Principles and Applications of CF2X Moieties as Unconventional Halogen Bond Donors in Medicinal Chemistry, Chemical Biology, and Drug Discovery.

As an orthogonal principle to the established (hetero)aryl halides, we herein highlight the usefulness of CF2X (X = Cl, Br, or I) moieties. Using tool compounds bearing CF2X moieties, we study their chemical/metabolic stability and their logP/solubility, as well as the role of XB in their small molecular crystal structures. Employing QM techniques, we analyze the observed interactions, provide insights into the conformational flexibilities and preferences in the potential interaction space. For their application in molecular design, we characterize their XB donor capacities and its interaction strength dependent on geometric parameters. Implementation of CF2X acetamides into our HEFLibs and biophysical evaluation (STD-NMR/ITC), followed by X-ray analysis, reveals a highly interesting binding mode for fragment 23 in JNK3, featuring an XB of CF2Br toward the P-loop, as well as chalcogen bonds. We suggest that underexplored chemical space combined with unconventional binding modes provides excellent opportunities for patentable chemotypes for therapeutic intervention.

Synthesis of Amino Acids Bearing Halodifluoromethyl Moieties and Their Application to p53-Derived Peptides Binding to Mdm2/Mdm4.

Introduction: 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(εNHCOCF2X) 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(εNHCOCF2X) 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.

Revisiting a challenging p53 binding site: a diversity-optimized HEFLib reveals diverse binding modes in T-p53C-Y220C.

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 1H,15N-HSQC NMR. We could identify four hits binding in the Y220C cleft, one hit binding covalently and four hits binding to an uncharacterized binding site. Compound 1151 could be crystallized showing a flip of C220 and thus opening subsite 3. Additionally, 4482 was identified to alkylate cysteines. Data shows that the diversity-optimized HEFLib leads to multiple diverse hits. The identified scaffolds can be used to further optimize interactions with T-p53C-Y220C and increase thermal stability.

Screening of a Halogen-Enriched Fragment Library Leads to Unconventional Binding Modes.

We conceived the Halogen-Enriched Fragment Library (HEFLib) to investigate the potential of halogen bonds in the early stages of drug discovery. As the number of competitive interactions increases with ligand size, we reasoned that a binding mode relying on halogen bonding is more likely for fragments than highly decorated molecules. Thus, fragments could feature unexplored binding modes. We screened the HEFLib against the human kinase DYRK1a and verified micromolar binding fragments via isothermal titration calorimetry (ITC). The crystal structure of one fragment revealed a noncanonical binding mode, despite the fragment's classical hinge binding motif. In addition, the fragment occupies a secondary binding site. Both binding modes feature a halogen bond, which we evaluated by ab initio calculations. Structure-affinity relationship (SAR) from a set of analogues improves the affinity, provides a promising fragment-growth vector, and highlights the benefits and applicability of halogen bonds in early lead development.

Discovery of Ircinianin Lactones B and C-Two New Cyclic Sesterterpenes from the Marine Sponge Ircinia wistarii.

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).

Quadruple Target Evaluation of Diversity-Optimized Halogen-Enriched Fragments (HEFLibs) Reveals Substantial Ligand Efficiency for AP2-Associated Protein Kinase 1 (AAK1).

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 via STD-NMR. Using ITC to verify these initial hits, we determined affinities for many of these fragments. The best binding fragments exhibit affinities in the one-digit micromolar range and ligand efficiencies up to 0.83 for AAK1. A small set of analogs was used to study structure-affinity relationships and hereby analyze the specific importance of each polar interaction. This data clearly suggests that the halogen bond is the most important interaction of fragment 9595 with AAK1.

Scaffold Effects on Halogen Bonding Strength.

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.

Using Surface Scans for the Evaluation of Halogen Bonds toward the Side Chains of Aspartate, Asparagine, Glutamate, and Glutamine.

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.

Targeting the Gatekeeper MET146 of C-Jun N-Terminal Kinase 3 Induces a Bivalent Halogen/Chalcogen Bond.

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%).

Experimental and Theoretical Evaluation of the Ethynyl Moiety as a Halogen Bioisostere.

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.

Evaluating the potential of halogen bonding in molecular design: automated scaffold decoration using the new scoring function XBScore.

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.

Halogen-enriched fragment libraries as chemical probes for harnessing halogen bonding in fragment-based lead discovery.

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.

Targeting histidine side chains in molecular design through nitrogen-halogen bonds.

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.

Principles and applications of halogen bonding in medicinal chemistry and chemical biology.

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.

Using halogen bonds to address the protein backbone: a systematic evaluation.

Halogen bonds are specific embodiments of the sigma hole bonding paradigm. They represent directional interactions between the halogens chlorine, bromine, or iodine and an electron donor as binding partner. Using quantum chemical calculations at the MP2 level, we systematically explore how they can be used in molecular design to address the omnipresent carbonyls of the protein backbone. We characterize energetics and directionality and elucidate their spatial variability in sub-optimal geometries that are expected to occur in protein-ligand complexes featuring a multitude of concomitant interactions. By deriving simple rules, we aid medicinal chemists and chemical biologists in easily exploiting them for scaffold decoration and design. Our work shows that carbonyl-halogen bonds may be used to expand the patentable medicinal chemistry space, redefining halogens as key features. Furthermore, this data will be useful for implementing halogen bonds into pharmacophore models or scoring functions making the QM information available for automatic molecular recognition in virtual high throughput screening.

Halogen-enriched fragment libraries as leads for drug rescue of mutant p53.

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.

Intramolecular signaling in tandem-GAF domains from PDE5 and PDE10 studied with a cyanobacterial adenylyl cyclase reporter.

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.

Tri- and tetrasubstituted pyrazole derivates: regioisomerism switches activity from p38MAP kinase to important cancer kinases.

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.

Addressing Methionine in Molecular Design through Directed Sulfur-Halogen Bonds.

Halogen bonds are directional interactions involving an electron donor as binding partner. Employing quantum chemical calculations, we explore how they can be used in molecular design to address the sulfur atom in a methionine residue in a previously neglected, directed manner. We characterize energetics and directionality of these halogen bonds and elucidate their spatial variability in suboptimal geometries that are expected to occur in protein-ligand complexes featuring a multitude of concomitant interactions. We derive simple rules allowing medicinal chemists and chemical biologists to easily determine preferred areas of interaction within a binding site and to exploit them for scaffold decoration and design. Our work shows that sulfur-halogen bonds may be used to expand the patentable medicinal chemistry space. We demonstrate their potential to increase binding affinities and suggest that they can significantly contribute to inducing and tuning subtype selectivities.