Indole Inhibitors Research Articles

Indole Inhibitors of MMP-13 for Arthritic Disorders.

Here, we described the design, by fragment merging and multiparameter optimization, of selective MMP-13 inhibitors that display an appropriate balance of potency and physicochemical properties to qualify as tool compounds suitable for in vivo testing. Optimization of potency was guided by structure-based insights, specifically to replace an ester moiety and introduce polar directional hydrogen bonding interactions in the core of the molecule. By introducing polar enthalpic interactions in this series of inhibitors, the overall beneficial physicochemical properties were maintained. These physicochemical properties translated to excellent drug-like properties beyond potency. In a murine model of rheumatoid arthritis, treatment of mice with selective inhibitors of MMP-13 resulted in a statistically significant reduction in the mean arthritic score vs control when dosed over a 14 day period.

Different Single-Enzyme Conformational Dynamics upon Binding Hydrolyzable or Nonhydrolyzable Ligands.

Single-molecule measurements of protein dynamics help unveil the complex conformational changes and transitions that occur during ligand binding and catalytic processes. Using high-resolution single-molecule nanocircuit techniques, we have investigated differences in the conformational dynamics and transitions of lysozyme interacting with three ligands: peptidoglycan substrate, substrate-based chitin analogue, and indole derivative inhibitors. While processing peptidoglycan, lysozyme followed one of the two mechanistic pathways for the hydrolysis of the glycosidic bonds: a concerted mechanism inducing direct conformational changes from open to fully closed conformations or a nonconcerted mechanism involving transient pauses in intermediate conformations between the open and closed conformations. In the presence of either chitin or an indole inhibitor, lysozyme was unable to access the fully closed conformation where catalysis occurs. Instead, lysozymes' conformational closures terminated at slightly closed, "excited" conformations that were approximately one-quarter of the full hinge-bending range. With the indole inhibitor, lysozyme reached this excited conformation in a single step without any evidence of rate-liming intermediates, but the same conformational motions with chitin involved three hidden, intermediate processes and features similar to the nonconcerted peptidoglycan mechanism. The similarities suggest that these hidden processes involve attempts to accommodate imperfectly aligned polysaccharides in the active site. The results provide a detailed glimpse of the enzyme-ligand interplay at the crux of molecular recognition, enzyme specificity, and catalysis.

Kinase inhibitors allosterically disrupt a regulatory interaction to enhance PKCα membrane translocation

The eukaryotic kinase domain has multiple intrinsically disordered regions whose conformation dictates kinase activity. Small molecule kinase inhibitors (SMKIs) rely on disrupting the active conformations of these disordered regions to inactivate the kinase. While SMKIs are selected for their ability to cause this disruption, the allosteric effects of conformational changes in disordered regions is limited by a lack of dynamic information provided by traditional structural techniques. In this study, we integrated multiscale molecular dynamics simulations with FRET sensors to characterize a novel allosteric mechanism that is selectively triggered by SMKI binding to the protein kinase Cα domain. The indole maleimide inhibitors BimI and sotrastaurin were found to displace the Gly-rich loop (G-loop) that normally shields the ATP-binding site. Displacement of the G-loop interferes with a newly identified, structurally conserved binding pocket for the C1a domain on the N lobe of the kinase domain. This binding pocket, in conjunction with the N-terminal regulatory sequence, masks a diacylglycerol (DAG) binding site on the C1a domain. SMKI-mediated displacement of the G-loop released C1a and exposed the DAG binding site, enhancing protein kinase Cα translocation both to synthetic lipid bilayers and to live cell membranes in the presence of DAG. Inhibitor chemotype determined the extent of the observed allosteric effects on the kinase domain and correlated with the extent of membrane recruitment. Our findings demonstrate the allosteric effects of SMKIs beyond the confines of kinase catalytic conformation and provide an integrated computational-experimental paradigm to investigate parallel mechanisms in other kinases.

Synthesis of indole inhibitors of silent information regulator 1 (SIRT1), and their evaluation as cytotoxic agents

A series of achiral indole analogues of the selective sirtuin inhibitor EX-527 (a racemic, substituted 1,2,3,4 tetrahydrocarbazole) was designed to stabilize the bioactive conformation, and synthesized. These new indoles were evaluated against the isolated sirtuin enzymes SIRT1 and SIRT2, and against a panel of nine human cell lines. Structure-activity relationship studies demonstrated the influence of the substituent at position 3 of the indole. The most potent SIRT1 inhibitor 3h, bearing an isopropyl substituent, was as potent as EX-527, and more selective for SIRT1 over SIRT2. Compound 3g, bearing a benzyl substituent, inhibited both sirtuins at micromolar concentration and was more cytotoxic than EX-527 on several cancer cell lines.

