The anti-mitochondrial antifungal assay for the discovery and development of new drugs

Introduction: New targets and drugs are constantly searched for to effectively combat fungal infections and diseases such as cancer. Mitochondria, as the main powerhouses of eukaryotic cells, must be regarded as important targets for the development of new therapies. This has lead to the development of a fungal assay that shows potential in the selection of new antifungal and anticancer drugs as well as the identification of compounds that are toxic to human mitochondria.

Areas covered: In this review the authors discuss the development of a potential method of drug discovery that targets mitochondrial function. The authors cover the application of new nanotechnology as well as fungal systematic research where the link between fungal fruiting structures, cell growth, increased mitochondrial activity and susceptibility to a variety of anti-mitochondrial drugs is assessed.

Expert opinion: This assay shows potential to select anti-mitochondrial drugs as a first screen. This should be followed up by more specific in vitro and in vivo tests to pinpoint the type of anti-mitochondrial activity exerted by these drugs, if any. This is because the possibility exists that compounds regarded as anti-mitochondrial may not inhibit mitochondrial function but other fruiting structure developmental stages and therefore yield false positives. To enhance our knowledge on how these drugs act at the structural level, the authors recommend Nano Scanning Auger Microscopy as the tool of choice.     

The anti-mitochondrial antifungal assay for the discovery and development of new drugs

Introduction: New targets and drugs are constantly searched for to effectively combat fungal infections and diseases such as cancer. Mitochondria, as the main powerhouses of eukaryotic cells, must be regarded as important targets for the development of new therapies. This has lead to the development of a fungal assay that shows potential in the selection of new antifungal and anticancer drugs as well as the identification of compounds that are toxic to human mitochondria. Areas covered: In this review the authors discuss the development of a potential method of drug discovery that targets mitochondrial function. The authors cover the application of new nanotechnology as well as fungal systematic research where the link between fungal fruiting structures, cell growth, increased mitochondrial activity and susceptibility to a variety of anti-mitochondrial drugs is assessed. Expert opinion: This assay shows potential to select anti-mitochondrial drugs as a first screen. This should be followed up by more specific in vitro and in vivo tests to pinpoint the type of anti-mitochondrial activity exerted by these drugs, if any. This is because the possibility exists that compounds regarded as anti-mitochondrial may not inhibit mitochondrial function but other fruiting structure developmental stages and therefore yield false positives. To enhance our knowledge on how these drugs act at the structural level, the authors recommend Nano Scanning Auger Microscopy as the tool of choice.     

Structure-activity relationship studies of phenanthridine-based Bcl-XL inhibitors

Despite their structural similarities, the natural products chelerythrine ( 5) and sanguinarine ( 6) target different binding sites on the pro-survival Bcl-X L protein. This paper details the synthesis of phenanthridine-based analogues of the natural products that were used to probe this difference in binding profiles. The inhibitory constants for these compounds were then measured in a fluorescence polarization assay against Bcl-X L and the tagged Bak-BH3 peptide. The results led to structure-activity relationship studies, which identified the structural motifs required for binding-site specificity as well as inhibitory activity. We also identified synthetic analogues of the natural products that display similar binding modes but with more potent IC 50 values.     

A multifunctional cross-validation high-throughput screening protocol enabling the discovery of new SHP2 inhibitors

The protein tyrosine phosphatase Src homology phosphotyrosyl phosphatase 2 (SHP2) is implicated in various cancers, and targeting SHP2 has become a promising therapeutic approach. We herein described a robust cross-validation high-throughput screening protocol that combined the fluorescence-based enzyme assay and the conformation-dependent thermal shift assay for the discovery of SHP2 inhibitors. The established method can effectively exclude the false positive SHP2 inhibitors with fluorescence interference and was also successfully employed to identify new protein tyrosine phosphatase domain of SHP2 (SHP2-PTP) and allosteric inhibitors. Of note, this protocol showed potential for identifying SHP2 inhibitors against cancer-associated SHP2 mutation SHP2-E76A. After initial screening of our in-house compound library (∼2300 compounds), we identified 4 new SHP2-PTP inhibitors (0.17% hit rate) and 28 novel allosteric SHP2 inhibitors (1.22% hit rate), of which SYK-85 and WS-635 effectively inhibited SHP2-PTP (SYK-85: IC₅₀ = 0.32 μmol/L; WS-635: IC₅₀ = 4.13 μmol/L) and thus represent novel scaffolds for designing new SHP2-PTP inhibitors. TK-147, an allosteric inhibitor, inhibited SHP2 potently (IC₅₀ = 0.25 μmol/L). In structure, TK-147 could be regarded as a bioisostere of the well characterized SHP2 inhibitor SHP-099, highlighting the essential structural elements for allosteric inhibition of SHP2. The principle underlying the cross-validation protocol is potentially feasible to identify allosteric inhibitors or those inactivating mutants of other proteins.KEY WORDS: SHP2, High-throughput screening, Enzyme assay, Thermal shift assay, Allosteric inhibitors     

