Alkenyl oxindole is a novel PROTAC moiety that recruits the CRL4DCAF11 E3 ubiquitin ligase complex for targeted protein degradation.

Alkenyl oxindoles have been characterized as autophagosome-tethering compounds (ATTECs), which can target mutant huntingtin protein (mHTT) for lysosomal degradation. In order to expand the application of alkenyl oxindoles for targeted protein degradation, we designed and synthesized a series of heterobifunctional compounds by conjugating different alkenyl oxindoles with bromodomain-containing protein 4 (BRD4) inhibitor JQ1. Through structure-activity relationship study, we successfully developed JQ1-alkenyl oxindole conjugates that potently degrade BRD4. Unexpectedly, we found that these molecules degrade BRD4 through the ubiquitin-proteasome system, rather than the autophagy-lysosomal pathway. Using pooled CRISPR interference (CRISPRi) screening, we revealed that JQ1-alkenyl oxindole conjugates recruit the E3 ubiquitin ligase complex CRL4DCAF11 for substrate degradation. Furthermore, we validated the most potent heterobifunctional molecule HL435 as a promising drug-like lead compound to exert antitumor activity both in vitro and in a mouse xenograft tumor model. Our research provides new employable proteolysis targeting chimera (PROTAC) moieties for targeted protein degradation, providing new possibilities for drug discovery.

Physicochemical properties, drug likeness, ADMET, DFT studies, and in vitro antioxidant activity of oxindole derivatives.

Poor pharmacokinetic and safety profiles create significant hurdles in the drug development process. This work focuses on a detailed understanding of drug discovery interplay among physicochemical, pharmacokinetic, toxicity endpoints, and antioxidant properties of oxindole derivatives. DFT compiutations were also performed at B3LYP/6-311G** level to evaluate the physicochemical properties, global reactivity features, and intramolecular interactions. The BOILED-Egg pharmacokinetic model envisaged gastrointestinal absorption, blood-brain barrier penetration, and no interaction with p-glycoprotein for compounds C1 and C2. The physicochemical evaluation revealed that C1 possesses superior drug-like properties fit for oral absorption. Both derivatives were predicted to have high plasma protein binding, efficient distribution, and inhibiting CYP 450 major isoforms but serve as substrates only for a few of them. Both molecules have mild to moderate clearance rates. Out of ten toxicity parameters, only hepatotoxicity was predicted. DFT results implied that the meta position of the -OH group made the possibility of charge transfer greater than -para positioned -OH, due to the ΔNmax (eV) values of molecules C1 and C2 being calculated at 2.596 and 2.477, respectively. Both C1 and C2 exhibited a concentration dependant DPPH and ABTS radical scavenging activity. The chemical structure-physicochemical-pharmacokinetic relationship identified the meta position as the favorite for the electron-withdrawing hydroxyl group. This provides useful insight to medicinal chemists to design 6-chlorooxindole derivatives with an acceptable drug-like and pharmacokinetic property.

Effect of ferroptosis inhibitors oxindole-curcumin hybrid compound and N,N-dimethylaniline derivatives on rotenone-induced oxidative stress.

Oxidative stress is common to multiple cell death pathways, including apoptosis. We recently identified several compounds that protect against ferroptosis, another cell death pathway associated with oxidative stress, suggesting potential efficacy against apoptosis. The present study assessed the protective efficacies of the ferroptosis inhibitors oxindole-curcumin hybrid compound GIF-2165X-G1, N,N-dimethylaniline derivatives GIF-2014 and GIF-2115, and ferrostatin-1 against rotenone-induced apoptosis. Treatment of mouse hippocampal HT22 cells with the mitochondrial transport chain inhibitor rotenone for 24 h reduced mitochondrial membrane potential, increased reactive oxygen species production, promoted nuclear fragmentation, and ultimately impaired cell viability, consistent with apoptosis. Ferroptosis inhibitor cotreatment did not prevent any of these rotenone-induced apoptotic processes but did suppress delayed cell death associated with loss of plasma membrane integrity. These results suggest that GIF-2165X-G1, GIF-2014, GIF-2115, and ferrostatin-1 are selective for ferroptosis and do not affect apoptosis. Thus, erastin-induced ferroptosis and rotenone-induced apoptosis are distinct cell death pathways despite the common involvement of mitochondrial oxidative stress. Further, the cytoprotective efficacies of chemical antioxidants may depend on the specific source of oxidative stress.