The activity prediction of indole inhibitors against HCV NS5B polymerase.

Non-structural viral protein 5B (NS5B) is a viral protein in hepatitis C virus. Although various inhibitors against NS5B have been found, the activity prediction of similar untested inhibitors is still highly desirable. In this respect, the Tchebichef moments (TMs) calculated from the images of molecular structures were regarded as the independent variables while the inhibitory activity (pIC50 ) was the dependent variable, and the predictive model was established by means of stepwise regression. The R-squared of leave-one-out cross-validation (Q2 ) for the training set and the R-squared of prediction ( ) for external independent test set were 0.919 and 0.927, respectively. The obtained model was also evaluated strictly. Compared with the multivariate curve resolution with alternating least squares (MCR-ALS) and the QSAR approaches derived from the literature, the proposed method is more accurate and reliable. This study not only provides an effective approach to predict the biological activity of RNA replication's inhibitors, but also extends the QSAR modeling technique.

Optimization of Phenyl Indole Inhibitors of the AAA+ ATPase p97

Optimization of the side-chain of a phenyl indole scaffold identified from a high-throughput screening campaign for inhibitors of the AAA+ ATPase p97 is reported. The addition of an N-alkyl piperazine led to high potency of this series in a biochemical assay, activity in cell-based assays, and excellent pharmaceutical properties. Molecular modeling based on a subsequently obtained cryo-EM structure of p97 in complex with a phenyl indole was used to rationalize the potency of these allosteric inhibitors.

Secreted Phospholipases A2 in Hereditary Angioedema With C1-Inhibitor Deficiency.

Hereditary angioedema (HAE) caused by deficiency (type I) or dysfunction (type II) of the C1 inhibitor protein (C1-INH-HAE) is a disabling, potentially fatal condition characterized by recurrent episodes of swelling. We have recently found that patients with C1-INH-HAE have increased plasma levels of vascular endothelial growth factors and angiopoietins (Angs), which have been associated with vascular permeability in several diseases. Among these and other factors, blood endothelial cells and vascular permeability can be modulated by extracellular or secreted phospholipases A2 (sPLA2s). We sought to investigate the enzymatic activity and biological functions of sPLA2 in patients with C1-INH-HAE. sPLA2s enzymatic activity was evaluated in the plasma from 109 adult patients with C1-INH-HAE and 68 healthy donors in symptom-free period and attacks. Plasma level of group IIA sPLA2 (hGIIA) protein was measured in selected samples. The effect of C1-INH-HAE plasma on endothelial permeability was examined in vitro using a vascular permeability assay. The role of hGIIA was determined using highly specific sPLA2 indole inhibitors. The effect of recombinant hGIIA on C1-INH activity was examined in vitro by functional assay. Plasma sPLA2 activity and hGIIA levels are increased in symptom-free C1-INH-HAE patients compared with controls. sPLA2 activity negatively correlates with C1-INH protein level and function. C1-INH-HAE plasma increases endothelial permeability in vitro, and this effect is partially reverted by a specific hGIIA enzymatic inhibitor. Finally, recombinant hGIIA inhibits C1-INH activity in vitro. sPLA2 enzymatic activity (likely attributable to hGIIA), which is increased in C1-INH-HAE patients, can promote vascular permeability and impairs C1-INH activity. Our results may pave the way for investigating the functions of sPLA2s (in particular, hGIIA) in the pathophysiology of C1-INH-HAE and may inform the development of new therapeutic targets.

Substituted indole Mcl-1 inhibitors: a patent evaluation (WO2015148854A1)

ABSTRACTThe myeloid cell leukemia 1 (Mcl-1) protein, an anti-apoptotic member of Bcl-2 family, plays a critical role in the development and maintenance of many cancers and is listed in the ‘top ten’ pathological factors across the diversity of human cancers. The patent described in this evaluation (WO2015148854A1) claimed substituted indole Mcl-1 inhibitors for the treatment of diseases and conditions (e.g., cancer) characterized by the over-expression or dysregulation of Mcl-1 proteins. A variety of 2-position substituents distinguished indole Mcl-1 inhibitors claimed in this patent from another two patents by AbbVie Inc. (WO2008131000A2 and WO2008130970A1). They exhibited low-nanomolar binding affinities and >100-fold selectivity over Bcl-2 and Bcl-xL in vitro, and low-micromolar killing abilities against a panel of tumour cell lines. Moreover, the compounds in this patent revealed that the structural basis for selective Mcl-1 inhibitors may not completely depend on the 5 known binding hot-spots, and conformational flexibility of Mcl-1 protein could contribute to the binding specificity.