The discovery of new cholesteryl ester transfer protein inhibitors

Cholesteryl ester transfer protein (CETP) has been an important but controversial target for elevating HDLc (high density lipoprotein cholesterol) and treating atherosclerosis. Significant progress toward inhibiting CETP has occurred on several fronts, including the development of an antisense inhibitor, irreversible small molecule inhibitors and reversible small molecule inhibitors. Several orally bioavailable, small molecule CETP inhibitors have shown potential to improve the HDLc to LDLc (low density lipoprotein cholesterol) ratio in various animal models at reasonable doses, and one of these compounds has shown efficacy in preventing atherosclerosis in a rabbit model. However, several more years of clinical testing will likely be needed to demonstrate that these clinical candidates can provide a potential therapeutic benefit to patients with coronary artery disease.     

Pharmacophore modeling and virtual screening for the discovery of new fatty acid amide hydrolase inhibitors

A predictive pharmacophore model has been generated from a series of diverse fatty acid amide hydrolase (FAAH) inhibitors and the optimal pharmacophore model applied in virtual screening. The pharmacophore model was based on a training set of 21 compounds carefully selected from the published literatures. The optimal model Hypo-1 included four features (two hydrogen-bond acceptor units, one aromatic hydrophobic unit and one aromatic ring unit) and two excluded volumes. Cross-validation of the model confirmed that Hypo-1 was not generated by chance correlation. A large test set of 55 compounds showed that Hypo-1 performed well in classifying highly active and less active FAAH inhibitors. Superimposition analysis of the FAAH X-ray crystal structure and the pharmacophore Hypo-1 further validated the adequacy of the model. Virtual screening generated a total of 976 hits from the Zinc Natural Products database, a hit rate of 1.04% and enrichment of 83.89. The acceptable hit rate further supports the use of Hypo-1 as a 3D query tool for virtual screening.     

A cell-based assay for screening spindle checkpoint inhibitors

In eukaryotes, the spindle checkpoint acts as a surveillance mechanism that ensures faithful chromosome segregation. The spindle checkpoint prevents premature separation of sister chromatids and the onset of anaphase until every chromosome is properly attached to the mitotic spindle. Tumorigenesis might result from generation of aneuploidy by dysfunction of the spindle checkpoint. Differences of the checkpoint system in normal cells versus tumor cells might provide a new opportunity in cancer drug development; therefore, efforts to identify the spindle checkpoint inhibitors have been fostered. Based on spindle checkpoint inhibitors being able to induce cells to exit mitotic arrest caused by microtubule drug treatment, we developed a cell-based assay to screen compounds that were potential spindle checkpoint inhibitors. This assay was validated with a known spindle checkpoint inhibitor and was easy to adapt to a large-scale screening. It also had the advantages of being high in sensitivity and low in cost.     

(Na+-K+)-ATPase inhibitors: Implications for new drug discovery

The (Na⁺-K⁺)-ATPase is a ubiquitous membrane-bound enzyme that actively transports Na⁺ out of the cell in exchange for a smaller ratio of extracellular K⁺. The current report focuses on the role of modifiers of (Na⁺-K⁺)-ATPase activity in the development of new pharmacological agents. More versatile biological test systems are proposed. Possible use of Na⁺-K⁺-ATPase activity modulators in treatment of hypertension and other diseases are discussed. It is concluded that the ubiquitous distribution and crucial role of the enzyme in normal and diseased cell function merits a critical biological and chemical reappraisal of the enzyme beyond the current narrow viewpoint dealing with the application of glycosides in the treatment of congestive heart failure.     

