Hinge Binding Motif Research Articles

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.

Novel purine analogues regulate IL-1β release via inhibition of JAK activity in human aortic smooth muscle cells

Purine analogues bearing a nitrate ester motif were previously discovered as cardioprotective and anti-inflammatory agents, but the anti-inflammatory mechanism remains to be established. We therefore investigated the anti-inflammatory effect of two purine analogues, MK118 bearing a nitrate ester moiety and the methyl-substituted analogue MK196 in Aortic Smooth Muscle Cells (AoSMCs), with emphasis on IL-1β release. The AoSMCs were stimulated with LPS with or without purine analogue, followed by ELISA, Olink proteomics, Western blot and real time PCR of NLRP3 inflammasome components. Both purine analogues inhibited the release of proteins involved in inflammation, such as TRAIL, CCL4, CSF1 and IL-1β in AoSMCs, as well as intracellular gene and protein expression of IL-1β and NLRP3 inflammasome components. MK196, but not MK118, also inhibited the LPS-induced release of IL-7, CXCL10, PD-L1, FLT3L and CCL20. We also showed that MK118 and possibly MK196 act via inhibition of JAKs. In silico studies showed that the purine moiety is a competent hinge binding motif and that the purine-piperazine scaffold is well accommodated in the lipophilic groove of JAK1-3. Both compounds establish interactions with catalytic amino acids in the active site of JAK1-3 and the terminal nitrate ester of MK118 was revealed as a promising pharmacophore.Our data suggest that MK118 and MK196 inhibit the release of proinflammatory proteins in AoSMCs, and targets JAK1-3 activation. Purine analogues also inhibit the expression of NLRP3 inflammasome genes and proteins and may in the future be evaluated for anti-inflammatory aspects on inflammatory diseases.

Recent Advances of Pyridinone in Medicinal Chemistry.

Pyridinones have been adopted as an important block in medicinal chemistry that could serve as hydrogen bond donors and acceptors. With the help of feasible synthesis routes via established condensation reactions, the physicochemical properties of such a scaffold could be manipulated by adjustment of polarity, lipophilicity, and hydrogen bonding, and eventually lead to its wide application in fragment-based drug design, biomolecular mimetics, and kinase hinge-binding motifs. In addition, most pyridinone derivatives exhibit various biological activities ranging from antitumor, antimicrobial, anti-inflammatory, and anticoagulant to cardiotonic effects. This review focuses on recent contributions of pyridinone cores to medicinal chemistry, and addresses the structural features and structure–activity relationships (SARs) of each drug-like molecule. These advancements contribute to an in-depth understanding of the potential of this biologically enriched scaffold and expedite the development of its new applications in drug discovery.

Identification of Pyrimidine-Based Lead Compounds for Understudied Kinases Implicated in Driving Neurodegeneration

The pyrimidine core has been utilized extensively to construct kinase inhibitors, including eight FDA-approved drugs. Because the pyrimidine hinge-binding motif is accommodated by many human kinases, kinome-wide selectivity of resultant molecules can be poor. This liability was seen as an advantage since it is well tolerated by many understudied kinases. We hypothesized that nonexemplified aminopyrimidines bearing side chains from well-annotated pyrimidine-based inhibitors with off-target activity on understudied kinases would provide us with useful inhibitors of these lesser studied kinases. Our strategy paired mixing and matching the side chains from the 2- and 4-positions of the parent compounds with modifications at the 5-position of the pyrimidine core, which is situated near the gatekeeper residue of the binding pocket. Utilizing this approach, we imparted improved kinome-wide selectivity to most members of the resultant library. Importantly, we also identified potent biochemical and cell-active lead compounds for understudied kinases like DRAK1, BMP2K, and MARK3/4.

Exploiting an Asp-Glu “Switch” in Glycogen Synthase Kinase 3 to Design Paralog Selective Inhibitors for Acute Myeloid Leukemia Treatment

Exploiting an Asp-Glu “Switch” in Glycogen Synthase Kinase 3 to Design Paralog Selective Inhibitors for Acute Myeloid Leukemia Treatment

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a Pyridopyridazinone pan-RAF Kinase Inhibitor.

Structure-based optimization of a set of aryl urea RAF inhibitors has led to the identification of Type II pan-RAF inhibitor GNE-9815 (7), which features a unique pyrido[2,3-d]pyridazin-8(7H)-one hinge-binding motif. With minimal polar hinge contacts, the pyridopyridazinone hinge binder moiety affords exquisite kinase selectivity in a lipophilic efficient manner. The improved physicochemical properties of GNE-9815 provided a path for oral dosing without enabling formulations. In vivo evaluation of GNE-9815 in combination with the MEK inhibitor cobimetinib demonstrated synergistic MAPK pathway modulation in an HCT116 xenograft mouse model. To the best of our knowledge, GNE-9815 is among the most highly kinase-selective RAF inhibitors reported to date.

