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Sebastian Vaas,1 Markus O Zimmermann,1 Theresa Klett,1 Frank M Boeckler1,2 1Department of Pharmacy and Biochemistry, Eberhard Karls Universität Tübingen, Laboratory for Molecular Design and Pharmaceutical Biophysics, Institute of Pharmaceutical Sciences, Tübingen, 72076, Germany; 2Institute for Bioinformatics and Medical Informatics (IBMI), Eberhard Karls Universität Tübingen, Tübingen, 72076, GermanyCorrespondence: Frank M Boeckler, Molecular Design and Pharmaceutical Biophysics, Institute of Pharmaceutical Sciences, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 8 (Haus B), Tübingen, D-72076, Germany, Tel +49 7071 29 74567, Fax +49 7071 29 5637, Email frank.boeckler@uni-tuebingen.deIntroduction: Therapeutic peptides are a significant class of drugs in the treatment of a wide range of diseases. To enhance their properties, such as stability or binding affinity, they are usually chemically modified. This includes, among other techniques, cyclization of the peptide chain by bridging, modifications to the backbone, and incorporation of unnatural amino acids. One approach previously established, is the use of halogenated aromatic amino acids. In principle, they are thereby enabled to form halogen bonds (XB). In this study, we focus on the -R-CF2X moiety (R = O, NHCO; X = Cl, Br) as an uncommon halogen bond donor. These groups enable more spatial variability in protein–protein interactions. The chosen approach via Fmoc-protected building blocks allows for the incorporation of these modified amino acids in peptides using solid-phase peptide synthesis.Results and Discussion: Using a competitive fluorescence polarization assay to monitor binding to Mdm4, we demonstrate that a p53-derived peptide with Lys24Nle(&epsiv;NHCOCF2X) exhibits an improved inhibition constant Ki compared to the unmodified peptide. Decreasing Ki values observed with the increasing XB capacity of the halogen atoms (F ≪ Cl < Br) indicates the formation of a halogen bond. By reducing the side chain length of Nle(&epsiv;NHCOCF2X) to Abu(γNHCOCF2X) as control experiments and through quantum mechanical calculations, we suggest that the observed affinity enhancement is related to halogen bond-induced intramolecular stabilization of the α-helical binding mode of the peptide or a direct interaction with His54 in human Mdm4.Graphical Abstract: Keywords: halogen bonding, solid-phase peptide synthesis, unnatural amino acids, fluorescence polarization assay, oncological target, MdmX

The cellular tumor antigen p53 is a key component in cell cycle control. The mutation Y220C heavily destabilizes the protein thermally but yields a druggable crevice. We have screened the diversity-optimized halogen-enriched fragment library against T-p53C-Y220C with STD-NMR and DSF to identify hits, which we validated by

Two new ircinianin-type sesterterpenoids, ircinianin lactone B and ircinianin lactone C (7 and 8), together with five known entities from the ircinianin compound family (1, 3–6) were isolated from the marine sponge Ircinia wistarii. Ircinianin lactones B and C (7 and 8) represent new ircinianin terpenoids with a modified oxidation pattern. Despite their labile nature, the structures could be established using a combination of spectroscopic data, including HRESIMS and 1D/2D NMR techniques, as well as computational chemistry and quantum-mechanical calculations. In a broad screening approach for biological activity, the class-defining compound ircinianin (1) showed moderate antiprotozoal activity against Plasmodium falciparum (IC50 25.4 μM) and Leishmania donovani (IC50 16.6 μM).

Fragment-based drug discovery is one of the most utilized approaches for the identification of novel weakly binding ligands, by efficiently covering a wide chemical space with rather few compounds and by allowing more diverse binding modes to be found. This approach has led to various clinical candidates and approved drugs. Halogen bonding, on the other hand, has gained traction in molecular design and lead optimization, but could offer additional benefits in early drug discovery. Screening halogen-enriched fragments (HEFLibs) could alleviate problems associated with the late introduction of such a highly geometry dependent interaction. Usually, the binding mode is then already dominated by other strong interactions. Due to the fewer competing interactions in fragments, the halogen bond should more often act as an anchor point for the binding mode. Previously, we proposed a fragment library with a focus on diverse binding modes that involve halogens for gaining initial affinity and selectivity. Herein, we demonstrate the applicability of these HEFLibs with a small set of diverse enzymes: the histone-lysine N-methyltransferase DOT1L, the indoleamine 2,3-dioxygenase 1 (IDO1), the AP2-associated protein kinase 1 (AAK1), and the calcium/calmodulin-dependent protein kinase type 1G (CAMK1G). We were able to identify various binding fragments