Discovery of indole inhibitors of chemokine receptor 9 (CCR9)

Irritable bowel diseases (IBD) such as Crohn’s disease (CD) and ulcerative colitis (UC) are serious chronic diseases affecting millions of patients worldwide. Studies of human chemokine biology has suggested C–C chemokine receptor 9 (CCR9) may be a key mediator of pro-inflammatory signaling. Discovery of agents that inhibit CCR9 may lead to new therapies for CD and UC patients. Herein we describe the evolution of a high content screening hit (1) into potent inhibitors of CCR9, such as azaindole 12.

Structure-Activity Study of Bioisosteric Trifluoromethyl and Pentafluorosulfanyl Indole Inhibitors of the AAA ATPase p97.

Exploratory SAR studies of a new phenyl indole chemotype for p97 inhibition revealed C-5 indole substituent effects in the ADPGlo assay that did not fully correlate with either electronic or steric factors. A focused series of methoxy-, trifluoromethoxy-, methyl-, trifluoromethyl-, pentafluorosulfanyl-, and nitro-analogues was found to exhibit IC50s from low nanomolar to double-digit micromolar. Surprisingly, we found that the trifluoromethoxy-analogue was biochemically a better match of the trifluoromethyl-substituted lead structure than a pentafluorosulfanyl-analogue. Moreover, in spite of their almost equivalent strongly electron-depleting effect on the indole core, pentafluorosulfanyl- and nitro-derivatives were found to exhibit a 430-fold difference in p97 inhibitory activities. Conversely, the electronically divergent C-5 methyl- and nitro-analogues both showed low nanomolar activities.

Abstract 2437: Crystal structures of CARM1 bound to sinefungin and diverse peptide substrates

Abstract Co-activator-associated arginine methyltransferase 1 (CARM1) is a protein arginine N-methyltransferase (PRMT) enzyme that has been implicated in a variety of cancers including AML (1) and breast (2), prostate (3), lung (4) and colorectal (5) carcinomas. CARM1 is known to methylate H3 histones and non-histone substrates including p300/CBP, AIB1/SRC-3 and PABP1 (6). To date, several crystal structures of CARM1 have been solved, including structures with small molecule inhibitors (7), but no ternary structures with nucleotide and peptide substrate have been reported. Here, the crystal structures of human CARM1 with the SAM mimic sinefungin (SFG) and three different peptide sequences from histone H3 and PAPB1 are presented, and both non-methylated and singly-methylated arginine residues are exemplified. Extensive interactions are seen with residues Glu266, Glu257, and His414 and the substrate arginine side chain. Two key hydrogen bonds are made by the side chain of Asn161 with the backbone carbonyl of the P’1 peptide residue and the backbone NH of the P’3 peptide residue. The carbonyl of the P1 peptide residue engages the protein through water mediated hydrogen bonds. These structures show how the CARM1 binding site is capable of accommodating a variety of peptide sequences. Comparisons to known CARM1 complexes with small molecules provide additional insights into inhibitor design. 1. Vu, L. P. et al, PRMT4 blocks myeloid differentiation by assembling a methyl-RUNX1-dependent repressor complex. Cell Rep 2013, 5 (6), 1625-38. 2. Al-Dhaheri, M. et al, CARM1 is an important determinant of ERalpha-dependent breast cancer cell differentiation and proliferation in breast cancer cells. Cancer Res 2011, 71 (6), 2118-28. 3. Kim, Y. R. et al, Differential CARM1 expression in prostate and colorectal cancers. BMC Cancer 2010, 10, 197. 4. Elakoum, R. et al, CARM1 and PRMT1 are dysregulated in lung cancer without hierarchical features. Biochimie 2014, 97, 210-8. 5. Ou, C. Y. et al, A coactivator role of CARM1 in the dysregulation of beta-catenin activity in colorectal cancer cell growth and gene expression. Mol Cancer Res 2011, 9 (5), 660-70. 6. Bedford M.T.; Clarke S.G. Protein arginine methylation in mammals: who, what, and why. Mol Cell 2009, 33, 1-13. 7. Sack, J.S et al, Structural basis for CARM1 inhibition by indole and pyrazole inhibitors. Biochem J 2011, 436, 331-9. Citation Format: Ann Boriack-Sjodin, Lei Jin, Suzanne L. Jacques, Allison Drew, Margaret Porter Scott, Scott Ribich, Oscar Moradei. Crystal structures of CARM1 bound to sinefungin and diverse peptide substrates. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2437. doi:10.1158/1538-7445.AM2015-2437

ChemInform Abstract: Discovery of HCV NS5B Thumb Site I Inhibitors: Core‐Refining from Benzimidazole to Indole Scaffold

AbstractReview: 45 refs.