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a 5-Fluoro-4-(3H)-quinazolinone Aryl Urea pan-RAF Kinase Inhibitor.

Optimization of a series of aryl urea RAF inhibitors led to the identification of type II pan-RAF inhibitor GNE-0749 (7), which features a fluoroquinazolinone hinge-binding motif. By minimizing reliance on common polar hinge contacts, this hinge binder allows for a greater contribution of RAF-specific residue interactions, resulting in exquisite kinase selectivity. Strategic substitution of fluorine at the C5 position efficiently masked the adjacent polar NH functionality and increased solubility by impeding a solid-state conformation associated with stronger crystal packing of the molecule. The resulting improvements in permeability and solubility enabled oral dosing of 7. In vivo evaluation of 7 in combination with the MEK inhibitor cobimetinib demonstrated synergistic pathway inhibition and significant tumor growth inhibition in a KRAS mutant xenograft mouse model.

Fragment Binding to Kinase Hinge: If Charge Distribution and Local pKa Shifts Mislead Popular Bioisosterism Concepts.

Medicinal‐chemistry optimization follows strategies replacing functional groups and attaching larger substituents at a promising lead scaffold. Well‐established bioisosterism rules are considered, however, it is difficult to estimate whether the introduced modifications really match the required properties at a binding site. The electron density distribution and pK a values are modulated influencing protonation states and bioavailability. Considering the adjacent H‐bond donor/acceptor pattern of the hinge binding motif in a kinase, we studied by crystallography a set of fragments to map the required interaction pattern. Unexpectedly, benzoic acid and benzamidine, decorated with the correct substituents, are totally bioisosteric just as carboxamide and phenolic OH. A mono‐dentate pyridine nitrogen out‐performs bi‐dentate functionalities. The importance of correctly designing pK a values of attached functional groups by additional substituents at the parent scaffold is rendered prominent.

Discovery of Potent and Selective PI3Kγ Inhibitors.

The selective inhibition of the lipid signaling enzyme PI3Kγ constitutes an opportunity to mediate immunosuppression and inflammation within the tumor microenvironment but is difficult to achieve due to the high sequence homology across the class I PI3K isoforms. Here, we describe the design of a novel series of potent PI3Kγ inhibitors that attain high isoform selectivity through the divergent projection of substituents into both the "selectivity" and "alkyl-induced" pockets within the adenosine triphosphate (ATP) binding site of PI3Kγ. These efforts have culminated in the discovery of 5-[2-amino-3-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl]-2-[(1S)-1-cyclopropylethyl]-7-(trifluoromethyl)-2,3-dihydro-1H-isoindol-1-one (4, IC50 = 0.064 μM, THP-1 cells), which displays >600-fold selectivity for PI3Kγ over the other class I isoforms and is a promising step toward the identification of a clinical development candidate. The structure-activity relationships identified throughout this campaign demonstrate that greater γ-selectivity can be achieved by inhibitors that occupy an "alkyl-induced" pocket and possess bicyclic hinge-binding motifs capable of forming more than one hydrogen bond to the hinge region of PI3Kγ.

Selective targeting of the αC and DFG-out pocket in p38 MAPK

The p38 MAPK cascade is a key signaling pathway linked to a multitude of physiological functions and of central importance in inflammatory and autoimmune diseases. Although studied extensively, little is known about how conformation-specific inhibitors alter signaling outcomes. Here, we have explored the highly dynamic back pocket of p38 MAPK with allosteric urea fragments. However, screening against known off-targets showed that these fragments maintained the selectivity issues of their parent compound BIRB-796, while combination with the hinge-binding motif of VPC-00628 greatly enhanced inhibitor selectivity. Further efforts focused therefore on the exploration of the αC-out pocket of p38 MAPK, yielding compound 137 as a highly selective type-II inhibitor. Even though 137 is structurally related to a recent p38 type-II chemical probe, SR-318, the data presented here provide valuable insights into back-pocket interactions that are not addressed in SR-318 and it provides an alternative chemical tool with good cellular activity targeting also the p38 back pocket.

Discovery of Tyrosine Kinase 2 (TYK2) Inhibitor (PF-06826647) for the Treatment of Autoimmune Diseases.

