of different bromodomains largely diverge in sequence and length12, 13. Crystal structures of bromodomains with bound target peptides at their recognition site17,18 show that the peptide protrudes into a hydrophobic pocket formed by helices αB and αC, and loop ZA. The shape of the pocket complements the bound peptide, while the top of the pocket usually contains a wellconserved asparagine residue, which engages in hydrogen bonding to the terminal acetyl group of the lysine side chain of the natural substrate16. Hence, specific interactions of bromodomains with their target proteins are not only defined by the shape of their binding pocket, but also by the electrostatic surface potential of crucial amino acids within and in close vicinity of the binding pocket. ACS Paragon Plus Environment

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We recently introduced 1 (Table 1) as a novel inhibitor of the BET family of proteins1, exploring an additional site beyond the substrate recognition pocket of the first bromodomain of the human bromodomain containing protein 4, BRD4(1). X-ray crystallographic structure analysis revealed that 1 engages in hydrogen bonding to the β-amide function of the conserved Asn140 (Fig. 1A), which is equally exploited by other inhibitors, such as (+)-JQ116. The pyrrole ring of 1 is further stabilized by a hydrogen bonding interaction with the carbonyl backbone of Pro821. The alkyl substituents of the 4-acyl pyrrole ring are fixed inside the binding pocket by hydrophobic interactions with Leu92, Leu94, and Ile146 of BRD4(1) making a perfect fit with the protein surface. An additional determinant for binding to BRD4(1) is the amide bond bridging the 4-acyl pyrrole and the aryl moiety of 1. The carbonyl oxygen of this amide function is involved in hydrogen bonding to a water molecule coordinated by Gln85. Lastly, the aryl moiety is involved in a T-shaped πstacking interaction with Trp81, which is reinforced by a hydrophobic lid formed by Leu92 on the opposing side of the aromatic ring of 1. Hence, this specificity site is termed the WL trap1. The terminal sulfonamide was shown to mainly fulfil spatial requirements of the protein surface and to increase the hydrophilicity of the compound. The study presented here aimed for the exploration of possibilities to alter the substituents on the backbone of 1. These modifications focused on the 4-acyl pyrrole, the amide bond, the aryl moiety as well as the sulfonamide substituent (Tables 1-3) in order to modulate binding to the established sites and to address additional structural features of the protein surface of BRD4(1). The structural characterization performed in this study offers a rational for developing novel chemotypes that can target the first bromodomain of BRD4.



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Results In this study the functional groups of 1 were considered with respect to effects on the hydrogen bonding potential, inductive effects on the aryl moiety and their effect on WL trap binding as well as on hydrophilicity. Molecular modeling experiments were carried out to select the different analogs (see Methods). When individual modifications were not available, we chose alternating combinations of modifications to assess their individual contributions. As all compounds presented here are based on 1 (termed XD14), they were assigned the letter code XD followed by consecutive numbers. All new compounds were subjected to co-crystallization with BRD4(1) and, where successful, subsequent structure analysis. We chose the approach of co-crystallization rather than soaking of apo crystals in order to circumvent a preselection for only those interactions which are not involved in crystal packing. In other words, templating of amino acid side chains by packing within an apo crystal is avoided this way. This was complemented by the determination of thermodynamic binding parameters by means of isothermal titration calorimetry (ITC) according to the previously described methodology1. Where ITC was not feasible due to low solubility of the compound in water, a Thermofluor assay based on RT-PCR was used to investigate the effects of compound modifications on binding to BRD4(1) by quantifying thermal shifts of the melting temperature of the protein. The few exceptions where quantifications failed are discussed below.

Bidentate hydrogen bonding to the acetyl-lysine binding Asn140 We initially focused on modifications of the 4-acyl pyrrole moiety of 1, in order to change the positioning or the hydrogen bonding of the compounds in the depth of the binding pocket of BRD4(1). The modifications we introduced to achieve that goal are summarized in Table 1.


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First, we shortened the aliphatic 3-ethyl substituent vicinal to the acyl group by one carbon atom, yielding the 3-methyl derivative 2 (XD27) in order to evaluate the importance of hydrophobic shape complementarity of the original ethyl group of 1. The crystal structure shows an equivalent positioning of the entire ligand inside the pocket (Fig. 1B), yet with an inverse orientation of the sulfonamide group at the perimeter of the binding cleft, now pointing in the direction of Trp81. As we also observed this effect with some other ligands bearing an aryl-sulfonamide (see below), it is likely that this promiscuous orientation is due to energetically rather equal hydrogen bonds either to surface-bound water molecules or Lys91, and thus presumably has no major effect on binding. However, shortening the aliphatic side chain of the pyrrole moiety slightly impairs the surface complementarity. This is well supported by ITC, where the thermodynamic parameters exhibit a shift towards a slightly weaker binding with a reduced enthalpy contribution (-14.6 to 11.7 kcal/mole) partly compensated by a more favorable entropy change (-19.5 to -10.6 cal/[K⋅mole]) (Table 1 and 4). Next, we aimed for establishing a second hydrogen bond between the acyl group of the ligand to Asn140 of BRD4(1). We therefore exchanged the terminal methyl group of the 4-acyl substituent by an amino group. The new derivative, 3 (XD28), indeed engages in a second hydrogen bonding with the side chain of Asn140 (Fig. 1C). However, the rigidity of the head group causes a tilt of the pyrrole ring by 7°, which propagates to a positional shift of the aromatic plane of the aryl moiety of 2 in the WL trap of BRD4(1). Thus, the perpendicular orientation is lost, hampering ligand recognition by the WL trap. To what extent this might compensate the gain of binding energy by the additional hydrogen bond to Asn140 was further assessed by ITC. Here, the enthalpic (ΔH) and entropic (ΔS) contributions are only negligibly changed, yet with an overall lower decrease of the Gibbs free energy (Table 4). This causes an increase of the KD from 237 nM (1) to 810 nM (2). We considered that the loss in binding affinity could also result from its potential aggregation in ACS Paragon Plus Environment


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solution prompted by intermolecular hydrogen bonding of two molecules of 3. The contribution of this effect to the heat changes observed in ITC is unclear. In order to allow for more rotational freedom, and thus to avoid a tilt of the ligand molecule inside the binding pocket of BRD4(1), we then added a terminal hydroxyl group to the methyl group of the 4-acyl substituent. X-ray crystallography showed that this 4-(2-hydroxyacetyl) derivative, 4 (XD29), is able to engage in hydrogen bonding with its new substituent to Asn140, but surprisingly it provoked an inversion of its head group, now providing a hydrogen acceptor, oxygen, instead of the hydrogen donating amino group (Fig. 1D). Indeed, the modified 4-acyl group is rotated counter-clockwise by roughly 150°. This causes a profound effect on the positioning of the ligand: The pyrrole moiety moves slightly up and is tilted by several degrees. The rigidity of the residual ligand accounts for a subsequent displacement of the aryl ring within the WL trap. As already observed for 3 (Fig. 1B), the perpendicular orientation toward Trp81 is suspended. The affinity of 4 is significantly reduced as compared to 1: the KD is increased to 8 µM as demonstrated by ITC (Table 1). As with 2 and 3, the sulfonamide is inverted again, now hydrogen bonding to Lys91 instead of a surface water molecule. Finally, in 5 (XD32) the ethyl group at the pyrrole ring was replaced by an isopropyl group. Although mediocre binding to BRD4(1) could be demonstrated with a KD of 7.2 µM (Table 1), the structure determination failed. Hence, the impact of the extension of the alkyl substituent at the pyrrole ring on surface matching with BRD4(1) remains elusive. In summary, changes of the 4-acyl group of 1 impede binding as soon as they cause a shift of the aromatic system of the aryl group between Trp81 and Leu92 (2 and 3). As long as this positioning remains unaffected, binding energies and affinity depend mainly on surface complementarity of the ligand and BRD4(1), as seen with 2.