Halogen bonds have become increasingly popular interactions in molecular design and drug discovery. One of the key features is the strong dependence of the size and magnitude of the halogen's σ-hole on the chemical environment of the ligand. The term σ-hole refers to a region of lower electronic density opposite to a covalent bond, e.g., the C-X bond. It is typically (but not always) associated with a positive electrostatic potential in close proximity to the extension of the covalent bond. Herein, we use a variety of 30 nitrogen-bearing heterocycles, halogenated systematically by chlorine, bromine, or iodine, yielding 468 different ligands that are used to exemplify scaffold effects on halogen bonding strength. As a template interaction partner, we have chosen N-methylacetamide representing the ubiquitously present protein backbone. Adduct formation energies were obtained at a MP2/TZVPP level of theory. We used the local maximum of the electrostatic potential on the molecular surface in close proximity to the σ-hole, V S,max, as a descriptor for the magnitude of the positive electrostatic potential characterizing the tuning of the σ-hole. Free optimization of the complexes gave reasonable correlations with V S,max but was found to be of limited use because considerable numbers of chlorinated and brominated ligands lost their halogen bond or showed significant secondary interactions. Thus, starting from a close to optimal geometry of the halogen bond, we used distance scans to obtain the best adduct formation energy for each complex. This approach provided superior results for all complexes exhibiting correlations with R2 > 0.96 for each individual halogen. We evaluated the dependence of V S,max from the molecular surface onto which the positive electrostatic potential is projected, altering the isodensity values from 0.001 au to 0.050 au. Interestingly, the best overall fit using a third-order polynomial function (R2 = 0.99, RMSE = 0.562 kJ/mol) with rather smooth transitions between all halogens was obtained for V S,max calculated from an isodensity surface at 0.014 au.

Automated test case generation techniques usually aim to maximize some coverage criteria. For object oriented languages, like Java, the branches that can be reached in source code, frequently depend on the internal object state. Meaning certain branches will only be taken, if fields inside the tested class are set to specific values. It is however not obvious how much of the internal object state can be controlled. In this paper, we analyzed a corpus of 110 open source systems to evaluate how settable their classes are. Meaning we looked for ways that fields inside classes can be written. For instance, we analyzed the source code to identify setter methods that can be used to set the values of a field. Our results show that 66.5% of fields can be set to a desired value, while 31.5% of fields may be only settable to particular values or require a more in depth analysis. Only for 2% of fields, we did not find any way to set their values.

Bioisosteric replacements are widely used in medicinal chemistry to improve physicochemical and ADME properties of molecules while retaining or improving affinity. Here, using the p53 cancer mutant Y220C as a test case, we investigate both computationally and experimentally whether an ethynyl moiety is a suitable bioisostere to replace iodine in ligands that form halogen bonds with the protein backbone. This bioisosteric transformation is synthetically feasible via Sonogashira cross-coupling. In our test case of a particularly strong halogen bond, replacement of the iodine with an ethynyl group resulted in a 13-fold affinity loss. High-resolution crystal structures of the two analogues in complex with the p53-Y220C mutant enabled us to correlate the different affinities with particular features of the binding site and subtle changes in ligand binding mode. In addition, using QM calculations and analyzing the PDB, we provide general guidelines for identifying cases where such a transformation is likely to improve ligand recognition.

We present a QM-derived empirical scoring function for the interaction between aromatic halogenated ligands and the carbonyl oxygen atom of the protein backbone. Applying this scoring function, we developed an algorithm that evaluates the potential of protein-bound ligands to form favorable halogen-bonding contacts upon scaffold decoration with chlorine, bromine, or iodine. Full recovery of all existing halogen bonds in the PDB involving the protein backbone was achieved with our protocol. Interestingly, the potential for introducing halogen bonds through scaffold decoration of unsubstituted aromatic carbon atoms appears to easily match the number of previously known halogen bonds. Our approach can thus be used as a blueprint for integration of halogen bonding into general empirical scoring functions, which at present ignore this interaction. Most importantly, we were able to identify a substantial number of protein-ligand complexes where the benefits and challenges of introducing a halogen bond by molecular design can be studied experimentally.

Using halogen-specific Connolly type molecular surfaces, we herein invented a new type of surface-based interaction analysis employed for the study of halogen bonding toward model systems of biologically relevant carboxylates (ASP/GLU) and carboxamides (ASN/GLN). Database mining and statistical assessment of the PDB revealed that such interactions are widely underrepresented at the moment. We observed important distance-dependent adaptions of the binding modes of halobenzenes from a preferential oxygen-directed to a bifurcated interaction geometry of the carboxylate. In addition, halogen···π contacts perpendicular to the nitrogen atom of the carboxamide become increasingly important for the lighter halogens. Our analysis on a MP2/TZVPP level of theory is backed by CCSD(T)/CBS reference calculations. To put the vast interaction energies into perspective, we also performed COSMO-RS calculations of the solvation free energy. Facilitating the visualization of our results mapped onto any binding site of choice, we aim to inspire more design studies showcasing these underrepresented interactions.

