Discovery and Evaluation of Enantiopure 9H-pyrimido[4,5-b]indoles as Nanomolar GSK-3ß Inhibitors with Improved Metabolic Stability.

Glycogen synthase kinase-3ß (GSK-3ß) is a potential target in the field of Alzheimer's disease drug discovery. We recently reported a new class of 9H-pyrimido[4,5-b]indole-based GSK-3ß inhibitors, of which 3-(3-((7-chloro-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)propanenitrile (1) demonstrated promising inhibitory potency. However, this compound underwent rapid degradation by human liver microsomes. Starting from 1, we prepared a series of amide-based derivatives and studied their structure-activity relationships against GSK-3ß supported by 1 µs molecular dynamics simulations. The biological potency of this series was substantially enhanced by identifying the eutomer configuration at the stereocenter. Moreover, the introduction of an amide bond proved to be an effective strategy to eliminate the metabolic hotspot. The most potent compounds, (R)-3-(3-((7-chloro-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)-3-oxopropanenitrile ((R)-2) and (R)-1-(3-((7-bromo-9Hpyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)propan-1-one ((R)-28), exhibited IC50 values of 480 nM and 360 nM, respectively, and displayed improved metabolic stability. Their favorable biological profile is complemented by minimal cytotoxicity and neuroprotective properties.

Design, Synthesis and Biological Evaluation of 7-Chloro-9H-pyrimido[4,5-b]indole-based Glycogen Synthase Kinase-3ß Inhibitors.

Glycogen synthase kinase-3ß (GSK-3ß) represents a relevant drug target for the treatment of neurodegenerative pathologies including Alzheimer's disease. We herein report on the optimization of a novel class of GSK-3ß inhibitors based on the tofacitinib-derived screen hit 3-((3R,4R)-3-((7-chloro-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)-4-methylpiperidin-1-yl)-3-oxopropanenitrile (1). We synthesized a series of 19 novel 7-chloro-9H-pyrimido[4,5-b]indole-based derivatives and studied their structure-activity relationships with focus on the cyanoacetyl piperidine moiety. We unveiled the crucial role of the nitrile group and its importance for the activity of this compound series. A successful rigidization approach afforded 3-(3aRS,7aSR)-(1-(7-chloro-9H-pyrimido[4,5-b]indol-4-yl)octahydro-6H-pyrrolo[2,3-c]pyridin-6-yl)-propanenitrile (24), which displayed an IC50 value of 130 nM on GSK-3ß and was further characterized by its metabolic stability. Finally, we disclosed the putative binding modes of the most potent inhibitors within the ATP binding site of GSK-3ß by 1 µs molecular dynamics simulations.

From 2-Alkylsulfanylimidazoles to 2-Alkylimidazoles: An Approach towards Metabolically More Stable p38α MAP Kinase Inhibitors.

In vitro and in vivo metabolism studies revealed that 2-alkylsulfanylimidazole ML3403 (4-(5-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)-N-(1-phenylethyl)pyridin-2-amine) undergoes rapid oxidation to the sulfoxide. Replacing the sulfur atom present in the two potent p38α mitogen-activated protein (MAP) kinase inhibitors ML3403 and LN950 (2-((5-(4-fluorophenyl)-4-(2-((3-methylbutan-2-yl)amino)pyridin-4-yl)-1H-imidazol-2-yl)thio)ethan-1-ol) by a methylene group resulted in 2-alkylimidazole derivatives 1 and 2, respectively, having a remarkably improved metabolic stability. The 2-alkylimidazole analogs 1 and 2 showed 20% and 10% biotransformation after 4 h of incubation with human liver microsomes, respectively. They display a 4-fold increased binding affinity towards the target kinase as well as similar in vitro potency and ex vivo efficacy relative to their 2-alkylsulfanylimidazole counterparts ML3403 and LN950. For example, 2-alkylimidazole 2, the analog of LN950, inhibits both the p38α MAP kinase as well as the LPS-stimulated tumor necrosis factor-α release from human whole blood in the low double-digit nanomolar range.