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Electron density of aryl moiety defines WL trap recognition In a next step we investigated the effects of a change in the electron density within the aromatic system of the aryl moiety of 1 on binding of the compound to the WL trap, in particular with respect to the potential of attracting Trp81 (Table 2). For those compounds where X-ray structure determination was successful, molecular electrostatic potential maps of the different substitutions are shown in Fig. 2, in order to assess the extent of changes in the electron density of the aryl ring of these ligands generated by their substitution patterns. As a first representative for this series of experiments, we chose to remove the hydroxyl group from the aromatic ring, thereby decreasing the electron density. This compound, termed 6 (XD33), is no longer as well positioned in the WL trap as 1 (Fig. 3A). The aryl plane is no longer orthogonally positioned toward Trp81, suffering from weakening effects on binding. This is illustrated by ITC quantification of binding: the KD is apparently increased by a factor of about 24, ΔG is impaired as well (Table 1), although due to the poor solubility of 6 possibly the system has left the thermodynamic regime, rendering the quantitative data with high variances (Supporting Information: 1D error surface projections). Thermal shift assays (Table 1) back our findings, as the melting temperature increase (ΔTm) of BRD4(1) is largely reduced from +6.7 K (1) to only +3.3 K (6). The same effect was observed for 7 (XD40) (Fig. 3B), where we combined two substitutions that were expected to have a similar impact on the aromatic electron density as had the absence of the hydroxyl group in 6: In para position to the sulfonamide group a methyl was added, which exerts a minor positive inductive effect on the ring, while an exchange of the diethyl sulfonamide by a methyl sulfonamide substituent evens out the electron feed by increasing the negative induction (Fig. 2). Indeed, the crystal structure analysis shows that the perpendicular positioning of the aryl



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plane in the WL trap is lost again, with a tilt of the aromatic plane by 18°. As expected from the structure, binding of 7 to BRD4(1) is weaker than 1, with less favorable thermodynamic parameters (Table 1) and 40-fold apparent reduction in affinity. These findings are supported by differential scanning fluorimetry (DSF, Table 1), which yielded a similar ΔTm of 3.1 K as compared to 6. Another ligand following this route is 8 (XD45) (Table 2). Here, the sulfonamide is exchanged by an amidic substitution with two ethyl groups at the amidic nitrogen, combined with the removal of the hydroxyl group from the aryl ring system. The thermodynamic parameters of 8 (Table 4) indicate that binding to BRD4(1) is impaired. However, as a crystal structure is not available the binding parameters cannot be related to the positioning inside the substrate recognition pocket of BRD4(1). In order to reduce the unfavorable entropy decrease during binding, the terminal ethyl groups on the sulfonamide of the lead structure were covalently linked, yielding 9 (XD31) (Table 2). This ligand suffered from an unexpected insolubility both in aqueous solution and in DMSO that prohibited further analysis. Reducing the unfavorable entropy decrease in the course of binding was finally achieved by fixing the aryl plane and the amide group by introducing an indoline in 10 (XD41) (Table 2). Although the binding affinity and the positioning inside the substrate recognition site of BRD4(1) were not affected by this modification (Fig. 3C) as compared to 7, the thermodynamic contributions of enthalpy and entropy are considerably changed (Table 4). Binding of 10 is less enthalpically favorable, ΔH drops from -10.4 to -3.0 kcal/mol, but greatly profits by a substantial gain in entropy with ΔS increasing from -11.7 to 13.6 cal/(K*mol). In order to further drain electron density from the aryl-π-system, the hydroxyl group at the phenyl ring of 1 was substituted by a chlorine atom in ortho position to the sulfonamide in 11 (XD34)


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(Table 2). As this ligand was insoluble in DMSO and water, we combined the electron drain of the ring, this time with a fluorine atom in identical position, with the introduction of a morpholinyl ring in order to limit the conformational freedom and modify the compound’s solubility. The potent electron-withdrawing effect on the π-system is shown in Fig. 2. This compound 12 (XD44), places its 4-acyl pyrrole head group deeply into the already well-described recognition pocket of BRD4(1) (Fig. 3D), whereas its tail, beginning at the aryl moiety, is shifted up along the ZA channel, accompanied by a rotation of the longitudinal ligand axis by 25°. The displacement of the aryl system from the WL trap allows for establishing new van der Waals contacts to BRD4(1), as 12 perfectly complements the protein surface represented by Trp81. In fact, the weakened interaction with the WL trap caused by the fluorine substitution appears to be a prerequisite for the movement of the sulfonamide moiety toward Trp81. A thermodynamic analysis of the BRD4(1)•12 complex demonstrates the quantitative effects of the aryl displacement and the new van der Waals contacts: As expected, the KD of 1.27 µM is measurably increased with respect to 1 (Table 2). However, it is less impaired than the affinities of 6 and 7 to BRD4(1), which range from 6 to 9 µM. This underlines the potential benefits of van der Waals interactions of the sulfonamide groups with the protein surface even at the expense of a hampered WL trap recognition. A mild increase of the electron density by adding an additional methyl substituent to the aromatic ring while keeping the hydroxyl group, as given with 13 (XD35) (Table 2, Fig. 2), changes neither the ligand position in the binding pocket (Fig. 3E) nor the thermodynamic binding parameters considerably as compared to 1 (Table 4), except for a slightly weaker binding that resembles 3. Hence, the potential of enhancing binding by slightly increasing the electron density at the aryl’s π-system seems limited. Notably, the thermal shift induced by 13 is less pronounced, with only +3.5 K. We already made a similar observation with 2. One explanation might in fact arise from small quantities of impurities in the compound preparations of both ligands (less than 5 %). ACS Paragon Plus Environment


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They usually do not affect fitting of ITC curves, when the competition is taken into account by the model. However, the usually applied large excess of ligand in thermal shift assays may indeed affect a precise determination of melting points as even a few percent of impurities may in this setup reach equimolar or higher concentrations compared to the protein and thus may interfere with complex stability. In addition, entropically favored binding often results in higher apparent Tm shifts than enthalpically favored binding even at elsewise identical affinities20. Admittedly, such conclusions can only be indirectly made from ITC data as the observed entropy is always a global one, summarizing all entropical effects within the entire system.

Fine-tuning of the distal pocket binding In order to follow the line of experiments, we then aimed for a more pronounced increase of the electron density of the aromatic system of 1 by exchanging the sulfonamide by an ether function, exemplified in 14 (XD26) (Table 3). This significantly increased the electron density of the aromatic aryl system as a consequence of the positive mesomeric effect. Simultaneously, the hydroxyl group was removed from the ring in order to mitigate the electron feed. Still, the aryl moiety of 14 possesses significantly more electron density than that of 1. This effect is precisely reflected in the electrostatic potential of the molecule (Fig. 4E), which shows a larger electron density opposite to the substituent compared to that of 1. The crystal structure reveals that the aryl ring is completely displaced from the WL trap towards the lateral surface of the binding pocket (Fig. 4A), while the amide bond adopts an inverse orientation as compared to 1. The molecule produces an extensive rearrangement of the hydrogen bonding network within BRD4(1). Remarkably, 14 addresses a peripheral site of BRD4(1) by directly hydrogen bonding to the terminal oxygen of Gln84. This interaction, however, is only possible upon displacement of the electron-rich aryl from the WL