Tyrosine kinase 2 (TYK2) is a member of the JAK kinase family that regulates signal transduction downstream of receptors for the IL-23/IL-12 pathways and type I interferon family, where it pairs with JAK2 or JAK1, respectively. On the basis of human genetic and emerging clinical data, a selective TYK2 inhibitor provides an opportunity to treat autoimmune diseases delivering a potentially differentiated clinical profile compared to currently approved JAK inhibitors. The discovery of an ATP-competitive pyrazolopyrazinyl series of TYK2 inhibitors was accomplished through computational and structurally enabled design starting from a known kinase hinge binding motif. With understanding of PK/PD relationships, a target profile balancing TYK2 potency and selectivity over off-target JAK2 was established. Lead optimization involved modulating potency, selectivity, and ADME properties which led to the identification of the clinical candidate PF-06826647 (22).

Structure-guided optimization of a novel class of ASK1 inhibitors with increased sp3 character and an exquisite selectivity profile

Apoptosis Signal-Regulating Kinase-1 (ASK1) is a known member of the Mitogen-Activated Protein Kinase Kinase Kinase (MAP3K) family and upon stimulation will activate the p38- and JNK-pathways leading to cardiac apoptosis, fibrosis, and hypertrophy. Using Structure-Based Drug Design (SBDD) in parallel with deconstruction of a published compound, a novel series of ASK1 inhibitors was optimized, which incorporated a saturated heterocycle proximal to the hinge-binding motif. This yielded a unique chemical series with excellent selectivity across the broader kinome, and desirable drug-like properties. The lead compound (10) is highly soluble and permeable, and exhibits a cellular EC50 = 24 nM and Kd < 1 nM. Of the 350 kinases tested, 10 has an IC50 ≤ 500 nM for only eight of them. This paper will describe the design hypotheses behind this series, key data points during the optimization phase, as well as a possible structural rationale for the kinome selectivity. Based on crystallographic data, the presence of an aliphatic cycle adjacent to the hinge-binder in the active site of the protein kinase showed up in <1% of the >5000 structures in the Protein Data Bank, potentially conferring the selectivity seen in this series.

Discovery of an Atropisomeric PI3Kβ Selective Inhibitor through Optimization of the Hinge Binding Motif.

A series of PI3Kβ selective inhibitors derived from a novel 4-(1H-benzo[d]imidazol-1-yl)quinoline chemotype has been rationally designed. Crucial to achieving the desired selectivity over the other class I PI3K isoforms, including the challenging δ-isoform, was the identification of a subset of substituted pyridine hinge binders. This work led to the discovery of (P)-14, a highly selective and orally bioavailable PI3Kβ inhibitor displaying an excellent pharmacokinetic profile in addition to great cellular potency in various PTEN-deficient tumor cell lines. Results from a dog toxicology study revealing structure-related, off-target ocular toxicity are also briefly discussed.

New Insights into 4-Anilinoquinazolines as Inhibitors of Cardiac Troponin I–Interacting Kinase (TNNi3K)

We report the synthesis of several related 4-anilinoquinazolines as inhibitors of cardiac troponin I–interacting kinase (TNNi3K). These close structural analogs of 3-((6,7-dimethoxyquinazolin-4-yl)amino)-4-(dimethylamino)-N-methylbenzenesulfonamide (GSK114) provide new understanding of structure–activity relationships between the 4-anilinoquinazoline scaffold and TNNi3K inhibition. Through a small focused library of inhibitors, we observed that the N-methylbenzenesulfonamide was driving the potency in addition to the more traditional quinazoline hinge-binding motif. We also identified a compound devoid of TNNi3K kinase activity due to the addition of a methyl group in the hinge binding region. This compound could serve as a negative control in the study of TNNi3K biology. Small molecule crystal structures of several quinazolines have been solved, supporting observations made about overall conformation and TNNi3K inhibition.

Identification of 2-Imidazopyridine and 2-Aminopyridone Purinones as Potent Pan-Janus Kinase (JAK) Inhibitors for the Inhaled Treatment of Respiratory Diseases.

Janus kinases (JAKs) have a key role in regulating the expression and function of relevant inflammatory cytokines involved in asthma and chronic obstructive pulmonary disease. Herein are described the design, synthesis, and pharmacological evaluation of a series of novel purinone JAK inhibitors with profiles suitable for inhaled administration. Replacement of the imidazopyridine hinge binding motif present in the initial compounds of this series with a pyridone ring resulted in the mitigation of cell cytotoxicity. Further systematic structure-activity relationship (SAR) efforts driven by structural biology studies led to the discovery of pyridone 34, a potent pan-JAK inhibitor with good selectivity, long lung retention time, low oral bioavailability, and proven efficacy in the lipopolysaccharide-induced rat model of airway inflammation by the inhaled route.