Design and Optimization of Novel Benzimidazole- and Imidazo[4,5-b]pyridine-Based ATM Kinase Inhibitors with Subnanomolar Activities.

The ATM kinase is a promising target in cancer treatment as an important regulator of the cellular response to DNA double-strand breaks. In this work, we present a new class of specific benzimidazole-based ATM inhibitors with picomolar potency against the isolated enzyme and favorable selectivity within relative PIKK and PI3K kinases. We could identify two promising inhibitor subgroups with significantly different physicochemical properties, which we developed simultaneously. These efforts lead to numerous highly active inhibitors with picomolar enzymatic activities. Furthermore, initial low cellular activities on A549 cells could be increased significantly in numerous examples resulting in cellular IC50 values in the subnanomolar range. Further characterization of the highly potent inhibitors 90 und 93 revealed promising pharmacokinetic properties and strong activities in organoids in combination with etoposide. Additionally, 93 showed no off-target activities within a kinome-representative mini kinase panel, with favorable selectivities within the PIKK- and PI3K-families.

Decisive role of water and protein dynamics in residence time of p38α MAP kinase inhibitors.

Target residence time plays a crucial role in the pharmacological activity of small molecule inhibitors. Little is known, however, about the underlying causes of inhibitor residence time at the molecular level, which complicates drug optimization processes. Here, we employ all-atom molecular dynamics simulations (~400 µs in total) to gain insight into the binding modes of two structurally similar p38α MAPK inhibitors (type I and type I½) with short and long residence times that otherwise show nearly identical inhibitory activities in the low nanomolar IC50 range. Our results highlight the importance of protein conformational stability and solvent exposure, buried surface area of the ligand and binding site resolvation energy for residence time. These findings are further confirmed by simulations with a structurally diverse short residence time inhibitor SB203580. In summary, our data provide guidance in compound design when aiming for inhibitors with improved target residence time.

Development of novel urea-based ATM kinase inhibitors with subnanomolar cellular potency and high kinome selectivity.

The ATM kinase is a key molecule regulating DNA damage response and can be targeted resulting in efficient radio- or chemosensitization. Due to the enormous size of this protein and the associated difficulties in obtaining high-quality crystal structures, we sought to develop an accurate in silico model to identify new targeting possibilities. We identified a urea group as the most beneficial chemical anchor point, which could undergo multiple interactions in the aspartate-rich hydrophobic region I of the atypical ATM kinase domain. Based on in silico data, we designed and synthesized a comprehensive set of novel urea-based inhibitors and characterized them in diverse biochemical assays. Using this strategy, we identified inhibitors with subnanomolar potency, which were further evaluated in cellular models, selectivity and early DMPK properties. Finally, the two lead compounds 34 and 39 exhibited subnanomolar cellular activity along with an excellent selectivity profile and favorable metabolic stability.

Gefitinib-Tamoxifen Hybrid Ligands as Potent Agents against Triple-Negative Breast Cancer.

Anticancer drug conjugates may benefit from simultaneous action at two targets potentially overcoming the drawbacks of current cancer treatment, such as insufficient efficacy, high toxicity, and development of resistance. Compared to a combination of two single-target drugs, they may offer an advantage of pharmacokinetic simplicity and fewer drug-drug interactions. Here, we report a series of compounds connecting tamoxifen or endoxifen with the EGFR-inhibitor gefitinib via a covalent linkage. These hybrid ligands retain both ER antagonist activity and EGFR inhibition. The most potent analogues exhibited single-digit nanomolar activities at both targets. The amide-linked endoxifen-gefitinib drug conjugates 17b and 17c demonstrated the most favorable anti-cancer profile in cellular viability assays on MCF7, MDA-MB-231, MDA-MB-468, and BT-549 breast cancer cells. Most importantly, in TNBC cells 17b and 17c displayed nanomolar IC50-values (380 nM - 970 nM) and were superior in their anti-cancer activity compared to their control compounds and combinations thereof.