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trap. The provided gain of binding energy, however, might be entirely consumed by the loss of the T-shaped π-stacking with Trp81. This is illustrated by thermal shift assays, where the ΔTm drops to +3 K (1 with +6.7 K), which is in the range of the weak binders 6 and 7 (Table 2). ITC data point into the same direction, although here again the poor solubility of the ligand does not allow for a reliable quantification of thermodynamic binding parameters other than the KD (Supporting Information: 1D error surface projections), which rises to approximately 6 µM (Table 2). By introducing a methyl benzyl ether at the sulfonamide with 15 (XD39) (Table 3), we aimed for reaching sites outside the ZA channel of the protein, while maintaining the already described interactions. As the X-ray structure determination failed, the impact of this modification cannot be assessed any further. For further probing the distal pocket surface for putative binding interfaces and the contribution of the WL trap to binding affinity, we next aimed for an additional carbon between the amidic nitrogen and the aryl ring of 1. The compound harboring this modification, 16 (XD47), features additional changes to the well-known lead structure (Table 3): the sulfonamide substituent was exchanged by a carbon amide group, now presenting at the terminus both hydrogen donor and acceptor capabilities. 16 should thus benefit from a higher flexibility in finding hydrogen bonding partners at the perimeter of the binding pocket of BRD4(1) at the expense of an extended, delocalized π-system along the otherwise mainly planar molecule. This effect is reflected in the electrostatic potential map of 16 (Fig. 4E). The crystal structure (Fig. 4B) shows that the amide bond is again inversely oriented as compared to 1. Due to the inversion of the involved keto group, the hydrogen bond of the oxygen to a conserved surface water molecule is lost. In addition, the methyl substituent at the amidic nitrogen displaces a usually tightly bound water molecule from the protein surface, causing a partial rearrangement of the hydrogen bonding network inside the pocket (Fig. 4C). More importantly, the elongation of the linker between both ring systems of the ACS Paragon Plus Environment


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ligand pushes the aryl ring out of the WL trap. In the course of this positioning, Trp81 is no longer available for π-stacking. Leu92, however, is still involved in a hydrophobic interaction with the aromatic ring of 16. The overall weaker binding is illustrated by a significant loss of binding enthalpy of 13 kcal/mol (15 with -1.8 kcal/mol, 1 with -14.8 kcal/mol, Table 4) as determined by ITC. The drop in binding enthalpy is the primary reason for the 50-fold increase in KD (12 µM), despite the beneficial entropy contribution due to the expulsion of the water molecule. Notably, the displacement of the terminal aryl group of 16 does not preclude providing a hydrogen acceptor to the ε-amino group of Lys 91, making a hydrogen bond we have observed in other ligands (1, 2, 4, 7, 10) which are well localized inside the WL trap. Consequently, the higher flexibility of the terminal parts of 16 as compared to the other ligands presented here is not used for addressing novel sites beyond the ZA channel, as given by 14. It is used instead for re-establishing energetically more favorable and hence previously described interactions1. Along with changes on the electron density of the aromatic ring, we finally probed the potential of reducing the entropy decrease upon ligand binding in order to enhance the Gibbs free energy for complex formation, which would directly impact binding affinity. The most evident subjects for such modifications are the two terminal ethyl groups of the sulfonamide substituent, whose inherent entropy might be reduced a priori by crosslinking them. Hence an azepane ring was introduced yielding 17 (XD42) (Table 3). To further exploit the surface complementarity to the binding pocket, we simultaneously introduced a propyl group replacing the 3-ethyl group at the pyrrole system. The crystal structure (Fig. 4D) shows a good positioning of the 4-acyl pyrrole moiety of 17 in the binding pocket of BRD4(1), with the surface complementarity being improved as expected. The distal part of the molecule including the aromatic system, however, is comparably poorly defined in the electron density (Fig. 5D), as 17 fails to engage in hydrogen bonds with BRD4(1) in this part of the pocket, making the positioning of the aryl ring within the WL trap no ACS Paragon Plus Environment

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longer ideal. Surprisingly, the solubility of 17 is considerably diminished as compared to 1, which leads to a highly uncertain quantification of binding parameters by ITC. Thermal shift assays highlight the potential of the modifications harbored by 17 with a ΔTm of +5.3 K compared to the apo-protein (Table 3). Among all new ligands presented in this study, 17 is in fact runner-up in thermal shift assays, right behind 3 (ΔTm +5.6 K). In line with our findings from the crystal structure the improvement of thermal stability can be attributed to the modification of the pyrrole.

Atom-based pharmacophores reveal hot-spot recognition features The extensive crystallographic data presented here can be combined with the binding affinity of each analog in order to study the contribution of the different substituents to the protein–ligand interaction. Despite the high conservation of secondary structure among the crystallized BRD4(1) complexes, a superposition of C-alpha carbons was carried out prior to QSAR and pharmacophore studies. Upon superposition, displacements in the binding mode of the inhibitors can be used to characterize differential interactions and binding mode. Differences among the ligands leading to changes in orientation are particularly beneficial for this approach, as each cell in the QSAR grid is analyzed independently to consider the positive or negative contribution its occupation by a specific probe (hydrogen bond donor/acceptor, hydrophobic or aromatic) has on binding. The resulting atom-based quantitative structure-activity relationship (QSAR) and ligandbased pharmacophore are shown in Figure 5. Several conclusions can be drawn from the QSAR analysis. First, the amidic linker is preferably accommodated placing the carbonyl oxygen towards the inner site of the pocket, as indicated by the blue cages in Fig. 5A. This conformation allows the different ligands to interact with the water molecule W5, as shown in Figure 4C, and is consistent with the large repulsive positive potential in this region of the molecular electron density



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map of molecules presenting the contrary conformation (e.g. 14, Fig. 4E). Second, the correct positioning of the aryl in the WL trap is of utmost importance when designing potent inhibitors: The interaction only contributes positively to binding affinity if the ligand is positioned perpendicular to Trp81, thus enabling the above-mentioned T-shaped π-stacking with the amino acid (Fig 5C). Furthermore, displaced interactions correlate with unfavorable contribution to ΔG (red boxes). The analysis of the combined contribution (Fig. 5E) also shows that there is no preferred conformation for the rotatable sulfonamide group, as indicated by the lack of boxes surrounding this moiety. The ligand-based pharmacophore shown in Fig. 5F serves as a summary of the relevant physicochemical features discussed in the present study and that are important to addressing BRD4: hydrogen-bond acceptor capabilities are required for the interaction with Asn140 and the inner region of the ZA channel, a hydrogen-bond donor group fixes the ligand to Pro82, hydrophobic extensions are needed in the deeper core of the recognition pocket and WPF shelf, whereas aromatic systems are important contributors in the center of the recognition pocket and the WL trap to fix the ligand.

