New Class Of Inhibitors Research Articles

Abstract 2632: Syrbactin, a novel proteasome inhibitor class, induces apoptosis and autophagy in neuroblastoma

Abstract Syrbactins belong to a new class of proteasome inhibitors which include syringolins and glidobactins. These small molecules are structurally distinct from other, well-established proteasome inhibitors, and bind the eukaryotic 20S proteasome by a novel mechanism. In this study, we examined the effects of syringolin A (SylA) and glidobactin A (GlbA) as well as two synthetic SylA-analogs (SylA-PEG and SylA-LIP) in human neuroblastoma (SK-N-SH), human multiple myeloma (MM1.S, MM1.RL, and U266), and human ovarian cancer (SKOV3) cells. While all four syrbactins inhibited cell proliferation in a dose-dependent manner, GlbA was most potent in both dexamethasone-sensitive MM1.S cells (IC50: 0.004 µM) and dexamethasone-resistant MM1.RL cells (IC50: 0.005 µM). Syrbactins also inhibited the chymotrypsin-like proteasome activity in a dose-dependent fashion, and GlbA was most effective in SK-N-SH cells (IC50: 0.015 µM). The GlbA-promoted inhibition of proteasomal activity in SK-N-SH cells resulted in the accumulation of ubiquitinated proteins and tumor suppressor protein p53, and led to apoptotic cell death. Strikingly, GlbA treatment also promoted the activation of Akt/PKB via phosphorylation at residue Ser473 and induced autophagy as judged by the presence of the lipidated form of microtubule-associated protein 1 light chain 3 (LC3) and autophagosomes. Collectively, our data suggest that syrbactins belong to a new and effective proteasome inhibitor class which promotes cell death. Proteasome inhibition is a promising strategy for targeted anticancer therapy and syrbactins are a new class of inhibitors which provide a structural platform for the development of novel, proteasome inhibitor-based drug therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2632.

Sulfonamide Linked Neoglycoconjugates−A New Class of Inhibitors for Cancer-Associated Carbonic Anhydrases

The contribution of membrane-bound carbonic anhydrases (CAs) to hypoxic tumor growth and progression in cancer implicates cancer-associated CAs as a promising drug target for oncology. In this paper, we present a new class of sulfonamide-linked neoglycoconjugate that was designed to selectively target and inhibit the extracellular domains of the cancer-relevant CA isozymes. We describe the application of novel, yet straightforward, chemistry toward the synthesis of inhibitors that comprise both S-glycosyl sulfenamides and S-glycosyl sulfonamides. We also present the CA inhibition profile of our new neoglycoconjugates, more specifically a library of 30 compounds (3-32) that were designed to optimize both SAR (structure-activity relationship) and SPR (structure-property relationship) characteristics. We show that our approach produces neutral, water-soluble, and potent inhibitors (K(i)s in the low nanomolar range) that target cancer-associated CAs.

Syrbactin class proteasome inhibitor-induced apoptosis and autophagy occurs in association with p53 accumulation and Akt/PKB activation in neuroblastoma

Syrbactins belong to a new class of proteasome inhibitors which include syringolins and glidobactins. These small molecules are structurally distinct from other, well-established proteasome inhibitors, and bind the eukaryotic 20S proteasome by a novel mechanism. In this study, we examined the effects of syringolin A (SylA) and glidobactin A (GlbA) as well as two synthetic SylA-analogs (SylA-PEG and SylA-LIP) in human neuroblastoma (SK-N-SH), human multiple myeloma (MM1.S, MM1.RL, and U266), and human ovarian cancer (SKOV-3) cells. While all four syrbactins inhibited cell proliferation in a dose-dependent manner, GlbA was most potent in both dexamethasone-sensitive MM1.S cells (IC 50: 0.004 μM) and dexamethasone-resistant MM1.RL cells (IC 50: 0.005 μM). Syrbactins also inhibited the chymotrypsin-like proteasome activity in a dose-dependent fashion, and GlbA was most effective in SK-N-SH cells (IC 50: 0.015 μM). The GlbA-promoted inhibition of proteasomal activity in SK-N-SH cells resulted in the accumulation of ubiquitinated proteins and tumor suppressor protein p53 and led to apoptotic cell death in a time-dependent manner. GlbA treatment also promoted the activation of Akt/PKB via phosphorylation at residue Ser 473 and induced autophagy as judged by the presence of the lipidated form of microtubule-associated protein 1 light chain 3 (LC3) and autophagosomes. Collectively, our data suggest that syrbactins belong to a new and effective proteasome inhibitor class which promotes cell death. Proteasome inhibition is a promising strategy for targeted anticancer therapy and syrbactins are a new class of inhibitors which provide a structural platform for the development of novel, proteasome inhibitor-based drug therapeutics.