Pharmacokinetic Optimization of Small Molecule Janus Kinase 3 Inhibitors to Target Immune Cells.

Modulation of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling is a promising method of treating autoimmune diseases, and the profound potency of clinical compounds makes this mode of action particularly attractive. Other questions that remain unanswered also include: What is the ideal selectivity between JAK1 and JAK3? Which cells are most relevant to JAK blockade? And what is the ideal tissue distribution pattern for addressing specific autoimmune conditions? We hypothesized that JAK3 selectivity is most relevant to low-dose clinical effects and interleukin-10 (IL-10) stimulation in particular, that immune cells are the most important compartment, and that distribution to inflamed tissue is the most important pharmacokinetic characteristic for in vivo disease modification. To test these hypotheses, we prepared modified derivatives of JAK3 specific inhibitors that target C909 near the ATP binding site based on FM-381, first reported in 2016; a compound class that was hitherto limited in uptake and exposure in vivo. These limits appear to be due to metabolic instability of side groups binding in the selectivity pocket. We identified derivatives with improved stability and tissue exposure. Conjugation to macrolide scaffolds with medium chain linkers was sufficient to stabilize the compounds and improve transport to organs while maintaining JAK3 affinity. These conjugates are inflammation targeted JAK3 inhibitors with long tissue half-lives and high exposure to activated immune cells.

Discovery and Development of First-in-Class ACKR3/CXCR7 Superagonists for Platelet Degranulation Modulation.

The atypical chemokine receptor 3 (ACKR3), formerly known as CXC-chemokine receptor 7 (CXCR7), has been postulated to regulate platelet function and thrombus formation. Herein, we report the discovery and development of first-in-class ACKR3 agonists, which demonstrated superagonistic properties with Emax values of up to 160% compared to the endogenous reference ligand CXCL12 in a ß-arrestin recruitment assay. Initial in silico screening using an ACKR3 homology model identified two hits, C10 (EC50 19.1 µM) and C11 (EC50 = 11.4 µM). Based on these hits, extensive structure-activity relationship studies were conducted by synthesis and testing of derivatives. It resulted in the identification of the novel thiadiazolopyrimidinone-based compounds 26 (LN5972, EC50 = 3.4 µM) and 27 (LN6023, EC50 = 3.5 µM). These compounds are selective for ACKR3 versus CXCR4 and show metabolic stability. In a platelet degranulation assay, these agonists effectively reduced P-selectin expression by up to 97%, suggesting potential candidates for the treatment of platelet-mediated thrombosis.

Development of the First Covalent Monopolar Spindle Kinase 1 (MPS1/TTK) Inhibitor.

Monopolar spindle kinase 1 (MPS1/TTK) is a key element of the mitotic checkpoint and clinically evaluated as a target in the treatment of aggressive tumors such as triple-negative breast cancer. While long drug-target residence times have been suggested to be beneficial in the context of therapeutic MPS1 inhibition, no irreversible inhibitors have been reported. Here we present the design and characterization of the first irreversible covalent MPS1 inhibitor, RMS-07, targeting a poorly conserved cysteine in the kinase's hinge region. RMS-07 shows potent MPS1 inhibitory activity and selectivity against all protein kinases with an equivalent cysteine but also in a broader kinase panel. We demonstrate potent cellular target engagement and pronounced activity against various cancer cell lines. The covalent binding mode was validated by mass spectrometry and an X-ray crystal structure. This proof of MPS1 covalent ligandability may open new avenues for the design of MPS1-specific chemical probes or drugs.

Discovery of a Potent and Highly Isoform-Selective Inhibitor of the Neglected Ribosomal Protein S6 Kinase Beta 2 (S6K2).