Specificity within different families of bromodomains To assess changes in specificity towards BRD4(1) we performed an ITC based specificity screening of exemplary ligands with other bromodomains (Table 5). According to phylogenetic relationships of bromodomains based on sequence similarity17, we chose BRD3(1) and BRD3(2) as members of the BET family to test the intra-family specificity. BRD3(1) resides in the same branch of BET family members as BRD4(1) and is one of the closest relatives to BRD4(1). BRD3(2) is a member of the neighboring branch of this family and is slightly more distantly related. We selected a subset of ligands which mirrors best the variety of introduced modifications of 1: 4 showed the


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most prominent change of all 4-acyl modifications in binding to BRD4(1) by inducing an inversion of the hydrogen donor/acceptor pair of Asn140 (Fig. 1) and thus is the most likely to produce significant differences in target recognition within the BET family. 12, 14, and 16 displayed alternative positioning within the WL trap of BRD4(1) as compared to the other modifications. Two observations stand out as noteworthy: First, 4 is the only modification which prefers the offtarget BRD3(1) over BRD4(1) (Table 5) with a ratio of KDs for BRD3(1) and BRD4(1) of 0.62 (1: 12.2). Second, 12, although impaired in specifically discriminating against BRD3(1) as compared to 1, discriminates against the neighbor branch of BET proteins, exemplified by BRD3(2), even better than 1 with an according KD ratio of 13.8 (1: 9.88). These findings underline the potential of modifications of the 4-acyl pyrrole backbone to alter specificities even within the BET family. In addition we checked affinities to BRPF1(1) as member of a distantly related bromodomain family in order to validate the inter-family discrimination of the most representative ligands. We chose 2 as an example for shortening and 5 for enlarging the aliphatic substitutions at the pyrrole. 3 was picked as an example for 4-acyl modifications harboring both hydrogen donor and acceptor. Finally, we also tested 18 (XD46), which we could identify in our previous work1 to be a suitable core fragment for numerous bromodomains and thus should be the least specific one.

The observed differences in the KDs (Table 1) clearly suggest that smaller aliphatic substitutions (2) at the pyrrole favor binding to BRPF1(1), while bulkier ones impede it (5), although both ligands show a largely reduced inter-family specificity in comparison with 1. Adding a hydrogen donor to the 4-acyl moiety (3) results in a reduced affinity, as well, but here the specificity of 1 is to a ACS Paragon Plus Environment


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certain extent maintained. Notably, the core fragment 18, lacking the aryl moiety and the sulfonamide of 1, is second-best in BRPF1(1) recognition, yet with the lowest potential to discriminate between different bromodomain families. The KD for BRD4(1), against which 18 was originally designed, is with 6.8 µM only 4-fold better than the one for BRPF1(1) with 29.8 µM. This supports the assumption that specificity is only gained when the backbone of 18 is extended.

Discussion The robustness of the initial modelling could be validated, as all ligands presented in this study bound tightly to Asn140, the main determinant for substrate recognition in BET family proteins. In addition, the majority of the derivatives occupy the WL trap defined by Trp81 and Leu92 of BRD4(1) in a similar mode as the lead compound 1. Lys91 plays an interesting role in ligand recognition: in some complexes (2, 4, 7, 10, and 16) it is involved in binding to the terminal substituent of the aryl ring and at other times not (3, 6, 12, 13, 14, and 17). In order to assess, whether recruitment of Lys91 by any of the ligands depends on templating by crystal contacts, we compared the crystal packings of all structures presented in this study. All structures packed in only two different unit cells belonging to the same space group (see Supporting Information). Apparently, there is no correlation between recruitment of Lys91 by the ligand and the respective unit cell of the crystal. In fact, availability of Lys91 for crystal contacts appears to be independent of a priori recruitment by the ligand but rather depends on the overall crystal packing. Accordingly, in all unit cells presented in this study we find Lys91 occasionally involved in ligand interactions and occasionally in crystal contacts in a mutually non-exclusive manner. This is exemplified by the complex of BRD4(1) and 4, where the ε-amino group of K91 is involved in binding to both 4 and Gln59 of a symmetry mate. Hence, templating by the ligand alone does not govern the ability of


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Lys91 to make up crystal contacts nor does a certain packing in general prevent Lys91 from binding to ligand moieties. We conclude, that the recruitment of Lys91 by any potential ligand will have only minor effects on ligand recognition and may thus be regarded less important for future studies. This certainly does not hold true for the conserved water molecules in the Kac binding pocket of BRD4(1). Notably, with 16 we managed to displace one of the conserved water molecules. To our knowledge, 16 is the first inhibitor of BET proteins with the capacity to do so. The potential of such a mechanism is evident, as compounds with this property are expected to have more favorable dissociation kinetics, resulting in a more stable complex. Although an improved recognition of the WL trap while simultaneously fortifying the direct interaction with Asn140 at the top of the substrate pocket was not achieved here, the potential of involving more distant sites of BRD4(1) could nevertheless be demonstrated. Accordingly, the previously unaddressed Gln84 could be successfully recruited by 14. Atkinson et al. lately achieved this with IBET295 (PDB ID code 4CLB), as well. However, while IBET295 reaches Gln84 by following the Trp81 plane with an extended alkyl moiety including six additional rotatable bonds before bending towards Gln84, 13 involves Gln84 on a straight path requiring only two rotatable bonds. In addition, modifications of the 4-acyl pyrrole backbone to enhance the surface complementarity showed early promise, as demonstrated for 17. Taken together, these features may not necessarily increase the affinity to the target; instead they may be needed for gaining specificity and thus reducing possible offtarget interactions. The results from the specificity screening presented here support this assumption: modifications of the aliphatic substituents of the pyrrole indeed significantly altered specificity for different bromodomain families as well as within the BET-family itself. Hence the track of improving the surface match of the pyrrole moiety to BRD4(1), as shown for 17, is worth being followed in the future. Further, the positioning of the aryl moieties of the derivatives of 1 ACS Paragon Plus Environment


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seems to be governed mainly by the electron density of the phenyl ring at the center of the molecules. The modulation of the aryl’s electron density turned out to be a key element for addressing additional sites at the distal part of the substrate recognition site of BRD4(1). Loosening the interaction with the WL trap allows and is required for a movement of the ligand to other sites. This could be well shown with the derivatives 12, 14, and 17. However, once a tight binding to the WL trap is desired, the most promising strategy is to maintain the apparently already optimal electron density of the aryl system of 1, and to elongate and solidify the terminal substituent. In this context, molecular electron density maps have proven valid when evaluating the effect of specific substitutions in the π-system of the aryl. Since the size and shape of the recognition pocket diverges among the different bromodomain families21, this strategy offers the opportunity to further address BET-specific sites like the aromatic plane of Trp81 or the amide function of Gln84. Concomitantly, the discrimination against other bromodomain families could be greatly improved by considering interaction hot-spots as revealed by atom-based QSAR and ligand-based pharmacophore techniques. For example, the QSAR study revealed the important contribution of an acceptor feature within the inner region of the ZA channel in BRD4(1). Additionally, the impact of a perpendicular T-shaped π-stacking with Trp81 became apparent. A common feature in the presented analogs is the singular hydrogen bond between the pyrrole and Pro82 of BRD4(1). Remarkably, recent molecular dynamics experiments with 4-acyl pyrrole derivatives stressed the relevance of this interaction also in other structurally related bromodomains21. In summary, the work presented in this study delivers resilient concepts for a deeper understanding of substrate recognition in BRD4(1) and, ultimately, for the enhancement of specific, rationally designed BRD4(1) inhibitors.


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Experimental section Molecular modeling Target preparation Docking calculations were performed with Glide 5.8 (Schrödinger, LLC, New York, NY, USA) using the crystal structure of BRD4(1) in complex with 1 (PDB ID: 4LYW). The target structure was prepared using the Protein Preparation Wizard with default parameters, Epik 2.3 (Schrödinger, LLC) for the prediction of protonation states, and hydrogen-only energy minimization. Additionally, five water molecules relevant for the recognition of 1 by BRD41 were considered for docking. The recognition site of BRD4(1) was subjected to a computational molecular binding analysis. Acceptor and donor hydrogen-bond constraints with Asn140 and Pro82, respectively, were set as physical constraints for the docking experiment.