Author(s): Fechner, Uli; de Graaf, Chris; Torda, Andrew E; Gussregen, Stefan; Evers, Andreas; Matter, Hans; Hessler, Gerhard; Richmond, Nicola J; Schmidtke, Peter; Segler, Marwin HS; Waller, Mark P; Pleik, Stefanie; Shea, Joan-Emma; Levine, Zachary; Mullen, Ryan; van den Broek, Karina; Epple, Matthias; Kuhn, Hubert; Truszkowski, Andreas; Zielesny, Achim; Fraaije, Johannes Hans; Gracia, Ruben Serral; Kast, Stefan M; Bulusu, Krishna C; Bender, Andreas; Yosipof, Abraham; Nahum, Oren; Senderowitz, Hanoch; Krotzky, Timo; Schulz, Robert; Wolber, Gerhard; Bietz, Stefan; Rarey, Matthias; Zimmermann, Markus O; Lange, Andreas; Ruff, Manuel; Heidrich, Johannes; Onlia, Ionut; Exner, Thomas E; Boeckler, Frank M; Bermudez, Marcel; Firaha, Dzmitry S; Holloczki, Oldamur; Kirchner, Barbara; Tautermann, Christofer S; Volkamer, Andrea; Eid, Sameh; Turk, Samo; Rippmann, Friedrich; Fulle, Simone; Saleh, Noureldin; Saladino, Giorgio; Gervasio, Francesco L; Haensele, Elke; Banting, Lee; Whitley, David C; Oliveira Santos, Jana Sopkova-de; Bureau, Ronan; Clark, Timothy; Sandmann, Achim; Lanig, Harald; Kibies, Patrick; Heil, Jochen; Hoffgaard, Franziska; Frach, Roland; Engel, Julian; Smith, Steven; Basu, Debjit; Rauh, Daniel; Kohlbacher, Oliver; Boeckler, Frank M; Essex, Jonathan W; Bodnarchuk, Michael S; Ross, Gregory A; Finkelmann, Arndt R; Goller, Andreas H; Schneider, Gisbert; Husch, Tamara; Schutter, Christoph; Balducci, Andrea; Korth, Martin; Ntie-Kang, Fidele; Gunther, Stefan; Sippl, Wolfgang; Mbaze, Luc Meva'a

p38α mitogen-activated protein (MAP) kinase is a main target in drug research concerning inflammatory diseases. Nevertheless, no inhibitor of p38α MAP kinase has been introduced to the market. This might be attributed to the fact that there is no inhibitor which combines outstanding activity in biological systems and selectivity. Herein an approach to the development of such inhibitors on the basis of the highly selective molecular probe Skepinone-L is described. Introduction of a "deep pocket" moiety addressing the DFG motif led to an increased activity of the compounds. Hydrophilic moieties, addressing the solvent-exposed area adjacent to hydrophilic region II, conserved a high activity of the compounds in a whole blood assay. Combined with their outstanding selectivity and low ATP competitiveness, these inhibitors are very interesting candidates for use in biological systems and in therapy.

The dimeric mammalian phosphodiesterases (PDEs) are regulated by N-terminal domains. In PDE5, the GAF-A subdomain of a GAF-tandem (GAF-A and -B) binds the activator cGMP and in PDE10 GAF-B binds cAMP. GAF-tandem chimeras of PDE5 and 10 in which the 36 aa linker helix between GAF-A and -B was swapped lost allosteric regulation of a reporter adenylyl cyclase. In 16 consecutive constructs we substituted the PDE10 linker with that from PDE5. An initial stretch of 10 amino acids coded for isoform specificity. A C240Y substitution uncoupled cyclase activity from regulation, whereas C240F, L or G did not. The C240Y substitution increased basal activity to stimulated levels. Notably, over the next 12 substitutions basal cyclase activity decreased linearly. Further targeted substitutions were based on homology modeling using the PDE2 structure. No combination of substitutions within the initial 10 linker residues caused loss of regulation. The full 10 aa stretch was required. Modeling indicated a potential interaction of the linker with a loop from GAF-A. To interrupt H-bonding a glycine substitution of the loop segment was generated. Despite reduction of basal activity, loss of regulation was maintained. Possibly, the orientation of the linker helix is determined by formation of the dimer at the initial linker segment. Downstream deflections of the linker helix may have caused loss of regulation.