New 5-Modified Pyrimidine Nucleoside Inhibitors of Mycobacterial Growth

The WHO has declared tuberculosis (TB) a global health emergency. Therefore, there is an urgent need to discover and develop new anti-TB drugs. Here we report a new category of 5-substituted pyrimidine nucleosides as potent inhibitors of Mycobacterium tuberculosis growth in vitro. A series of 2'-deoxy-, 3'-azido-2',3'-dideoxy-, and 3'-amino-2',3'-dideoxypyrimidine nucleoside analogues bearing lengthy flexible alkyloxymethyl substituents exhibited marked inhibitory activity against M tuberculosis in vitro. 5-Dodecyloxymethyl-2'-deoxyuridine was found to be a potent inhibitor of M. tuberculosis propagation in vitro. In contrast, monophosphates of the tested nucleosides were devoid of antimycobacterial activity. This new class of inhibitors seems to be a promising chemotherapeutic agent against TB and merits further studies.

New 5-Modified Pyrimidine Nucleoside Inhibitors of Mycobacterial Growth

The WHO has declared tuberculosis (TB) a global health emergency. Therefore, there is an urgent need to discover and develop new anti-TB drugs. Here we report on a new category of 5-substituted pyrimidine nucleosides as potent inhibitors of Myco-bacterium tuberculosis growth in vitro. A series of 2'-deoxy-, 3'-azido-2',3'-dideoxy-, and 3'-amino-2',3'-dideoxypyrimidine nucleoside analogues bearing lengthy flexible alkyloxymethyl substituents exhibited marked inhibitory activity against M. tuberculosis in vitro. 5-Dodecyloxymethyl-2'-deoxyuridine was found to be a potent inhibitor of M. tuberculosis propagation in vitro. In contrast, monophosphates of the tested nucleosides were devoid of antimycobacterial activity. This new class of inhibitors seems to be a promising chemotherapeutic agent against TB and merits further studies.

Activity of Isatine-Sulfadimidine Derivatives against 2009 Pandemic H1N1 Influenza Virus in Cell Culture

The development of antiviral drugs has provided crucial new means to mitigate or relieve the debilitating effects of many viral pathogens. New classes of inhibitors are essential to combat swine influenza viral infection. A series of isatine-sulfadimidine derivatives were screened for antiviral activity against swine influenza A/California/07/2009 (H1N1) virus in Madin-Darby canine kidney (MDCK) cell culture. Cytotoxicity of the synthesized compounds was also tested in uninfected MDCK cells. All the compounds inhibit the influenza A (H1N1) in MDCK cells. The most active compounds, SPIII-5Br and SPIII-5H, inhibited virus-induced cytopathology by 50% at 27 and 30 microM, respectively, with 50% cytotoxicity occurring at a much higher dose (975-1,000 microM). The positive control compound ribavirin inhibits the replication of the virus at 18 microM and cytotoxic concentration was found to be >1,000 microM. SPIII-5Br and SPIII-5H exhibited potency in the same range as ribavirin and are suitable candidate molecules for further investigation.

The Coumarin-Binding Site in Carbonic Anhydrase Accommodates Structurally Diverse Inhibitors: The Antiepileptic Lacosamide As an Example and Lead Molecule for Novel Classes of Carbonic Anhydrase Inhibitors

Coumarins constitute a general and totally new class of inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), binding at the entrance of the active site cavity. We report here that the coumarin-binding site in CAs may interact with diverse compounds, such as the antiepileptic drug lacosamide, which inhibits mammalian CAs I-XV, with inhibition constants in range of 331 nM to 4.56 microM. Its X-ray crystal structure in adduct with CA II reveals the molecular basis for this inhibition. Lacosamide was found in the coumarin-binding site, making favorable van der Waals interactions with Thr200, Asn67, Gln92, and Phe131. No interactions with the Zn(II) ion were evidenced in the CA II-lacosamide adduct. The coumarin-binding site may thus accommodate structurally diverse compounds which possess an inhibition mechanism distinct of that of sulfonamides. This finding opens new possibilities for designing CA inhibitors/activators with various biomedical applications.

Outsmarting a Mastermind

Moellering et al. recently reported that a cell-permeable "stapled" synthetic peptide of the Notch coactivator Mastermind is a potent dominant-negative inhibitor of oncogenic Notch signaling in T cell acute lymphoblastic leukemia. This new class of inhibitor may find broad utility in blocking protein-protein interactions that underlie many human diseases.