Design of selective PI3Kδ inhibitors using an iterative scaffold-hopping workflow.

PI3Kδ mediates key immune cell signaling pathways and is a target of interest for multiple indications in immunology and oncology. Here we report a structure-based scaffold-hopping strategy for the design of chemically diverse PI3Kδ inhibitors. Using this strategy, we identified several scaffolds that can be combined to generate new PI3Kδ inhibitors with high potency and isoform selectivity. In particular, an oxindole-based scaffold was found to impart exquisite selectivity when combined with several hinge binding motifs.

A Facile Access to Novel (5+5) Annellated Heterocycles: Synthesis of a Furopyrrole, an Imidazoimidazole and a Pyrroloimidazole

We describe the synthesis of an ethyl 3-aryl-6H-furo[2,3-b]pyrrole-5-carboxylate 2, a 5-aryl-1H-imidazo[1,2-a]imidazole 3, and an ethyl-1-aryl-1,4-dihydropyrrolo[2,3-d]imidazole-5-carboxylate 4 as new hinge-binding motifs for PI3K kinase inhibitors. A key reaction for the formation of 2 and 4 is the Hemmetsberger–Knittel cyclization of acryloazides. The core of 3 is accessible through reaction of an α-haloketone with 2-aminoimidazole.

Identification of Selective Dual ROCK1 and ROCK2 Inhibitors Using Structure-Based Drug Design.

A HTS campaign identified compound 1, an excellent hit-like molecule to initiate medicinal chemistry efforts to optimize a dual ROCK1 and ROCK2 inhibitor. Substitution (2-Cl, 2-NH2, 2-F, 3-F) of the pyridine hinge binding motif or replacement with pyrimidine afforded compounds with a clean CYP inhibition profile. Cocrystal structures of an early lead compound were obtained in PKA, ROCK1, and ROCK2. This provided critical structural information for medicinal chemistry to drive compound design. The structural data indicated the preferred configuration at the central benzylic carbon would be ( R), and application of this information to compound design resulted in compound 16. This compound was shown to be a potent and selective dual ROCK inhibitor in both enzyme and cell assays and efficacious in the retinal nerve fiber layer model after oral dosing. This tool compound has been made available through the AbbVie Compound Toolbox. Finally, the cocrystal structures also identified that aspartic acid residues 176 and 218 in ROCK2, which are glutamic acids in PKA, could be targeted as residues to drive both potency and kinome selectivity. Introduction of a piperidin-3-ylmethanamine group to the compound series resulted in compound 58, a potent and selective dual ROCK inhibitor with excellent predicted drug-like properties.

7-Azaindole: A Versatile Scaffold for Developing Kinase Inhibitors.

The majority of kinase inhibitors have been developed as ATP competitors to interact with a hinge region in ATP binding sites of kinases. 7-Azaindole has been found as an excellent hinge binding motif by making two hydrogen bonds with the kinase hinge region. Vemurafenib, a B-RAF kinase (serine-threonine kinase [STK]) inhibitor approved by the U.S. Food and Drug Administration (FDA) for the treatment of melanoma, was created from this simple 7-azaindole fragment by successful use of structure-based drug design techniques. The huge potential of 7-azaindole as a hinge-binding motif has encouraged many researchers to employ it as a kinase privileged fragment. This paper will review recent examples of 7-azaindole-based kinase inhibitors, and discusses their binding interactions with the kinase hinge regions.

Kinase Crystal Miner: A Powerful Approach to Repurposing 3D Hinge Binding Fragments and Its Application to Finding Novel Bruton Tyrosine Kinase Inhibitors

Protein kinases represent an important target class for drug discovery because of their role in signaling pathways involved in disease areas such as oncology and immunology. A key element of many ATP-competitive kinase inhibitors is their hinge-binding motif. Here, we describe Kinase Crystal Miner (KCM)-a new approach developed at Boehringer Ingelheim (BI) that harvests the existing crystallographic information on kinase-inhibitor co-crystal structures from internal and external databases. About 1000 unique three-dimensional kinase inhibitor hinge binding motifs have been extracted from structures covering more than 180 different protein kinases. These hinge binding motifs along with their attachment vectors have been combined in the KCM for the purpose of scaffold hopping, kinase screening deck design, and interactive structure-based design. Prospective scaffold hopping using the KCM identified two potent and selective Bruton tyrosine kinase (BTK) inhibitors with hinge binding fragments novel to BTK.