Isorhynchophylline ameliorates the progression of osteoarthritis by inhibiting the NF-κB pathway.

Osteoarthritis (OA), a progressive and degenerative joint disease, is characterized by cartilage degradation, synovitis, subchondral bone remodeling and osteophyte formation. Isorhynchophylline (IRN) is an oxindole alkaloid isolated from the traditional Chinese herb Uncaria rhynchophylla. In this study, we evaluated the protective effects of IRN on human OA chondrocytes. IRN treatment dose-dependently decreased the interleukin-1ß (IL-1ß)-induced expressions of nitric oxide (NO; p < 0.001), prostaglandin E2 (PGE2; p < 0.001), tumor necrosis factor alpha (TNF-α; p < 0.001), interleukin-6 (IL-6; p < 0.001), cyclooxygenase-2 (COX-2; p < 0.001) and inducible nitric oxide synthase (iNOS; p < 0.001) in chondrocytes. Meanwhile, the production of metalloproteinase 13 (MMP13; p < 0.001) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5; p < 0.001) was inhibited by IRN treatment. Molecular docking studies revealed that IRN directly interacted with the nuclear factor kappa B (NF-κB) complex, which was associated with a reduced level of NF-κB nuclear translocation and the inhibition of NF-κB signaling activity. Furthermore, administration of IRN generated marked in vivo protective effects during OA development. Collectively, our results demonstrate that IRN may exhibit therapeutic benefits against OA, potentially by ameliorating the inflammative and degenerative progression of OA via inhibiting the NF-κB pathway.

RK-270D and E, Oxindole Derivatives from Streptomyces sp. with Anti-Angiogenic Activity.

A chemical investigation of a culture extract from Streptomyces sp. RK85-270 led to the isolation and characterization of two new oxindoles, RK-270D (1) and E (2). The structures of 1 and 2 were determined by analyzing spectroscopic and spectrometric data from 1D and 2D NMR and High-resolution electrospray ionization mass spectrometry (HRESIMS) experiments. Compound 1 exhibited anti-angiogenic activities against human umbilical vein endothelial cells (HUVECs) without cytotoxicity. Results of Western blot analysis revealed that 1 inhibits VEGF-induced angiogenesis in the HUVECs via VEGFR2/ p38 MAPK-mediated pathway.

The main oxidative inactivation pathway of the plant hormone auxin.

Inactivation of the phytohormone auxin plays important roles in plant development, and several enzymes have been implicated in auxin inactivation. In this study, we show that the predominant natural auxin, indole-3-acetic acid (IAA), is mainly inactivated via the GH3-ILR1-DAO pathway. IAA is first converted to IAA-amino acid conjugates by GH3 IAA-amidosynthetases. The IAA-amino acid conjugates IAA-aspartate (IAA-Asp) and IAA-glutamate (IAA-Glu) are storage forms of IAA and can be converted back to IAA by ILR1/ILL amidohydrolases. We further show that DAO1 dioxygenase irreversibly oxidizes IAA-Asp and IAA-Glu into 2-oxindole-3-acetic acid-aspartate (oxIAA-Asp) and oxIAA-Glu, which are subsequently hydrolyzed by ILR1 to release inactive oxIAA. This work established a complete pathway for the oxidative inactivation of auxin and defines the roles played by auxin homeostasis in plant development.

Structural insights of oxindole based kinase inhibitors as anticancer agents: Recent advances.

