A Chemoproteomic Approach to Query the Degradable Kinome Using a Multi-kinase Degrader.

Heterobifunctional molecules that recruit E3 ubiquitin ligases, such as cereblon, for targeted protein degradation represent an emerging pharmacological strategy. A major unanswered question is how generally applicable this strategy is to all protein targets. In this study, we designed a multi-kinase degrader by conjugating a highly promiscuous kinase inhibitor with a cereblon-binding ligand, and used quantitative proteomics to discover 28 kinases, including BTK, PTK2, PTK2B, FLT3, AURKA, AURKB, TEC, ULK1, ITK, and nine members of the CDK family, as degradable. This set of kinases is only a fraction of the intracellular targets bound by the degrader, demonstrating that successful degradation requires more than target engagement. The results guided us to develop selective degraders for FLT3 and BTK, with potentials to improve disease treatment. Together, this study demonstrates an efficient approach to triage a gene family of interest to identify readily degradable targets for further studies and pre-clinical developments.

Discovery of Potent Antiallodynic Agents for Neuropathic Pain Targeting P2X3 Receptors.

Antagonism of the P2X3 receptor is one of the potential therapeutic strategies for the management of neuropathic pain because P2X3 receptors are predominantly localized on small to medium diameter C- and Aδ-fiber primary afferent neurons, which are related to the pain-sensing system. In this study, 5-hydroxy pyridine derivatives were designed, synthesized, and evaluated for their in vitro biological activities by two-electrode voltage clamp assay at hP2X3 receptors. Among the novel hP2X3 receptor antagonists, intrathecal treatment of compound 29 showed parallel efficacy with pregabalin (calcium channel modulator) and higher efficacy than AF353 (P2X3 receptor antagonist) in the evaluation of its antiallodynic effects in spinal nerve ligation rats. However, because compound 29 was inactive by intraperitoneal administration in neuropathic pain animal models due to low cell permeability, the corresponding methyl ester analogue, 28, which could be converted to compound 29 in vivo, was investigated as a prodrug concept. Intravenous injection of compound 28 resulted in potent antiallodynic effects, with ED50 values of 2.62 and 2.93 mg/kg in spinal nerve ligation and chemotherapy-induced peripheral neuropathy rats, respectively, indicating that new drug development targeting the P2X3 receptor could be promising for neuropathic pain, a disease with high unmet medical needs.

Insights of a Lead Optimization Study and Biological Evaluation of Novel 4-Hydroxytamoxifen Analogs as Estrogen-Related Receptor γ (ERRγ) Inverse Agonists.

We evaluated the in vitro pharmacology as well as the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of chemical entities that not only were shown to be highly selective agonists for ERRγ but also exhibited enhanced pharmacokinetic profile compared with 3 (GSK5182). 6g and 10b had comparable potency to 3 and were far more selective for ERRγ over the ERRα, -ß, and ERα. The in vivo pharmacokinetic profiles of 6g and 10b were further evaluated, as they possessed superior in vitro ADMET profiles compared to the other compounds. Additionally, we observed a significant increase of fully glycosylated NIS protein, key protein for radioiodine therapy in anaplastic thyroid cancer (ATC), in 6g- or 10b-treated CAL62 cells, which indicated that these compounds could be promising enhancers for restoring NIS protein function in ATC cells. Thus, 6g and 10b possess advantageous druglike properties and can be used to potentially treat various ERRγ-related disorders.

Suppression of the metastatic spread of breast cancer by DN10764 (AZD7762)-mediated inhibition of AXL signaling.

Breast cancer is the most common malignant disease occurring in women and represents a substantial proportion of the global cancer burden. In these patients, metastasis but not the primary tumor is the main cause of breast cancer-related deaths. Here, we report the novel finding that DN10764 (AZD7762, a selective inhibitor of checkpoint kinases 1 and 2) can suppress breast cancer metastasis. In breast cancer cells, DN10764 inhibited cell proliferation and GAS6-mediated AXL signaling, consequently resulting in suppressed migration and invasion. In addition, DN10764 induced caspase 3/7-mediated apoptosis in breast cancer cells and inhibited tube formation of human umbilical vein endothelial cells. Finally, DN10764 significantly suppressed the tumor growth and metastasis of breast cancer cells in in vivo metastasis models. Taken together, these data suggest that therapeutic strategies targeting AXL in combination with systemic therapies could improve responses to anti-cancer therapies and reduce breast cancer recurrence and metastases.

Synthesis and biological evaluation of novel 4-hydroxytamoxifen analogs as estrogen-related receptor gamma inverse agonists.

Estrogen-related receptor gamma (ERRγ) has recently been recognized as an attractive target for treating inflammation, cancer, and metabolic disorders. Herein, we discovered and demonstrated the in vitro pharmacology as well as the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of chemical entities that could act as highly selective inverse agonists for ERRγ. The results were comparable to those for GSK5182 (4), a leading ERRγ inverse agonist ligand. Briefly, the half-maximal inhibitory concentration (IC50) range of the synthesized compounds for ERRγ was 0.1-10 µM. Impressively, compound 24e exhibited potency comparable to 4 but was more selective for ERRγ over three other subtypes: ERRα, ERRß, and estrogen receptor α. Furthermore, compound 24e exhibited a superior in vitro ADMET profile compared to the other compounds. Thus, the newly synthesized class of ERRγ inverse agonists could be lead candidates for developing clinical therapies for ERRγ-related disorders.