Identification of indole inhibitors of human hematopoietic prostaglandin D2 synthase (hH-PGDS).

Human H-PGDS has shown promise as a potential target for anti-allergic and anti-inflammatory drugs. Here we describe the discovery of a novel class of indole inhibitors, identified through focused screening of 42,000 compounds and evaluated using a series of hit validation assays that included fluorescence polarization binding, 1D NMR, ITC and chromogenic enzymatic assays. Compounds with low nanomolar potency, favorable physico-chemical properties and inhibitory activity in human mast cells have been identified. In addition, our studies suggest that the active site of hH-PGDS can accommodate larger structural diversity than previously thought, such as the introduction of polar groups in the inner part of the binding pocket.

Discovery of HCV NS5B thumb site I inhibitors: Core-refining from benzimidazole to indole scaffold

Hepatitis C virus (HCV) NS5B RNA-depended-RNA-polymerase (RdRp) is an essential enzyme in HCV viral replication and has no functional equivalent in mammalian cells. Several classes of nucleoside and non-nucleoside inhibitors, targeting the different allosteric sites, have demonstrated efficacy in clinical trials. Compared to other allosteric sites, thumb site I is a more compelling allosteric target with a significant number of inhibitors in clinical trials. Among them, indole analogues are the most important series of NS5B thumb site I inhibitors with considerable antiviral activity. This review focuses on the discovery and development of indole inhibitors targeting on NS5B thumb site I. Five fundamental principles, the general structure–activity relationships (SARs) model of indole scaffold, were summarized, which could pave the way for further structural optimization of indole-based anti-HCV agents.

RpoS and indole signaling control the virulence of Vibrio anguillarum towards gnotobiotic sea bass (Dicentrarchus labrax) larvae.

Quorum sensing, bacterial cell-to-cell communication with small signal molecules, controls the virulence of many pathogens. In contrast to other vibrios, neither the VanI/VanR acylhomoserine lactone quorum sensing system, nor the three-channel quorum sensing system affects virulence of the economically important aquatic pathogen Vibrio anguillarum. Indole is another molecule that recently gained attention as a putative signal molecule. The data presented in this study indicate that indole signaling and the alternative sigma factor RpoS have a significant impact on the virulence of V. anguillarum. Deletion of rpoS resulted in increased expression of the indole biosynthesis gene tnaA and in increased production of indole. Both rpoS deletion and the addition of exogenous indole (50–100 µM) resulted in decreased biofilm formation, exopolysaccharide production (a phenotype that is required for pathogenicity) and expression of the exopolysaccharide synthesis gene wbfD. Further, indole inhibitors increased the virulence of the rpoS deletion mutant, suggesting that indole acts downstream of RpoS. Finally, in addition to the phenotypes found to be affected by indole, the rpoS deletion mutant also showed increased motility and decreased sensitivity to oxidative stress.

Converting potent indeno[1,2-b]indole inhibitors of protein kinase CK2 into selective inhibitors of the breast cancer resistance protein ABCG2.

A series of indeno[1,2-b]indole-9,10-dione derivatives were synthesized as human casein kinase II (CK2) inhibitors. The most potent inhibitors contained a N(5)-isopropyl substituent on the C-ring. The same series of compounds was found to also inhibit the breast cancer resistance protein ABCG2 but with totally different structure-activity relationships: a N(5)-phenethyl substituent was critical, and additional hydrophobic substituents at position 7 or 8 of the D-ring or a methoxy at phenethyl position ortho or meta also contributed to inhibition. The best ABCG2 inhibitors, such as 4c, 4h, 4i, 4j, and 4k, behaved as very weak inhibitors of CK2, whereas the most potent CK2 inhibitors, such as 4a, 4p, and 4e, displayed limited interaction with ABCG2. It was therefore possible to convert, through suitable substitutions of the indeno[1,2-b]indole-9,10-dione scaffold, potent CK2 inhibitors into selective ABCG2 inhibitors and vice versa. In addition, some of the best ABCG2 inhibitors, which displayed a very low cytotoxicity, thus giving a high therapeutic ratio, and appeared not to be transported, constitute promising candidates for further investigations.