The ribosomal protein S6 kinase beta 2 (S6K2) is thought to play an important role in malignant cell proliferation, but is understudied compared to its closely related homolog S6 kinase beta 1 (S6K1). To better understand the biological function of S6K2, chemical probes are needed, but the high similarity between S6K2 and S6K1 makes it challenging to selectively address S6K2 with small molecules. We were able to design the first potent and highly isoform-specific S6K2 inhibitor from a known S6K1-selective inhibitor, which was merged with a covalent inhibitor engaging a cysteine located in the hinge region in the fibroblast growth factor receptor kinase (FGFR) 4 via a nucleophilic aromatic substitution (SNAr) reaction. The title compound shows a high selectivity over kinases with an equivalently positioned cysteine, as well as in a larger kinase panel. A good stability towards glutathione and Nα-acetyl lysine indicates a non-promiscuous reactivity pattern. Thus, the title compound represents an important step towards a high-quality chemical probe to study S6K2-specific signaling.

Development of a Selective Dual Discoidin Domain Receptor (DDR)/p38 Kinase Chemical Probe.

Discoidin domain receptors 1 and 2 (DDR1/2) play a central role in fibrotic disorders, such as renal and pulmonary fibrosis, atherosclerosis, and various forms of cancer. Potent and selective inhibitors, so-called chemical probe compounds, have been developed to study DDR1/2 kinase signaling. However, these inhibitors showed undesired activity on other kinases such as the tyrosine protein kinase receptor TIE or tropomyosin receptor kinases, which are related to angiogenesis and neuronal toxicity. In this study, we optimized our recently published p38 mitogen-activated protein kinase inhibitor 7 toward a potent and cell-active dual DDR/p38 chemical probe and developed a structurally related negative control. The structure-guided design approach used provided insights into the P-loop folding process of p38 and how targeting of non-conserved amino acids modulates inhibitor selectivity. The developed and comprehensively characterized DDR/p38 probe, 30 (SR-302), is a valuable tool for studying the role of DDR kinase in normal physiology and in disease development.

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.

Bioisosteric Replacement of Arylamide-Linked Spine Residues with N-Acylhydrazones and Selenophenes as a Design Strategy to Novel Dibenzosuberone Derivatives as Type I 1/2 p38α MAP Kinase Inhibitors.

The recent disclosure of type I 1/2 inhibitors for p38α MAPK demonstrated how the stabilization of the R-spine can be used as a strategy to greatly increase the target residence time (TRT) of inhibitors. Herein, for the first time, we describe N-acylhydrazone and selenophene residues as spine motifs, yielding metabolically stable inhibitors with high potency on enzymatic, NanoBRET, and whole blood assays, improved metabolic stability, and prolonged TRT.

Pyridinylimidazoles as GSK3ß Inhibitors: The Impact of Tautomerism on Compound Activity via Water Networks.

Glycogen synthase kinase-3ß (GSK3ß) is involved in many pathological conditions and represents an attractive drug target. We previously reported dual GSK3ß/p38α mitogen-activated protein kinase inhibitors and identified N-(4-(4-(4-fluorophenyl)-2-methyl-1H-imidazol-5-yl)pyridin-2-yl)cyclopropanecarboxamide (1) as a potent dual inhibitor of both target kinases. In this study, we aimed to design selective GSK3ß inhibitors based on our pyridinylimidazole scaffold. Our efforts resulted in several novel and potent GSK3ß inhibitors with IC50 values in the low nanomolar range. 5-(2-(Cyclopropanecarboxamido)pyridin-4-yl)-4-cyclopropyl-1H-imidazole-2-carboxamide (6g) displayed very good kinase selectivity as well as metabolical stability and inhibited GSK3ß activity in neuronal SH-SY5Y cells. Interestingly, we observed the importance of the 2-methylimidazole's tautomeric state for the compound activity. Finally, we reveal how this crucial tautomerism effect is surmounted by imidazole-2-carboxamides, which are able to stabilize the binding via enhanced water network interactions, regardless of their tautomeric state.

Pyridinylimidazoles as dual glycogen synthase kinase 3ß/p38α mitogen-activated protein kinase inhibitors.