Nanoparticle-encapsulated P2X7 receptor antagonist in a pH-sensitive polymer as a potential local drug delivery system to acidic inflammatory environments.

We have developed nanoparticles of anti-inflammatory P2X7 receptor antagonist encapsulated in a pH-sensitive polymer, poly(tetrahydropyran-2-yl methacrylate) (poly(THPMA)), as a potential local drug delivery system to target to acidic inflammatory environments, in which P2X7 receptors are implicated in the pathology of inflammation via the activation of immune cells. The nanoparticles were prepared using single emulsion methods, also their size and shape were confirmed by microscopy and spectroscopy, etc. The profiles of the pH-dependent degradation, release of antagonist and biological activities were investigated. The nanoparticles that encapsulated the 3,5-dichloropyridine derivative (2) with poly(THPMA), were observed to be more slowly cleaved than the blank nanoparticles. Moreover, the free P2X7 receptor antagonists potently inhibited the receptor activation, whereas the nanoparticles of the 3,5-dichloropyridine derivative (2) encapsulated poly(THPMA) exhibited much lower P2X7 antagonistic activity through sustained encapsulation. Thus, the nanoparticles of the 3,5-dichloropyridine derivative (2) encapsulated poly(THPMA) may be utilized to develop a pH-sensitive local drug delivery system for controlled release of anti-inflammatory therapeutics in acidic physiological environments.

Benserazide, the first allosteric inhibitor of Coxsackievirus B3 3C protease.

Coxsackievirus B3 is the main cause of human viral myocarditis and cardiomyopathy. Virally encoded Coxsackievirus 3C protease (3C(pro)) plays an essential role in viral proliferation. Here, benserazide was discovered as a novel inhibitor from a drug library screen targeting Coxsackievirus 3C(pro) using a FRET-based enzyme assay. Benserazide, whose chemical structure has no electrophilic functional groups, was characterized as a non-competitive inhibitor by enzyme kinetic studies. A molecular docking study with benserazide and its analogs indicated that a novel putative allosteric binding site was involved. Specifically, a 2,3,4-trihydroxybenzyl moiety was determined to be a key pharmacophore for the enzyme's inhibitory activity. We suggest that the putative allosteric binding site may be a novel target for future therapeutic strategies.

Discovery of novel 2,5-dioxoimidazolidine-based P2X(7) receptor antagonists as constrained analogues of KN62.

Novel 2,5-dioxoimidazolidine-based conformationally constrained analogues of KN62 (1) were developed as P2X7 receptor (P2X7R) antagonists using a rigidification strategy of the tyrosine backbone of 1. SAR analysis of the 2,5-dioxoimidazolidine scaffold indicated that piperidine substitution at the N3 position and no substitution at N1 position were preferable. Further optimization of the substituents at the piperidine nitrogen and the spacer around the skeleton resulted in several superior antagonists to 1, including 1-adamantanecarbonyl analogue 21i (IC50 = 23 nM in ethidium uptake assay; IC50 = 14 nM in IL-1ß ELISA assay) and (3-CF3-4-Cl)benzoyl analogue (-)-21w (54 nM in ethidium uptake assay; 9 nM in IL-1ß ELISA assay), which was more potent than the corresponding (+) isomer. Compound 21w displayed potent inhibitory activity in an ex vivo model of LTP-induced pain signaling in the spinal cord and significant anti-inflammatory activity in in vivo models of carrageenan-induced paw edema and type II collagen-induced joint arthritis.

Design and synthesis of potent and selective P2X3 receptor antagonists derived from PPADS as potential pain modulators.

Pyridoxalphosphate-6-azophenyl-2',4'-disulfonate (7a, PPADS), a nonselective P2X receptor antagonist, was extensively modified to develop more stable, potent, and selective P2X3 receptor antagonists as potential antinociceptive agents. Based on the results of our previous report, all strong anionic groups in PPADS including phosphate and sulfonate groups were changed to carboxylic acids or deleted. The unstable azo (-NN-) linkage of 7a was transformed to more stable carbon-carbon, ether or amide linkages through the synthesis of the 5-hydroxyl-pyridine moieties with substituents at 2 position via a Diels-Alder reaction. This resulted in the retention of antagonistic activity (IC50 = 400 ∼ 700 nM) at the hP2X3 receptor in the two-electrode voltage clamp (TEVC) assay system on the Xenopus oocytes. Introduction of bulky aromatic groups at the carbon linker, as in compounds 13 h-n, dramatically improved the selectivity profiles of hP2X3 when compared with mP2X1 and hP2X7 receptors. Among the substituents tested at the 2-position, the m-phenoxybenzyl group showed optimum selectivity and potency at the hP2X3 receptor. In searching for effective substituents at the 4- and 3-positions, we found that compound 36j, with 4-carboxaldehyde, 3-propenoic acid and 2-(m-phenoxy)benzyl groups, was the most potent and selective hP2X3 receptor antagonist with an IC50 of 60 nM at hP2X3 and marginal antagonistic activities of 10 µM at mP2X1 and hP2X7. Furthermore, using an ex-vivo assay system, we found that compound 36j potently inhibited pain signaling in the rat dorsal horn with 20 µM 36j displaying 65% inhibition while 20 µM pregabalin, a clinically available drug, showed only 31% inhibition.