Small-molecule kinase inhibitors are being continuously explored as new anticancer therapeutics. Kinases are the phosphorylating enzymes which regulate numerous cellular functions such as proliferation, differentiation, migration, metabolism, and angiogenesis by activating several signalling pathways. Kinases have also been frequently found to be deregulated and overexpressed in cancerous tissues. Therefore, modulating the kinase activity by employing small molecules has emerged as a strategic approach for cancer treatment. On the other hand, oxindole motifs have surfaced as privileged scaffolds with significant multi-kinase inhibitory activity. The present review summarises recent advances in the development of oxindole based kinase inhibitors. The role of distinguished structural frameworks of oxindoles, such as 3-alkenyl oxindoles, spirooxindoles, 3-iminooxindoles and similar hydrazone derivatives have been described based on their kinase inhibition potential. Furthermore, the design strategies, mechanism of actions, structure activity relationships (SARs) and their mode of interaction with target protein have been critically highlighted.

Achieving effective and selective CK1 inhibitors through structure modification.

Casein kinase 1 (CK1) is an extensively expressed serine/threonine kinase family, with six highly conserved isoforms of human CK1. Due to its involvement in many biological processes, CK1 is a promising target for several pathological states, including circadian sleep disorder, neurodegenerative diseases, cancer and inflammation. However, due to the structural similarities between the six CK1 members, the design of CK1 inhibitors is intricate. So far, no effective CK1 inhibitors are reported to reach clinical trials; thus, approaches to obtaining both selective and effective CK1 inhibitors are in great demand. Here we analyze several CK1 inhibitors that provide successful experience for structure-based drug design and rational structure modification, which could provide references for further drug design.

Potency and Selectivity Optimization of Tryptophanol-Derived Oxazoloisoindolinones: Novel p53 Activators in Human Colorectal Cancer.

To search for novel p53 activators, four series of novel (S)- and (R)-tryptophanol-derived oxazoloisoindolinones were synthesized in a straightforward manner and their antiproliferative activity was evaluated in the human colorectal cancer HCT116 cell line. Structural optimization of the hit compound SLMP53-1 led to the identification of a (R)-tryptophanol-derived isoindolinone that was found to be six-fold more active, with increased selectivity for HCT116 cells with p53 and with low toxicity in normal cells. Binding studies with MDM2 showed that the antiproliferative activity of tryptophanol-derived isoindolinones does not involve inhibition of the main negative regulator of the p53 protein. Molecular docking simulations showed that although these molecules establish hydrophobic interactions with MDM2, they do not possess the required features to bind MDM2.

Recent research progress of Uncaria spp. based on alkaloids: phytochemistry, pharmacology and structural chemistry.

Medicinal plants are well-known in affording clinically useful agents, with rich medicinal values by combining with disease targets through various mechanisms. Plant secondary metabolites as lead compounds lay the foundation for the discovery and development of new drugs in disease treatment. Genus Uncaria from Rubiaceae family is a significant plant source of active alkaloids, with anti-hypertensive, sedative, anti-Alzheimer's disease, anti-drug addiction and anti-inflammatory effects. This review summarizes and discuss the research progress of Uncaria based on alkaloids in the past 15 years, mainly in the past 5 years, including biosynthesis, phytochemistry, pharmacology and structural chemistry. Among, focusing on representative compounds rhynchophylline and isorhynchophylline, the pharmacological activities surrounding the central nervous system and cardiovascular system are described in detail. On the basis of case studies, this article provides a brief overview of the synthesis and analogues of representative compounds types. In summary, this review provides an early basis for further searching for new targets and activities, discussing the mechanisms of pharmacological activity and studying the structure-activity relationships of active molecules.

Candida antarctica lipase-B-catalyzed kinetic resolution of 1,3-dialkyl-3-hydroxymethyl oxindoles.