Compounds simultaneously inhibiting two targets that are involved in the progression of the same complex disease may exhibit additive or even synergistic therapeutic effects. Here we unveil 2,4,5-trisubstituted imidazoles as dual inhibitors of p38α mitogen-activated protein kinase and glycogen synthase kinase 3ß (GSK3ß). Both enzymes are potential therapeutic targets for neurodegenerative disorders, like Alzheimer's disease. A set of 39 compounds was synthesized and evaluated in kinase activity assays for their ability to inhibit both target kinases. Among the synthesized compounds, potent dual-target-directed inhibitors showing IC50 values down to the low double-digit nanomolar range, were identified. One of the best balanced dual inhibitors presented in here is N-(4-(2-ethyl-4-(4-fluorophenyl)-1H-imidazol-5-yl)pyridin-2-yl)cyclopropanecarboxamide (20c) (p38α, IC50 = 16 nM; GSK3ß, IC50 = 35 nM) featuring an excellent metabolic stability and an appreciable isoform selectivity over the closely related GSK3α. Our findings were rationalized by computational docking studies based on previously published X-ray structures.

Fast Iterative Synthetic Approach toward Identification of Novel Highly Selective p38 MAP Kinase Inhibitors.

p38 mitogen-activated protein kinases are key mediators of environmental stress response and are promising targets for treatment of inflammatory diseases and cancer. Numerous efforts have led to the discovery of several potent inhibitors; however, so far no highly selective type-II inhibitors have been reported. We previously identified VPC-00628 as a potent and selective type-II inhibitor of p38α/ß with few off-targets. Here we analyzed the chemical building blocks of VPC-00628 that played a key role in achieving potency and selectivity through targeting an inactive state of the kinases induced by a unique folded P-loop conformation. Using a rapid, systematic combinatorial synthetic approach, we identified compound 93 (SR-318) with excellent potency and selectivity for p38α/ß, which potently inhibited the TNF-α release in whole blood. SR-318 therefore presents a potent and selective type-II inhibitor of p38α/ß that can be used as a chemical probe for targeting this particular inactive state of these two p38 isoforms.

Discovery of potent p38α MAPK inhibitors through a funnel like workflow combining in silico screening and in vitro validation.

This work describes the rational discovery of novel chemotypes of p38α MAPK inhibitors using a funnel approach consisting of several computer-aided drug discovery methods and biological experiments. Among the identified hits, four compounds belonging to different chemical families showed IC50 values lower than 10 µM. In particular, the 1,4-benzodioxane derivative 5 turned out to be a potent and efficient p38α MAPK inhibitor having IC50 = 0.07 µM, and LEexp and LipE values of 0.38 and 4.8, respectively; noteworthy, the compound had also a promising kinase selectivity profile and the capability to suppress p38α MAPK effects in human immune cells. Overall, the collected findings highlight that the applied strategy has been successful in generating chemical novelty in the inhibitor kinase field, providing suitable chemical candidates for further inhibitor optimization.

Structural Optimization of a Pyridinylimidazole Scaffold: Shifting the Selectivity from p38α Mitogen-Activated Protein Kinase to c-Jun N-Terminal Kinase 3.

Starting from known p38α mitogen-activated protein kinase (MAPK) inhibitors, a series of inhibitors of the c-Jun N-terminal kinase (JNK) 3 was obtained. Altering the substitution pattern of the pyridinylimidazole scaffold proved to be effective in shifting the inhibitory activity from the original target p38α MAPK to the closely related JNK3. In particular, a significant improvement for JNK3 selectivity could be achieved by addressing the hydrophobic region I with a small methyl group. Furthermore, additional structural modifications permitted to explore structure-activity relationships. The most potent inhibitor 4-(4-methyl-2-(methylthio)-1H-imidazol-5-yl)-N-(4-morpholinophenyl)pyridin-2-amine showed an IC50 value for the JNK3 in the low triple digit nanomolar range and its binding mode was confirmed by X-ray crystallography.