Synthesis and structure-activity relationships of carboxylic acid derivatives of pyridoxal as P2X receptor antagonists.

Carboxylic acid derivatives of pyridoxal were developed as potent P2X(1) and P2X(3) receptor antagonists with modifications of a lead compound, pyridoxal-5'-phosphate-6-azophenyl-2',5'-disulfonate (5b, iso-PPADS). The designing strategies included the modifications of aldehyde, phosphate or sulfonate groups of 5b, which may be interacted with lysine residues of the receptor binding pocket, to weak anionic carboxylic acid groups. The corresponding carboxylic acid analogs of pyridoxal-5'-phosphate (1), 13 and 14, showed parallel antagonistic potencies. Also, most of 6-azophenyl derivatives (24-28) of compound 13 or 14 showed potent antagonistic activities similar to that of 5b at human P2X(3) receptors with 100 nM range of IC(50) values in two-electrode voltage clamp (TEVC) assay system on the Xenopus oocyte. The results indicated that aldehyde and phosphoric or sulfonic acids in 5b could be changed to a carboxylic acid without affecting antagonistic potency at mouse P2X(1) and human P2X(3) receptors.

Structure-activity relationships and optimization of 3,5-dichloropyridine derivatives as novel P2X(7) receptor antagonists.

Screening of a library of chemical compounds showed that the dichloropyridine-based analogue 9 was a novel P2X(7) receptor antagonist. To optimize its activity, we assessed the structure-activity relationships (SAR) of 9, focusing on the hydrazide linker, the dichloropyridine skeleton, and the hydrophobic acyl (R(2)) group. We found that the hydrazide linker and the 3,5-disubstituted chlorides in the pyridine skeleton were critical for P2X(7) antagonistic activity and that the presence of hydrophobic polycycloalkyl groups at the R(2) position optimized antagonistic activity. In the EtBr uptake assay in hP2X(7)-expressing HEK293 cells, the optimized antagonists, 51 and 52, had IC(50) values of 4.9 and 13 nM, respectively. The antagonistic effects of 51 and 52 were paralleled by their ability to inhibit the release of the pro-inflammatory cytokine, IL-1ß, by LPS/IFN-γ/BzATP stimulation of THP-1 cells (IC(50) = 1.3 and 9.2 nM, respectively). In addition, 52 strongly inhibited iNOS/COX-2 expression and NO production in THP-1 cells, further indicating that this compound blocks inflammatory signaling and suggesting that the dichloropyridine analogues may be useful in developing P2X(7) receptor targeted anti-inflammatory agents.

Synthesis of potent chemical inhibitors of dynamin GTPase.

Dynamin is a key regulatory protein in clathrin mediated endocytosis. Compared to genetic or immunological tools, small chemical dynamin inhibitors such as dynasore have the potential to study the dynamic nature of endocytic events in cells. Dynasore inhibits dynamin GTPase activity and transferrin uptake at IC(50) approximately 15 microM but use in some biological applications requires more potent inhibitor than dynasore. Here, we chemically modified the side chains of dynasore and found that two derivatives, DD-6 and DD-11 more potently inhibited transferrin uptake (IC(50): 4.00 microM for DD-6, 2.63 microM for DD-11) and dynamin GTPase activity (IC(50): 5.1 microM for DD-6, 3.6 microM for DD-11) than dynasore. The effect was reversible and they were washed more rapidly out than dynasore. TIRF microscopy showed that they stabilize the clathrin coats on the membrane. Our results indicated that new dynasore derivatives are more potent inhibitor of dynamin, displaying promise as leads for the development of more effective analogues for broader biological applications.

Design and synthesis of 1,4-dihydropyridine derivatives as BACE-1 inhibitors.

BACE-1 has been shown to be an attractive therapeutic target in Alzheimer's disease (AD). Using a 1,4-dihydropyridine (DHP) scaffold, we synthesized new inhibitors of BACE-1 by modifying the known BACE inhibitor 2 containing a hydroxyethylamine (HEA) motif. Using structure-based drug design based on computer-aided molecular docking, the isophthalamide ring of 2 was replaced with a 1,4-dihydropyridine ring as a brain-targeting strategy. Several of the new dihydropyridine derivatives were synthesized and their BACE-1-inhibitory activities were evaluated using a cell-based, reporter gene assay system that measures the cleavage of alkaline phosphatase (AP)-APP fusion protein by BACE-1. Most of the 1,4-DHP analogs showed BACE-1-inhibitory activities with IC50 values in the range 8-30 microM, suggesting that the 1,4-DHP skeleton may be utilized to develop brain-targeting BACE-1 inhibitors.