Candida antarctica (CAL-B) lipase-catalyzed resolution of 1,3-dialkyl-3-hydroxymethyl oxindoles has been performed to obtain (R)-1,3-dialkyl-3-acetoxymethyl oxindoles with up to 99% ee and (S)-1,3-dialkyl-3-hydroxymethyl oxindoles with up to 78% ee using vinyl acetate as acylating agent and acetonitrile as solvent transforming (S)-3-allyl-3-hydroxymethyl oxindole to (3S)-1'-benzyl-5-(iodomethyl)-4,5-dihydro-2H-spiro[furan-3,3'-indolin]-2'-one. The optically active 3-substituted-3-hydroxymethyl oxindoles and spiro-oxindoles are among the key synthons in the synthesis of potentially biologically active molecules.

Novel 2-indolinone thiazole hybrids as sunitinib analogues: Design, synthesis, and potent VEGFR-2 inhibition with potential anti-renal cancer activity.

Novel 2-indolinone thiazole hybrids were designed and synthesized as VEGFR-2 inhibitors based on sunitinib, an FDA-approved anticancer drug. The proposed structures of the prepared 2-indolinone thiazole hybrids were confirmed based on their spectral data and CHN analyses. The target compounds were screened in vitro for their anti-VEGFR-2 activity. All tested compounds exhibited a potent submicromolar inhibition of VEGFR-2 kinase with IC50 values ranging from 0.067 to 0.422 µM, relative to sunitinib reference drug (IC50 = 0.075 ± 0.002 µM). Compounds 5, 15a, 15b, 17, 19c displayed excellent VEGFR-2 inhibitory activity, comparable or nearly equipotent to sunitinib. Compound 13b stood out as the most potent against VEGFR-2 showing IC50 value of 0.067 ± 0.002 µM, lower than that of sunitinib. In addition, the most potent derivatives were assessed for their anticancer activity against two renal cancer cell lines. Compound 13b (IC50 = 3.9 ± 0.13 µM) was more potent than sunitinib (IC50 = 4.93 ± 0.16 µM) against CAKI-1 cell line. Moreover, thiazole 15b displayed excellent anticancer activity against CAKI-1 cell line (IC50 = 3.31 ± 0.11 µM), superior to that of sunitinib (IC50 = 4.93 ± 0.16 µM). Thiazole 15b was also equipotent to sunitinib (IC50 = 1.23 ± 0.04 µM) against A498 cell line. Besides, compound 15b revealed a safety profile much better than that of sunitinib against normal human renal cells. Furthermore, a docking study revealed a proper fitting of the most active compounds into the ATP binding site of VEGFR-2, rationalizing their potent anti-VEGFR-2 activity.

Activating Effect of 3-Benzylidene Oxindoles on AMPK: From Computer Simulation to High-Content Screening.

AMP-activated protein kinase (AMPK) is currently the subject of intensive study and active discussions. AMPK performs its functions both at the cellular level, providing the switch between energy-consuming and energy-producing processes, and at the whole body level, particularly, regulating certain aspects of higher nervous activity and behavior. Control of such a 'main switch' compensates dysfunctions and associated diseases. In the present paper, we studied the binding of 3-benzylidene oxindoles to the kinase domain of the AMPK α-subunit, which is thought to prevent its interaction with the autoinhibitory domain and thus result in the AMPK activation. For this purpose, we developed the cellular test system based on the AMPKAR plasmid, which implements the FRET effect, synthesized a number of 3-benzylidene oxindole compounds and simulated their binding to various sites of the kinase domain. The most probable binding site for the studied compounds was established by the correlation of calculated and experimental data. The obtained results allow to analyze various classes of AMPK activators using virtual and high-content screening.

Design, synthesis and biological evaluation of 2-indolinone derivatives as PAK1 inhibitors in MDA-MB-231 cells.