The effect of temperature and hydrodynamics on carbon steel corrosion and its inhibition in oxygenated acid–salt solution

Corrosion of carbon steel in hydrochloric acid (HCl)–sodium sulphate (Na2SO4) solution mixture was investigated using rotating cylinder electrode (RCE) for a range of rotation velocity, 0–2000rpm, solution temperature of 32–52°C, and different oxygen concentrations. The corrosion rat was determined by using both weight loss method and electrochemical polarization technique. Different acid and salt concentrations were used ranged from 0.01 to 0.2M for salt and 0.5 to 5% for acid. The conjoint effect of increased oxygen concentration and high rotational velocity was studied based on experimental measurements of O2 concentration. The effects of operating conditions on indole and cetyl trimethyl ammonium bromides (CTAB) inhibition efficiency were also studied and discussed. The results showed that increasing the rotational velocity leads to an increase in the corrosion rate depending on the concentration of salt and acid. Increasing the temperature and acid concentrations leads to an increase in the corrosion rate while the corrosion rate exhibited unstable trend with salt concentration leads to change of corrosion. It is found that increasing O2 concentration leads to a considerable increase in the corrosion rates especially at high rotational velocity. Indole and CTAB inhibitors exhibited very good inhibition efficiency in most conditions investigated with the former exhibited better inhibition efficiency arriving up to 87% at low rotational velocities. The inhibition efficiency of both inhibitors was found to decrease with increasing velocity. In addition, indole inhibitor reveals excellent inhibition efficiency even at high temperatures while CTAB efficiency decreased appreciably with temperature increase.

Paramagnetic Relaxation Assisted Docking of a Small Indole Compound in the HIV-1 gp41 Hydrophobic Pocket

The hydrophobic pocket contained within the gp41 coiled coil is an important target for small molecules designed to inhibit HIV-1 fusion. While various screening experiments have identified molecules purported to bind in this pocket, few have confirmed details of the interaction, instead relying on computational docking to predict the binding mode. This is made more challenging by the fact that residues lining the hydrophobic pocket are highly flexible, as is typical for a protein-protein interaction site, limiting the predictive power of computational tools. In this study, we report on an NMR method to define the binding mode of 1-5i, a compound in a series of newly developed indole inhibitors. We show that paramagnetic relaxation enhancement of ligand protons due to an MTSL group positioned close to the binding pocket could be applied quantitatively to distinguish between more than 30 different computational poses, selecting a single pose that agreed with the NMR data. In this pose, important hydrophobic and polar contacts occur with pocket lysine, tryptophan, and glutamine residues, including putative hydrogen bonds between the ligand carboxylate and the lysine ε-amino group. A study of the ligand orientation suggests directions for optimization.

Discovery of (7R)-14-Cyclohexyl-7-{[2-(dimethylamino)ethyl](methyl) amino}-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylic Acid (MK-3281), a Potent and Orally Bioavailable Finger-Loop Inhibitor of the Hepatitis C Virus NS5B Polymerase

Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. The polymerase of HCV is responsible for the replication of viral genome and has been a prime target for drug discovery efforts. Here, we report on the further development of tetracyclic indole inhibitors, binding to an allosteric site on the thumb domain. Structure-activity relationship (SAR) studies around an indolo-benzoxazocine scaffold led to the identification of compound 33 (MK-3281), an inhibitor with good potency in the HCV subgenomic replication assay and attractive molecular properties suitable for a clinical candidate. The compound caused a consistent decrease in viremia in vivo using the chimeric mouse model of HCV infection.

Molecular modeling based approach, synthesis and in vitro assay to new indole inhibitors of hepatitis C NS3/4A serine protease

In an attempt to identify potential HCV NS3 protease inhibitors lead compounds, a series of novel indoles (10a-g) was designed. Molecular modeling study, including fitting to a 3D-pharmacophore model of the designed molecules (10a-g), with HCV NS3 protease hypothesis using catalyst program was fulfilled. Also, the molecular docking into the NS3 active site was examined using Discovery Studio 2.5 software. Several compounds showed significant high simulation docking score and fit values. The designed compounds with high docking score and fit values were synthesized and biologically evaluated in vitro using an NS3 protease binding assay. It appears that most of the tested compounds reveal promising inhibitory activity against NS3 protease. Of these, compounds 10a and 10b demonstrated potent HCV NS3 protease inhibitors with IC(50) values of 9 and 12μg/mL, respectively. The experimental serine protease inhibitor activities of compounds 10a-g were consistent with their molecular modeling results. Inhibitors from this class have promising characteristics for further development as anti-HCV agents.