Ligand library preparation We collected analogs of 1 from PurchasableBoX, comprising over 35 million commercially available compounds with drug-like properties23. A similarity search with Tanimoto coefficient ≥ 0.6 yielded 2,599 compounds, which were merged with 5,874 molecules identified by performing a substructure search based on the inner core of 1, the compound 181. The resulting collection consisted of 7,574 unique compounds, which were processed using LigPrep 2.5 (Schrödinger, LLC) and used for the molecular docking experiments. Additionally, tens of rationally designed molecules covering a wide range of modifications on 18 were included for further assessment, despite their commercial availability.

Docking experiments ACS Paragon Plus Environment


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Compounds were virtually tested in the active site of BRD4(1) using the eXtra Precision (XP) algorithm as implemented in Glide. Acceptor and donor hydrogen-bond constraints to Asn140 and Pro82, respectively, were imposed during the screening. As the ultimate purpose of the study was to explore the recognition site of BRD4(1), the final selection of compounds was based both on predicted binding affinity and the introduction of diverse, novel chemical substitutions.

Molecular electrostatic potential surfaces The starting conformation for the model compounds in Figure 3 had a 90° dihedral angle between the S-N bond in the sulfonamide moiety and the ring plane, whereas for Figure 5E, the atomic coordinates from the X-ray crystal of each ligand were used. The initial poses were then subjected to a full-atom energy and geometry optimization at the DFT(B3LYP)/6-31G** level using Jaguar (Schrödinger, LLC) and the electrostatic potential surface computed.

Atom-based QSAR and ligand-based pharmacophore The co-crystallized complexes of ligands studied herein were used to derive a pharmacophore model with up to 7 feature sites using Phase (Schrödinger, LLC), based on ΔG as descriptor for activity (16 was omitted). The resulting pharmacophore hypothesis was subsequently used to build a one-factor atom-based QSAR model.

Availability of the selected candidates 1, 2, 5, 6, 7, 8, 9, 10, 11, 13, 14, 16, and 18 were provided by Enamine Ltd. (Kiev, Ukraine), as well as the custom synthesis of 3; whereas 15 was provided by InterChim (Montluçon Cedex, France). 12 and 17 were provided by AKos GmbH (Lörrach, Germany). All compounds were provided with


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purity ≥ 95 %. Additionally, we designed and carried out the organic synthesis of 4 with a purity of ≥ 95 % as verified by HPLC.



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Synthesis of 4 4-(2-Chloroacetyl)-N-(5-(N,N-diethylsulfamoyl)-2-hydroxyphenyl)-3-ethyl-5-methyl-1H-pyrrole-2carboxamide

To a suspension of finely powdered dry CaCl2 (355 mg, 3.20 mmol, 4.5 eq.) in dry methanol (4.0 ml) was added AgNO3 (241 mg, 1.42 mmol, 2.0 eq.) and 4-acetyl-N-(5-(N,N-diethylsulfamoyl)-2hydroxyphenyl)-3-ethyl-5-methyl-1H-pyrrole-2-carboxamide (1, 300 mg, 0.71 mmol). At 70°C, a solution of I2 (198 mg, 0.78 mmol, 1.1 eq.) in dry methanol (1.5 ml) was added slowly. After 2 h at this temperature, the mixture was diluted with methanol (30 ml) and not soluble solids were filtered off. After evaporation of the solvent, the crude product was filtered over silica gel (ethyl acetate) and recrystallized from cyclohexane/ethyl acetate to provide the desired compound (301 mg, 0.66 mmol, 93%) as beige solid. 1

H-NMR (499.500 MHz, (CD3)2CO): δ [ppm] = 1.11 (t, J=7.1 Hz, 6H), 1.28 (t, J=7.4 Hz, 3H), 2.68

(s, 3H), 3.13 (q, J=7.6 Hz, 2H), 3.21 (q, J=7.3 Hz, 4H), 4.68 (s, 2H), 7.11 (d, J=8.5 Hz, 1H), 7.44 (dd, J=8.5, 2.2 Hz, 1H), 8.62 (br. s., 1H), 8.76 (d, J=1.9 Hz, 1H), 10.43 (br. s., 1H), 11.09 (br. s., 1H). 13

C-NMR (125.599 MHz, (CD3)2CO): δ [ppm] = 14.6, 15.0, 15.6, 19.8, 42.8, 50.3, 115.1, 119.1, 119.2,

119.9, 120.0, 124.0, 128.1, 131.8, 132.3, 138.7, 159.7, 187.8. MS-analysis (neg. APCI): m/z = 454.12 [M-H]-, 419.15 [M-HCl]-, 378.15 [M-C(O)CH2Cl]-. HRMS (C20H27N3O5SCl, [M+H]+): calc.: 456.13590, found: 456.13590, deviation: 0.2 ppm.


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2-(5-((5-(N,N-Diethylsulfamoyl)-2-hydroxyphenyl)carbamoyl)-4-ethyl-2-methyl-1H-pyrrol-3-yl)-2oxoethylacetate

To

4-(2-chloroacetyl)-N-(5-(N,N-diethylsulfamoyl)-2-hydroxyphenyl)-3-ethyl-5-methyl-1H-

pyrrole-2-carboxamide (50.0 mg, 0.11 mmol) in dry DMF (11 ml) KOAc (32.4 mg, 0.33 mmol, 3.0 eq.) and KI (18.3 mg, 0.11 mmol, 1.0 eq.) were added. After 45 h at r.t. the mixture was diluted with ethyl acetate (50 ml) and washed with brine (5×20 ml). The organic phase was dried over Na2SO4, evaporated and filtrated over silica gel (ethyl acetate) to provide the product (52.0 mg, 10.8 mmol, 99%) as beige solid. 1


(s, 3H), 2.66 (s, 3H), 3.11 (q, J=7.6 Hz, 2H), 3.19 (q, J=7.0 Hz, 4H), 5.09 (s, 2H), 7.12 (d, J=8.5 Hz, 1H), 7.44 (dd, J=8.4, 2.4 Hz, 1H), 8.58 (s, 1H), 8.81 (d, J=2.5 Hz, 1H), 10.47 (s, 1H), 11.14 (br. s., 1H). 13


119.9, 122.5, 124.1, 128.2, 132.0, 132.2, 138.4, 150.0, 159.9, 170.5, 189.6. MS-Analysis (pos. ESI): m/z = 981.33 [M2+Na]+, 502.16 [M+Na]+. HRMS (C22H29N3O7SNa, [M+Na]+): calc.: 502.16239, found: 502.16200, deviation: 0.8 ppm.



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N-(5-(N,N-diethylsulfamoyl)-2-hydroxyphenyl)-3-ethyl-4-(2-hydroxy-acetyl)-5-methyl-1H-pyrrole2-carboxamide (4)

2-(5-((5-(N,N-Diethylsulfamoyl)-2-hydroxyphenyl)carbamoyl)-4-ethyl-2-methyl-1H-pyrrol-3yl)-2-oxoethylacetate (38.0 mg, 79.2 μmol) was dissolved in a solution of Na2CO3 (9.2 mg, 87.2 μmol, 1.1 eq.) in MeOH (2 ml). After 16 h at r.t. the reaction mixture was evaporated. The obtained residue was dissolved in ethyl acetate (10 ml) and washed with brine (2×5 ml). After drying over Na2SO4 and evaporation of the solvent, the crude product was purified by column chromatography (2×20 cm SiO2, ethyl acetate) and afterwards trituration in very little DCM. The desired compound 4 (26.8 mg, 58.8 μmol, 74%) was obtained as off-white solid and had a purity of ≥ 95 % as proven by HPLC. 1


(s, 3 ), 3.20 (q, J=7.6 Hz, 2H), 3.24 (q, J=7.3 Hz, 4H), 4.05 (br. s., 1H), 4.55 (s, 2H), 7.11 (d, J=8.5 Hz, 1H), 7.46 (dd, J=8.5, 2.5 Hz, 1H), 8.62 (d, J=2.5 Hz, 1H), 9.38 (s, 1H), 10.29 (br. s., 1H), 11.82 (br. s., 1H). 13


122.0, 125.0, 128.4, 132.8, 135.1, 139.8, 151.9, 161.0, 196.3. MS-Analysis (pos. APCI): m/z = 438.17 [M+H]+. HRMS (C20H28N3O6S, [M+H]+): calc.: 438.16988, found: 438.16980, deviation: 0.2 ppm.