P21-activated kinase 1 (PAK1) plays a vital role in the proliferation, survival and migration of cancer cells, which has emerged as a promising drug target for cancer therapy. In this study, a series of 2-indolinone derivatives were designed and synthesized through a structure-based strategy. A potent PAK1 inhibitor (ZMF-005) was discovered, which presented an IC50 value of 0.22 µM against PAK1 with potent antiproliferative activity. Furthermore, we predicted the binding mode of ZMF-005 and PAK1 by molecule docking and dynamic (MD) simulation. In addition, ZMF-005 was documented to induce significant apoptosis and suppress migration in MDA-MB-231 cells. Collectively, these findings revealed that ZMF-005 is a novel potent PAK1 inhibitor for breast cancer treatment.

Targeting fungal virulence factor by small molecules: Structure-based discovery of novel secreted aspartic protease 2 (SAP2) inhibitors.

Secreted aspartic protease 2 (SAP2), a kind of virulence factor, is an emerging new antifungal target. Using docking-based virtual screening and structure-based inhibitor design, a series of novel SAP2 inhibitors were successfully identified. Among them, indolone derivative 24a showed potent SAP2 inhibitory activity (IC50 = 0.92 µM). It blocked fungi biofilm and hypha formation by down-regulating the expression of genes SAP2, ECE1, ALS3 and EFG1. As a virulence factor inhibitor, compound 24a was inactive in vitro and showed potent in vivo efficacy in a murine model of invasive candidiasis. It represents a promising lead compound for the discovery of novel antifungal agents.

Flavin-Dependent Monooxygenases NotI and NotI' Mediate Spiro-Oxindole Formation in Biosynthesis of the Notoamides.

The fungal indole alkaloids are a unique class of complex molecules that have a characteristic bicyclo[2.2.2]diazaoctane ring and frequently contain a spiro-oxindole moiety. While various strains produce these compounds, an intriguing case involves the formation of individual antipodes by two unique species of fungi in the generation of the potent anticancer agents (+)- and (-)-notoamide A. NotI and NotI' have been characterized as flavin-dependent monooxygenases that catalyze epoxidation and semi-pinacol rearrangement to form the spiro-oxindole center within these molecules. This work elucidates a key step in the biosynthesis of the notoamides and provides an evolutionary hypothesis regarding a common ancestor for production of enantiopure notoamides.

Substituted oxindol-3-ylidenes as AMP-activated protein kinase (AMPK) inhibitors.

AMP-activated protein kinase (AMPK) is a central metabolic regulator that promotes cancer growth and survival under hypoxia and plays a role in the maintenance of cancer stem cells. A major challenge to interrogating the potential of targeting AMPK in cancer is the lack of potent and selective small molecule inhibitors. Compound C has been widely used as an AMPK inhibitor, but it lacks potency and has a poor selectivity profile. The multi-kinase inhibitor, sunitinib, has demonstrated potent nanomolar inhibition of AMPK activity and has scope for modification. Here, we have designed and synthesized several series of oxindoles to determine the structural requirements for AMPK inhibition and to improve selectivity. We identified two potent, novel oxindole-based AMPK inhibitors that were designed to interact with the DFG motif in the ATP-binding site of AMPK, this key feature evades interaction with the common recptor tyrosine kinase targets of sunitinib. Cellular engagement of AMPK by these oxindoles was confirmed by the inhibition of phosphorylation of acetyl-CoA carboxylase (ACC), a known substrate of AMPK, in myeloid leukemia cells. Interestingly, although AMPK is highly expressed and activated in K562 cells these oxindole-based AMPK inhibitors did not impact cell viability or result in significant cytotoxicity. Our studies serve as a platform for the further development of oxindole-based AMPK inhibitors with therapeutic potential.

Refactoring the Concise Biosynthetic Pathway of Cyanogramide Unveils Spirooxindole Formation Catalyzed by a P450 Enzyme.