Solid-supported membrane technology for the investigation of the influenza A virus M2 channel activity

Influenza A virus encodes an integral membrane protein, A/M2, that forms a pH-gated proton channel that is essential for viral replication. The A/M2 channel is a target for the anti-influenza drug amantadine, although the effectiveness of this drug has been diminished by the appearance of naturally occurring point mutations in the channel pore. Thus, there is a great need to discover novel anti-influenza therapeutics, and, since the A/M2 channel is a proven target, approaches are needed to screen for new classes of inhibitors for the A/M2 channel. Prior in-depth studies of the activity and drug sensitivity of A/M2 channels have employed labor-intensive electrophysiology techniques. In this study, we tested the validity of electrophysiological measurements with solid-supported membranes (SSM) as a less labor-intensive alternative technique for the investigation of A/M2 ion channel properties and for drug screening. By comparing the SSM-based measurements of the activity and drug sensitivity of A/M2 wild-type and mutant channels with measurements made with conventional electrophysiology methods, we show that SSM-based electrophysiology is an efficient and reliable tool for functional studies of the A/M2 channel protein and for screening compounds for inhibitory activity against the channel.

Deciphering the Mechanism of Carbonic Anhydrase Inhibition with Coumarins and Thiocoumarins

Coumarin derivatives were recently shown to constitute a totally new class of inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), being hydrolyzed within the CA active site to 2-hydroxycinnamic acids. We explore here a new series of variously substituted coumarins and a thiocoumarin for their interaction with 13 mammalian CA isoforms, detecting low nanomolar and isoform selective inhibitors. The mechanism of action of this class of inhibitors is delineated in detail by resolving the X-ray crystal structure of CA II in complex with trans-2-hydroxy-cinnamic acid, the in situ hydrolysis product of simple coumarin. Thiocoumarins also act as efficient CAIs, similarly to coumarins. The versatility of the (thio)coumarin chemistry, the cis-trans isomerization evidenced here, and easy derivatization of the (thio)coumarin rings, coupled with the nanomolar inhibition range of several isozymes, afford isoform-selective CAIs with various biomedical applications, which render these classes of compounds superior to the clinically used sulfonamides.

Attacking the flu: New prospects for the rational design of antivirals

Attacking the flu: New prospects for the rational design of antivirals

Inhibitors for Human Glutaminyl Cyclase by Structure Based Design and Bioisosteric Replacement

The inhibition of human glutaminyl cyclase (hQC) has come into focus as a new potential approach for the treatment of Alzheimer's disease. The hallmark of this principle is the prevention of the formation of Abeta(3,11(pE)-40,42), as these Abeta-species were shown to be of elevated neurotoxicity and likely to act as a seeding core leading to an accelerated formation of Abeta-oligomers and fibrils. Starting from 1-(3-(1H-imidazol-1-yl)propyl)-3-(3,4-dimethoxyphenyl)thiourea, bioisosteric replacements led to the development of new classes of inhibitors. The optimization of the metal-binding group was achieved by homology modeling and afforded a first insight into the probable binding mode of the inhibitors in the hQC active site. The efficacy assessment of the hQC inhibitors was performed in cell culture, directly monitoring the inhibition of Abeta(3,11(pE)-40,42) formation.

Personalized Cancer Therapy With Selective Kinase Inhibitors: An Emerging Paradigm in Medical Oncology

Selective kinase inhibitors have emerged as an important class of anticancer agents, with demonstrated clinical efficacy and generally favorable toxicity profiles in several common disease settings where conventional treatments have previously provided only modest benefit. Consequently, a substantial effort is now underway to identify additional therapeutically relevant kinase targets and to develop and test inhibitors of those proteins in a variety of human malignancies. However, it has also become clear that the clinical benefit associated with these agents is typically limited to a subset of treated patients, who in many cases are defined by a specific genomic lesion within their tumor cells--frequently, an activating mutation within the gene encoding the target kinase. This discovery has prompted efforts to stratify patients before treatment with kinase inhibitors based on specific genomic biomarkers, with the goal of optimizing clinical outcomes through the effective personalization of treatment (ie, matching the right patients with the right therapies). With recent advances in our understanding of the relationship between tumor genotypes and cancer cell sensitivity to kinase inhibition, together with improved technologies for rapidly genotyping tumor biopsies for relevant lesions, the implementation of personalized cancer care with this exciting new class of inhibitors is now becoming a reality. In this review, we summarize recent developments in this area, and we highlight some of the logistical challenges posed by this emerging paradigm in medical oncology.

Terpenoids. III: Synthesis and biological evaluation of 23-hydroxybetulinic acid derivatives as novel inhibitors of glycogen phosphorylase

A series of 23-hydroxybetulinic acid derivatives were prepared and tested in vitro as a new class of inhibitors of glycogen phosphorylase (GP). Within this series of compounds, 12b (IC50=3.5μM) is the most potent GPa inhibitor. The preliminary SAR results of the 23-hydroxybetulinic acid derivatives are discussed.