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Isothermal titration calorimetry ITC experiments were performed with a Microcal VP-ITC microcalorimeter (Malvern Instruments, Herrenberg, Germany) at 25 °C using ligand concentrations between 10 and 80 μM in the sample cell and BRD4 concentrations between 100 and 800 μM in the injection syringe. Concentrations were determined gravimetrically (ligands) and by the native absorption coefficient at 280 nm wavelength (protein). The protein concentration was additionally cross-validated by titration against a gravimetrically determined amount of 1. Data were obtained in discrete titration experiments with an injection volume of 12 μl per injection. For initial approximations of binding constants, the heats per injection were calculated as integrals and after normalization against the molar concentrations plotted against the molar ratio as implemented in Microcal Origin. Finally, data were fitted according to a binary interaction model with

= ∙ ∆ ∙ ∙ 1 + + − 1 + + − 4 /

as function to calculate the overall sum of heat of the titration and with ∆ = +

!"# "$

∙ +

%#&

− − 1

(According to Microcal’s manual “ITC Data Analysis in Origin” (September 1998)) as function describing the sum of heat of each individual injection. A correction term was included to compensate for displacement of volume of the sample cell in the course of subsequent injections according to the manufacturer’s manual “ITC Data Analysis in Origin” (Microcal). Where binding affinities were too low to produce sigmoidal binding curves (Wiseman parameter c < 5) the parameter N was fixed initially at a value of 1 to allow for calculation of the other binding parameters. For a high precision quantification of binding parameters raw data were processed with NITPIC24 for automated baseline correction and peak integration. Data of multiple titrations of



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the same ligand were then globally analyzed with SEDPHAT25, again with a binary interaction model as basis for the fitting procedure. The concentration errors of the components were corrected by introducing concentration correction factors and incompetent fractions as implemented in SEDPHAT. The validity of the fits was assessed subsequently by one-dimensional error surface projections in SEDPHAT at P-levels of 0.95 and 0.99 (Supporting Information).

Differential Scanning Fluorimetry (DSF) Thermal shift assays with DSF were performed with a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Munich, Germany). Protein was buffered in PBS-Buffer with additionally 360 mm NaCl at a final concentration of 20 µM. SYPRO Orange (fluorescence dye) was added at a dilution of 1 in 1,000 and ligands were added with a final concentration of 200 µM in a 96 well plate in 25 µl. Measurements were performed using the machines FRET channel (excitation filter 450-490 nm, emission filter 560-580 nm). The temperature gradient was set from 25 °C to 95 °C with steps of 0.5 °C per 10 seconds. ΔTm was calculated as the difference between the inflection point of the observed melting curves from sample and reference wells without ligand. Each Tm given in Table 1 is the mean value of three independently measured samples.

Protein preparation, crystallization, and structure determination BRD4(1) was expressed and purified as described previously1. Co-crystals with the individual ligands were obtained in the Index HT Screen (Hampton Research) at a protein concentration of 10 mg/ml and a ligand concentration of 2 mm added directly to the protein prior to crystallization from a 100 mm stock solution in DMSO. Data were collected at 100K. In-house data collection was done using a Rigaku HF-007 rotating anode X-ray generator at λ=1.54179 Å equipped with Vari-


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MaxHF optics and a Saturn944 CCD detector and a mar345 image plate, respectively. Synchrotron data collection was done at the PXI beamline at the Swiss Light Source at λ=1.000 Å with a Pilatus6M detector. Data processing and reduction was done with iMOSFLM26-28 (version 7.1.3), POINTLESS, SCALA, and AIMLESS29-31, or with XDS, XSCALE, XDSCONV32 and XPREP33. BRD4ligand complexes crystallized in space group P212121 with the cell constants indicated in Supporting Table 1. The structures were solved by molecular replacement with PHASER34 with apo BRD4 as search model (internal data) yielding one molecule per asymmetric unit. Compounds were modeled into 2Fo-Fc electron density maps using AFITT-CL35 (version 2.1.0, OpenEye Scientific Software, Inc., Santa Fe, NM, USA). Model building and real space refinement was done with COOT36, and reciprocal space refinement against the calculated data was done with REFMAC537,38 as implemented in the CCP4 suite (version 6.4.0). Final structure validation was done with PROCHECK/SFCHECK39. The relevant statistics of data collection and model refinement are provided in the Supporting Information. All illustrations of X-ray models were created with PyMOL40 (version 1.5.0.4).



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ASSOCIATED CONTENT Supporting Information. X-ray data collection and model refinement statistics, molecular formula strings of all novel ligands, and ITC thermograms including 1D-error-surface-projections for validation of fits for the provided ITC data.

AUTHOR INFORMATION Corresponding Author * Daniel Wohlwend: Albert-Ludwigs-Universität Freiburg, Institut für Biochemie, Albertstr. 21, D79104 Freiburg

Present Addresses † Gerhard Weitzel: Bachem AG, Hauptstrasse 144, CH-4416 Bubendorf

Author Contributions MH purified BRD4(1) and did ITC, DSF, crystallization and X-ray data collection, and he helped drafting the manuscript. XL designed and carried out the computational studies and helped drafting the manuscript. GW and DO designed and carried out the synthesis of 3. S. Gerhardt helped solving the crystal structures of BRD4(1)•13 and BRD4(1)•1. BB, OE, and S. Günther helped designing the study and drafting the manuscript. DW carried out ITC experiments, crystallization, X-ray data collection, solved the crystal structures and prepared the manuscript.


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Funding Sources The work of M.H. has been financially supported by the German Research Community (DFG) (WO2012/1-1). S. Günther has received funding from the Excellence Initiative of the German Federal and State Governments through the Junior Research Group Program (ZUK 43). The work of X.L. has been funded by the DFG (GU1225/3-1). D. O. and G. W. received funding from the DFG (SFB 992).

ABBREVIATIONS BRD4(1), first bromodomain of bromodomain-containing protein 4; ITC, isothermal titration calorimetry; DSF, differential scanning fluorimetry

PDB ID CODES Atomic coordinates and structure factors were deposited in the Protein Data Bank: BRD4(1)•2, PDB ID 5D25; BRD4(1)•3, PDB ID 5D26; BRD4(1)•4, PDB ID 5D3H; BRD4(1)•6, PDB ID 5D3J; BRD4(1)•7, PDB ID 5D3N; BRD4(1)•10, PDB ID 5D3P; BRD4(1)•12, PDB ID 5D3S; BRD4(1)•13, PDB ID 5D3L; BRD4(1)•14, PDB ID 5D24; BRD4(1)•16, PDB ID 5D3T; BRD4(1)•17, PDB ID 5D3R. REFERENCES (1) Lucas, X.; Wohlwend, D.; Hügle, M.; Schmidtkunz, K.; Gerhardt, S.; Schüle, R.; Jung, M.; Einsle, O.; Günther, S. 4-Acyl pyrroles: mimicking acetylated lysines in histone code reading. Angew. Chem. Int. Ed., 2013, 52, 14055-14059.