Cyanogramide (1) from the marine actinomycete Actinoalloteichus cyanogriseus WH1-2216-6 features a unique spirooxindole skeleton and exhibits significant bioactivity to efficiently reverse drug resistance in tumor cells. The biosynthetic gene cluster of 1 in A. cyanogriseus WH1-2216-6 was identified and refactored by promoter engineering for heterologous expression in Streptomyces coelicolor YF11, thereby enabling the production of 1 and five new derivatives. Interesting, four of them, including 1, were identified as enantiomeric mixtures in different ratios. The functions of tailoring enzymes, including two methyltransferases (CyaEF), and three cytochrome P450 monooxygenases (CyaGHI) were confirmed by gene inactivation and feeding experiments, leading to the elucidation of a concise biosynthetic pathway for 1. Notably, CyaH was biochemically verified to catalyze the formation of the spirooxindole skeleton in 1 through an unusual carbocation-mediated semipinacol-type rearrangement reaction.

Synthesis, Evaluation and Proposed Binding Pose of Substituted Spiro-Oxindole Dihydroquinazolinones as IRAP Inhibitors.

Insulin-regulated aminopeptidase (IRAP) is a new potential macromolecular target for drugs aimed for treatment of cognitive disorders. Inhibition of IRAP by angiotensin IV (Ang IV) improves the memory and learning in rats. The majority of the known IRAP inhibitors are peptidic in character and suffer from poor pharmacokinetic properties. Herein, we present a series of small non-peptide IRAP inhibitors derived from a spiro-oxindole dihydroquinazolinone screening hit (pIC50 5.8). The compounds were synthesized either by a simple microwave (MW)-promoted three-component reaction, or by a two-step one-pot procedure. For decoration of the oxindole ring system, rapid MW-assisted Suzuki-Miyaura cross-couplings (1 min) were performed. A small improvement of potency (pIC50 6.6 for the most potent compound) and an increased solubility could be achieved. As deduced from computational modelling and MD simulations it is proposed that the S-configuration of the spiro-oxindole dihydroquinazolinones accounts for the inhibition of IRAP.

Heterobinuclear copper(II)­platinum(II) complexes with oxindolimine ligands: Interactions with DNA, and inhibition of kinase and alkaline phosphatase proteins.

Two mononuclear copper(II) compounds, [Cu(isad)(H2O)Cl]Cl 1 and [Cu(isah)(H2O)Cl]Cl 2, and its corresponding heterobinuclear species containing also platinum(II), [CuCl(isad)Pt(NH3)Cl2] 3 and [CuCl(isah)Pt(NH3)Cl2] 4 (where isad and isah are oxindolimine ligands, (E)-3-(2-(3-aminopropylamino)ethylimino)indolin-2-one, and (E)-3-(3-amino-2-hydroxypropylimino)indolin-2-one, respectively), have been previously synthesized and characterized by different spectroscopic techniques in our laboratory. Cytotoxicity assays performed with B16F10 murine cancer cells, and MES-SA human uterine sarcoma cells, showed IC50 values lower or in the same order of cisplatin. Herein, in order to better elucidate their probable modes of action, possible interaction and damage to DNA, as well as their effect on the activity of crucial proteins were verified. Both mononuclear complexes and the binuclear compound 4 displayed a significant cleavage activity toward plasmid DNA, while compound 3 tends to protect DNA from oxidative damage, avoiding degradation. Complementary experiments indicated a significant inhibition activity toward cyclin-dependent kinase (CDK1/cyclinB) activity in the phosphorylation of histone H1, and only moderate inhibition concerning alkaline phosphatase. Results also revealed that the reactivity is reliant on the ligand structure and on the nature of the metal present, in a synergistic effect. Simulation studies complemented and supported our results, indicating different bindings of the binuclear compounds to DNA. Therefore, the verified cytotoxicity of these complexes comprises multiple modes of action, including modification of DNA conformation, scission of DNA strands by reactive oxygen species, and inhibition of selected proteins that are crucial to the cellular cycle.