Design, synthesis and structure–activity relationship of 2-(3′,4′,5′-trimethoxybenzoyl)-benzo[ b]furan derivatives as a novel class of inhibitors of tubulin polymerization

The biological importance of microtubules in mitosis and cell division makes them an interesting target for the development of anticancer agents. Small molecules such as benzo[ b]furans are attractive as inhibitors of tubulin polymerization. Thus, a new class of inhibitors of tubulin polymerization based on the 2-(3′,4′,5′-trimethoxybenzoyl)-benzo[ b]furan molecular skeleton, with electron-donating (Me, OMe or OH) or electron-withdrawing (F, Cl and Br) substituents on the benzene ring, was synthesized and evaluated for antiproliferative activity, inhibition of tubulin polymerization and cell cycle effects. Adding a methyl group at the C-3 position resulted in increased activity. The most promising compound in this series was 2-(3′,4′,5′-trimethoxybenzoyl)-3-methyl-6-ethoxy-benzo[ b]furan, which inhibits cancer cell growth at nanomolar concentrations and interacts strongly with tubulin by binding to the colchicine site.

2-Arylamino-4-Amino-5-Aroylthiazoles. “One-Pot” Synthesis and Biological Evaluation of a New Class of Inhibitors of Tubulin Polymerization

The essential role of microtubules in mitosis makes them a major target of compounds useful for cancer therapy. In our search for potent antitumor agents, a novel series of 2-anilino-4-amino-5-aroylthiazoles was synthesized and evaluated for antiproliferative activity, inhibition of tubulin polymerization, and cell cycle effects. SAR was elucidated with various substitutions on the phenylamino and aroyl moiety at the 2- and 5-positions, respectively, of the 4-aminothiazole skeleton. Tumor cell exposure to several of these compounds led to the arrest of HeLa cells in the G2/M phase of the cell cycle and induction of apoptosis.

Non-nucleoside inhibitors of HCV polymerase NS5B. Part 2: Synthesis and structure–activity relationships of benzothiazine-substituted quinolinediones

A new series of benzothiazine-substituted quinolinediones were evaluated as inhibitors of HCV polymerase NS5B. SAR studies on this series revealed a methyl sulfonamide group as a high affinity feature. Analogues with this group showed submicromolar potencies in the HCV cell based replicon assay. Pharmacokinetic and toxicology studies were also performed on a selected compound ( 34) to evaluate in vivo properties of this new class of inhibitors of HCV NS5B polymerase.

A new class of UDP-3-O-(R-3-hydroxymyristol)-N-acetylglucosamine deacetylase (LpxC) inhibitors for the treatment of Gram-negative infections: PCT application WO 2008027466

Background: Human infections due to Gram-negative bacteria cause significant morbidity and mortality. Identification of new strategies, molecular targets, and agents for the treatment of Gram-negative bacterial infections are needed urgently. Lipid A is a necessary component of the lipopolysaccharide-containing outer membrane of Gram-negative bacteria. The zinc-dependent hydrolase UDP-3-O-(R-3-hydroxymyristol)-N-acetylglucosamine deacetylase (LpxC) involved in the first committed step in the biosynthetic pathway of lipid A has no sequence homology to any known mammalian enzymes and has emerged as an attractive Gram-negative antibacterial molecular target. Most previously described LpxC inhibitors contain a hydroxamic acid, which can lead to low specificity vs. other metal-dependent enzymes and can consequently result in unwanted side effects. Objective: This review examines a new reported class of nonhydroxamic LpxC inhibitors for the treatment of Gram-negative infections. Methods: The new class of inhibitor is compared with several previously reported LpxC inhibitors. Conclusion: The LpxC inhibitors disclosed in PCT application WO 2008027466 contain hydantoins in place of the hydroxamic acids commonly found in most previously described inhibitors. These molecules could represent a means of treating Gram-negative infections via a more selective inhibition of LpxC.

Identification of bisindolylmaleimides and indolocarbazoles as inhibitors of HCV replication by tube-capture-RT-PCR

We devised a screening method for hepatitis C virus (HCV) inhibitors by exploiting the JFH1 viral culture system. The viral RNA released in the medium was adsorbed onto PCR plates, and real-time RT-PCR was performed by directly adding the one-step RT-PCR reaction mixture to the wells. The “tube-capture-RT-PCR” method obviates the need for labor-intensive RNA isolation and should allow high-throughput screening of HCV inhibitors. To substantiate the validity of the assay for drug screening, a pilot screen of an inhibitor library composed of 95 compounds was performed. In addition to the known inhibitors of HCV replication included in the library, the assay identified the PKC inhibitor bisindolylmaleimide I (BIM I) as an HCV replication inhibitor. BIM I was also effective in reducing the viral protein level in genotype 1b and 2a subgenomic replicon cells, indicating inhibition of HCV replication. Further assays revealed that a broad range of bisindolylmaleimides and indolocarbazoles inhibit HCV, but no correlation was found between the PKC inhibition pattern and anti-HCV activity. These series of compounds represent new classes of inhibitors that may warrant further development.

Research Highlights

Research Highlights