(2) Biel, M.; Wascholowski, V.; Giannis, A. Epigenetics - an epicenter of gene regulation: histones and histone-modifying enzymes. Angew. Chem. Int. Ed., 2005, 44, 3186-3216.


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(3) Bannister, A. J.; Schneider, R.; Kouzarides, T. Histone methylation: dynamic or static? Cell, 2002, 109, 801-806.

(4) Klose, R. J.; Zhang, Y. Regulation of histone methylation by demethylimination and demethylation. Nat. Rev. Mol. Cell Biol., 2007, 8, 307-318.

(5) Tian, X.; Fang, J. Current perspectives on histone demethylases. Acta Biochim. Biophys. Sin. (Shanghai), 2007, 39, 81-88.

(6) Imhof, A.; Becker, P. B. Modifications of the histone N-terminal domains. Evidence for an "epigenetic code"? Mol. Biotechnol., 2001, 17, 1-13.

(7) Jenuwein, T.; Allis, C. D. Translating the histone code. Science, 2001, 293, 1074-1080.

(8) Kouzarides, T. Chromatin modifications and their function. Cell, 2007, 128, 693-705.

(9) Muller, S.; Filippakopoulos, P.; Knapp, S. Bromodomains as therapeutic targets. Expert Rev. Mol. Med., 2011, 13, e29.

(10) Waldmann, T.; Schneider, R. Targeting histone modifications--epigenetics in cancer. Curr. Opin. Cell Biol., 2013, 25, 184-189.



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(11) Tamkun, J. W.; Deuring, R.; Scott, M. P.; Kissinger, M.; Pattatucci, A. M.; Kaufman, T. C.; Kennison, J. A. brahma: a regulator of Drosophila homeotic genes structurally related to the yeast transcriptional activator SNF2/SWI2. Cell, 1992, 68, 561-572.

(12) Zeng, L.; Zhou, M. M. Bromodomain: an acetyl-lysine binding domain. FEBS Lett. 2002, 513, 124-128.

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(15) Ciro, M.; Prosperini, E.; Quarto, M.; Grazini, U.; Walfridsson, J.; McBlane, F.; Nucifero, P.; Pacchiana, G.; Capra, M.; Christensen, J.; Helin, K. ATAD2 is a novel cofactor for MYC, overexpressed and amplified in aggressive tumors. Cancer Res., 2009, 69, 8491-8498.

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(17) Filippakopoulos, P., Picaud, S.; Mangos, M.; Keates, T.; Lambert, J.-P.; Barsyte-Lovejoy, D.; Felletar, I.; Volkmer, R.; Müller, S.; Pawson, T.; Gingras, A.-C.; Arrowsmith, C.-H.; Knapp, S. Histone recognition and large-scale structural analysis of the human bromodomain family. Cell, 2012, 149, 214-231.

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FIGURES

Figure 1. X-ray crystal structures of compounds with modified 4-acyl pyrrole bound to BRD4(1). The protein is shown as cartoon (light blue) with solvent accessible surface (light grey, the WL trap in green), waters bridging protein side chains and ligand molecules (both in ball-and-stick, Fo-Fc-map of ligand electron density contoured at 1.3 σ) as red spheres, hydrogen bonds as dashed lines. (A) The lead compound 11 inside the binding pocket is shown as reference. (B) 2 is well positioned inside the binding pocket. (C) Introducing an amide as the 4-acyl substituent in 3 provides a second hydrogen bond to Asn140. The T-shaped π-stacking of the aryl ring and Trp81 is contorted by 10°. (D) Adding a hydroxyl function to the methyl group of the 4-acyl substituent in



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4 results in a second hydrogen bond to the β-amide function of Asn140 with inverted donors and acceptors.


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Figure 2. Molecular electrostatic potential maps reveal the effect of substitutions in the electrostatic potential of the aryl ring in analogs of 1. 1 is modeled as N,N-diethyl-4-hydroxybenzene-1sulfonamide, 6 as N,N-diethylbenzenesulfonamide, 7 as N,4-dimethylbenzene-1-sulfonamide, 12 as 4-(2-fluorobenzenesulfonyl)morpholine , and 13 as N,N-diethyl-4-hydroxy-2-methylbenzene-1sulfonamide. Note, that the amidic substitutions of all ligands have been omitted for the calculation as they do not generate changes to the electrostatic potentials.



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Figure 3. Modifications of the aryl’s electron density of derivatives of 1 modulate the positioning of the ring in the WL trap of BRD4(1). X-ray crystal structures of the compounds in complex with BRD4(1). Representations of BRD4(1) and the ligands are analogous to Figure 1. (A) 6, lacking the hydroxyl group at the aryl ring, loses the orthogonal orientation of the aryl with respect to Trp81. It is rotated by 9°. (B) Substitution of the hydroxyl group by a methyl group while replacing the


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alkyl chains at the sulfonamide by a methyl group in 7 causes a contortion of the T-shaped πstacking by 18°. (C) Closing the ring between the aryl moiety and the amidic nitrogen by introducing an indoline in 10 does not significantly affect the positioning inside the pocket. (D) The change of the o-hydroxyl function to a p-fluoro substituent and the replacement of the ethyl groups of the sulfonamide group by a morpholine ring in 12 cause a tilt of the aryl ring along the ZA channel by 25°. (E) Binding of 13, harboring a p-methyl at the aryl ring, to BRD4(1) is not significantly modified as compared to the lead compound 1.



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Figure 4. Enhancing the surface complementarity and addressing additional sites at the distal binding pocket of BRD4(1). Representations of the X-ray crystal structures of the substrate recognition site of BRD4(1) in complex with new derivatives of 1 are analogous to Figure 1, except for (C). (A) Structure of 14 bound to BRD4(1). The amide group in the center of 14 is inversely oriented, with the oxygen pointing towards the solvent. The aryl plane is displaced along the ZA channel. The terminal amino group engages in hydrogen bonding to Gln84 and Gln85. (B) Structure of 16 bound to BRD4(1). Again, the amide function of the ligand is inversely oriented. The methyl group of the amide function displaces a tightly surface-bound water molecule. The aryl moiety locates outside of the WL trap allowing the terminal amide function to reach to Lys91. (C) Superposition of BRD4(1)•1 (green) and BRD4(1)•16 (orange). Water W5, highlighted with a red circle, is displaced by the methyl substituent at the amide linker of 16, resulting in a reorganization of the hydrogen bond network (dashed lines) between waters (spheres), amino acids (lines), and the ligand (ball-and-stick). (D) Structure of 17 bound to BRD4(1). An increase in surface complementarity is achieved by introducing a propyl substituent at the pyrrole ring and a terminal azepane ACS Paragon Plus Environment

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ring. The aryl ring is rotated along the ZA channel by 12°. (E) Molecular electrostatic potential maps of 1, 10, 14, and 16.



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Figure 5. Hot-spot recognition features in BRD4(1) derived from atom-based QSAR and ligandbased pharmacophore studies. Partial view along the ZA channel of the binding pocket of BRD4(1) in complex with 3. Asn140 and Trp81 are highlighted. Boxes indicate probes with favorable (blue) and unfavorable (red) contribution to ΔG. Atom-based QSAR contributions on ΔG of (A) hydrogen-bond acceptor, (B) hydrogen-bond donor, (C) aromatic ring, and (D) hydrophobic probes, and (E) their combined effect. (F) Ligand-based pharmacophore for BRD4(1), comprising hydrogen-bond acceptor (red spheres), hydrogen-bond donor (blue sphere), hydrophobic (green spheres), and aromatic ring (brown doughnuts) features.


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TABLES Table 1: Modifications on 1 which change binding to Asn140.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table 2: Modifications on 1 which alter WL trap recognition.