In the course of searching for new p38α MAP kinase inhibitors, we found that the regioisomeric switch from 3-(4-fluorophenyl)-4-(pyridin-4-yl)-1-(aryl)-1H-pyrazol-5-amine to 4-(4-fluorophenyl)-3-(pyridin-4-yl)-1-(aryl)-1H-pyrazol-5-amine led to an almost complete loss of p38α inhibition, but they showed activity against important cancer kinases. Among the tested derivatives, 4-(4-fluorophenyl)-3-(pyridin-4-yl)-1-(2,4,6-trichlorophenyl)-1H-pyrazol-5-amine (6a) exhibited the best activity, with IC(50) in the nanomolar range against Src, B-Raf wt, B-Raf V600E, EGFRs, and VEGFR-2, making it a good lead for novel anticancer programs.

We target the gatekeeper MET146 of c-Jun N-terminal kinase 3 (JNK3) to exemplify the applicability of X···S halogen bonds in molecular design using computational, synthetic, structural and biophysical techniques. In a designed series of aminopyrimidine-based inhibitors, we unexpectedly encounter a plateau of affinity. Compared to their QM-calculated interaction energies, particularly bromine and iodine fail to reach the full potential according to the size of their σ-hole. Instead, mutation of the gatekeeper residue into leucine, alanine, or threonine reveals that the heavier halides can significantly influence selectivity in the human kinome. Thus, we demonstrate that, although the choice of halogen may not always increase affinity, it can still be relevant for inducing selectivity. Determining the crystal structure of the iodine derivative in complex with JNK3 (4X21) reveals an unusual bivalent halogen/chalcogen bond donated by the ligand and the back-pocket residue MET115. Incipient repulsion from the too short halogen bond increases the flexibility of Cε of MET146, whereas the rest of the residue fails to adapt being fixed by the chalcogen bond. This effect can be useful to induce selectivity, as the necessary combination of methionine residues only occurs in 9.3% of human kinases, while methionine is the predominant gatekeeper (39%).

Halogen bonding has recently experienced a renaissance, gaining increased recognition as a useful molecular interaction in the life sciences. Halogen bonds are favorable, fairly directional interactions between an electropositive region on the halogen (the σ-hole) and a number of different nucleophilic interaction partners. Some aspects of halogen bonding are not yet understood well enough to take full advantage of its potential in drug discovery. We describe and present the concept of halogen-enriched fragment libraries. These libraries consist of unique chemical probes, facilitating the identification of favorable halogen bonds by sharing the advantages of classical fragment-based screening. Besides providing insights into the nature and applicability of halogen bonding, halogen-enriched fragment libraries provide smart starting points for hit-to-lead evolution.

Halogen bonds are directional noncovalent interactions that can be used to target electron donors in a protein binding site. In this study, we employ quantum chemical calculations to explore halogen···nitrogen contacts involving histidine side chains. We characterize the energetics on the MP2 level of theory using SCS-MP2 and CCSD(T)/CBS as reference calculations and elucidate their energy profile in suboptimal geometries. We derive simple rules allowing medicinal chemists and chemical biologists to easily determine preferred areas of interaction in a binding site and exploit them for scaffold decoration and design. Our work shows that nitrogen-halogen bonds are valuable interactions that are this far underexploited in patent applications, lead structure, and clinical candidate selection. We highlight their potential to increase binding affinities and suggest that they can significantly contribute to inducing and tuning subtype selectivities.

Halogen bonding has been known in material science for decades, but until recently, halogen bonds in protein–ligand interactions were largely the result of serendipitous discovery rather than rational design. In this Perspective, we provide insights into the phenomenon of halogen bonding, with special focus on its role in drug discovery. We summarize the theoretical background defining its strength and directionality, provide a systematic analysis of its occurrence and interaction geometries in protein–ligand complexes, and give recent examples where halogen bonding has been successfully harnessed for lead identification and optimization. In light of these data, we discuss the potential and limitations of exploiting halogen bonds for molecular recognition and rational drug design.

The destabilizing p53 cancer mutation Y220C creates a druggable surface crevice. We developed a strategy exploiting halogen bonding for lead discovery to stabilize the mutant with small molecules. We designed halogen-enriched fragment libraries (HEFLibs) as starting points to complement classical approaches. From screening of HEFLibs and subsequent structure-guided design, we developed substituted 2-(aminomethyl)-4-ethynyl-6-iodophenols as p53-Y220C stabilizers. Crystal structures of their complexes highlight two key features: (i) a central scaffold with a robust binding mode anchored by halogen bonding of an iodine with a main-chain carbonyl and (ii) an acetylene linker, enabling the targeting of an additional subsite in the crevice. The best binders showed induction of apoptosis in a human cancer cell line with homozygous Y220C mutation. Our structural and biophysical data suggest a more widespread applicability of HEFLibs in drug discovery.