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Table 3: Modifications on 1 addressing additional sites outside the ZA channel of BRD4(1).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Table 4: Binding parameters of BRD4(1) interacting with derivatives of 1 as determined by ITC and DSF. Best binders highlighted in green, intermediate binders in yellow, weak binders in red. Ligand 1 2 3 4 5 6 7 8 10 12 13 14 15 16 17

KD

∆G

∆H

∆S

[µM]

[kcal/mol]

[kcal/mol]

[cal/(mol*K)]

0.24 0.50 0.81 7.9 7.2 5.7 9.2 6.8 7.4 1.3 0.88 5.8 3.9 12 n.d.

-9.03 -8.59 -8.31 -6.96 -7.01 -7.15 -6.87 -7.05 -7.00 -8.04 -8.26 -7.14 -7.38 -6.71 n.d.

-14.8 -11.7 -11.7 -6.49 -9.81 -18.7 -10.4 -7.90 -2.96 -9.77 -6.67 -11.8 -5.90 -1.77 n.d.

-19.5 -10.6 -11.3 1.56 -9.36 -38.6 -11.7 -2.86 13.6 -5.80 5.34 -15.6 4.98 16.6 n.d.

∆Tm [K]

6.7 2.1 5.6 4.4 n.d. 3.3 3.1 n.d. n.d. 2.8 3.5 3.0 n.d. 2.4 5.3


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Table 5: Specificity screening of chosen representatives of the derivatives of 1 against BRD3(1) as representative of the same branch and BRD3(2) as representative of the neighbor branch of the BET family, and BRPF1 as distantly related bromodomain. All affinity constants were determined by ITC. Affinity as KD [µM] Ligand

Bromodomain family discrimination

BRD4(1) BRD3(1) BRD3(2) BRPF1(1) KD1 KD2 KD3 KD4

Intra-BET

Intra-BET

Extra-BET

-first BDs-

'( ⁄'(

-second BDs-

12.2

'(* ⁄'(

-distant relative-

9.88

159

'(* ⁄'(

1

0.24

2.9

2.4

38

2

0.50

n.d.

n.d.

5.9

-

-

11.8

3

0.81

n.d.

n.d.

48

-

-

59.3

4

7.9

4.9

11

n.d.

0.620

1.41

-

5

7.2

n.d.

n.d.

36

-

-

5.04

12

1.3

6.1

18

n.d.

4.79

13.8

-

14

5.8

41

23

n.d.

n.d.

6.95

4.01

16

12

50

19

n.d.

4.12

1.59

-

18

6.8

n.d.

n.d.

30

-

-

4.37



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Table of Contents artwork


52

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Figure 1. X-ray crystal structures of compounds with modified 4-acyl pyrrole bound to BRD4(1). The protein is shown as cartoon (light blue) with solvent accessible surface (light grey, the WL trap in green), waters bridging protein side chains and ligand molecules (both in ball-and-stick, Fo-Fc-map of ligand electron density contoured at 1.3 σ) as red spheres, hydrogen bonds as dashed lines. (A) The lead compound 11 inside the binding pocket is shown as reference. (B) 2 is well positioned inside the binding pocket. (C) Introducing an amide as the 4-acyl substituent in 3 provides a second hydrogen bond to Asn140. The Tshaped π-stacking of the aryl ring and Trp81 is contorted by 10°. (D) Adding a hydroxyl function to the methyl group of the 4-acyl substituent in 4 results in a second hydrogen bond to the β-amide function of Asn140 with inverted donors and acceptors. 119x81mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. Molecular electrostatic potential maps reveal the effect of substitutions in the electrostatic potential of the aryl ring in analogs of 1. 1 is modeled as N,N‐diethyl‐4‐hydroxybenzene‐1‐sulfonamide, 6 as N,N‐ diethylbenzenesulfonamide, 7 as N,4‐dimethylbenzene‐1‐sulfonamide, 12 as 4‐(2‐ fluorobenzenesulfonyl)morpholine , and 13 as N,N‐diethyl‐4‐hydroxy‐2‐methylbenzene‐1‐sulfonamide. Note, that the amidic substitutions of all ligands have been omitted for the calculation as they do not generate changes to the electrostatic potentials. 88x92mm (300 x 300 DPI)


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Figure 3. Modifications of the aryl’s electron density of derivatives of 1 modulate the positioning of the ring in the WL trap of BRD4(1). X-ray crystal structures of the compounds in complex with BRD4(1). Representations of BRD4(1) and the ligands are analogous to Figure 1. (A) 6, lacking the hydroxyl group at the aryl ring, loses the orthogonal orientation of the aryl with respect to Trp81. It is rotated by 9°. (B) Substitution of the hydroxyl group by a methyl group while replacing the alkyl chains at the sulfonamide by a methyl group in 7 causes a contortion of the T-shaped π-stacking by 18°. (C) Closing the ring between the aryl moiety and the amidic nitrogen by introducing an indoline in 10 does not significantly affect the positioning inside the pocket. (D) The change of the o-hydroxyl function to a p-fluoro substituent and the replacement of the ethyl groups of the sulfonamide group by a morpholine ring in 12 cause a tilt of the aryl ring along the ZA channel by 25°. (E) Binding of 13, harboring a p-methyl at the aryl ring, to BRD4(1) is not significantly modified as compared to the lead compound 1. 180x191mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Enhancing the surface complementarity and addressing additional sites at the distal binding pocket of BRD4(1). Representations of the X-ray crystal structures of the substrate recognition site of BRD4(1) in complex with new derivatives of 1 are analogous to Figure 1, except for (C). (A) Structure of 14 bound to BRD4(1). The amide group in the center of 14 is inversely oriented, with the oxygen pointing towards the solvent. The aryl plane is displaced along the ZA channel. The terminal amino group engages in hydrogen bonding to Gln84 and Gln85. (B) Structure of 16 bound to BRD4(1). Again, the amide function of the ligand is inversely oriented. The methyl group of the amide function displaces a tightly surface-bound water molecule. The aryl moiety locates outside of the WL trap allowing the terminal amide function to reach to Lys91. (C) Superposition of BRD4(1)•1 (green) and BRD4(1)•16 (orange). Water W5, highlighted with a red circle, is displaced by the methyl substituent at the amide linker of 16, resulting in a reorganization of the hydrogen bond network (dashed lines) between waters (spheres), amino acids (lines), and the ligand (balland-stick). (D) Structure of 17 bound to BRD4(1). An increase in surface complementarity is achieved by introducing a propyl substituent at the pyrrole ring and a terminal azepane ring. The aryl ring is rotated along the ZA channel by 12°. (E) Molecular electrostatic potential maps of 1, 10, 14, and 16. 96x53mm (300 x 300 DPI)


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Figure 5. Hot-spot recognition features in BRD4(1) derived from atom-based QSAR and ligand-based pharmacophore studies. Partial view along the ZA channel of the binding pocket of BRD4(1) in complex with 3. Asn140 and Trp81 are highlighted. Boxes indicate probes with favorable (blue) and unfavorable (red) contribution to ∆G. Atom-based QSAR contributions on ∆G of (A) hydrogen-bond acceptor, (B) hydrogenbond donor, (C) aromatic ring, and (D) hydrophobic probes, and (E) their combined effect. (F) Ligand-based pharmacophore for BRD4(1), comprising hydrogen-bond acceptor (red spheres), hydrogen-bond donor (blue sphere), hydrophobic (green spheres), and aromatic ring (brown doughnuts) features. 77x33mm (300 x 300 DPI)


4-Acyl Pyrrole Derivatives Yield Novel Vectors for Designing Inhibitors

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