A new structural class of proteasome inhibitors identified by microbial screening using yeast-based assay

A yeast-based growth interference assay was developed utilizing a yeast strain in which expression of Xenopus cyclin A1 was induced to elevate cell division cycle 28 (Cdc28) kinase activity. Since the hyperactivation of Cdc28 kinase in yeast results in a growth-arrest phenotype, compounds which could rescue the cyclin A1-induced growth arrest might be potential new, antitumor drug candidates acting on the cyclin-dependent, kinase-mediated, cell cycle regulation pathway. In the course of our microbial screening program, the new Streptomyces metabolites, belactosins, were identified. As reported previously, belactosin A induced cell cycle arrest at G2/M phase in human cancer cells. However, the molecular mechanism of action was unknown. We herein demonstrate the proteasome inhibition by belactosin A. Belactosin A did not inhibit yeast Cdc28 kinase and human cyclin-dependent kinase in vitro. On the other hand, it inhibited the chymotrypsin-like activity of the rabbit 20S proteasome. From the initial SAR studies, we identified a hydrophobic belactosin A derivative, KF33955, which exhibited a 100-fold greater growth-inhibitory activity against HeLa S3 cells than belactosin A, presumably due to its higher cell permeability. The biochemical analysis using KF33955 suggested that the proteasome inhibitory activity of KF33955 were irreversible and required the beta-lactone moiety to inhibit the proteasome. KF33955 increased the intracellular levels of protein ubiquitination in NIH3T3 cells. In addition, KF33955 treatment resulted in the accumulation of known proteasome substrates in HeLa S3 cells. These results identify belactosin A as a useful lead compound to target proteasome for the treatment of disease whose etiology is dependent on the unregulated ubiquitin-proteasome pathway.     

Recent advances and new directions in the discovery and development of cyclin-dependent kinase inhibitors

The eukaryotic cell division cycle is coordinated by cyclin-dependent protein kinases (CDKs) and cyclin subunits specific for the different phases of the cycle. These complexes phosphorylate target substrates, including the retinoblastoma susceptibility gene product (pRb) and related proteins. Cellular neoplastic transformations are accompanied by loss of regulation of cell cycle checkpoints, frequently through aberrant expression of CDKs and cyclins, as well as loss or mutation of their negative regulators. Consequently, one strategy in the development of mechanism-based anticancer therapeutics has been to halt malignant cellular proliferation through inhibition of the enzymatic activity of CDKs. The development of inhibitors selective for the ATP binding sites of particular protein kinases is a comparatively recent medicinal chemistry endeavor. Advances relevant to CDK inhibition are reviewed critically and alternative approaches to CDK inhibition, as well as applications of CDK inhibitors to therapeutic areas other than oncology, are also discussed.     

Web support for the more efficient discovery of kinase inhibitors

Kinases have a crucial role in cell signaling and are important drug targets, given that aberrant kinase activity has been linked to most disease areas. Therefore, kinase inhibitors (KIs) have significant potential as new therapeutics. In recent years, an increasing amount of computational resources have been developed to design ideal scaffold and selective KIs more efficiently. Thus, in this review, we systematically examine the computational tools used in KI research, and discuss and compare the characteristics and limitations of these resources. Such a discussion will facilitate the design of new KIs and provide a learning platform for nonspecialists.     

Pharmacophore modeling technique applied for the discovery of proteasome inhibitors

Introduction: The 26S proteasome has many important roles in the biological functions of the cells, and proteasome inhibitors have multiple and complex activities on cells. These compounds can be natural or synthesized. Most synthetic derivatives have been rationally designed, synthesized and optimized to obtain the best selectivity and increase the activity. The design of chemical entities with desired molecular identification, which plays an important role in biological systems, is provided by pharmacophore modeling. Indeed, pharmacophore models can be established either in a ligand-based manner or in a receptor-based manner.

Areas covered: The authors discuss the application of pharmacophore modeling techniques to proteasome inhibitors development. Furthermore, the article reviews the classification of the currently discovered proteasome inhibitors where the principal mechanism of action and clinical application are represented.

Expert opinion: In the era of new drug development, database of compounds should be thoroughly evaluated with a combination of methods that consider both pharmacophore- and ligand-based virtual screening. The concept of pharmacophore helps to discover new active compounds and to evaluate their activity. The nature of proteasome inhibitor pharmacophore affects the secondary active-site specificity; indeed, increasing specificity decreases the cytotoxicity of the proteasome inhibitors. It is hypothesized that the balanced simultaneous modulation of a few druggable targets may have superior efficacy and fewer side effects than single-target or combination therapies for the treatment of human cancers. The discovery of new compounds should aim to find more active compounds that improve the compliance of patients.     

Contemporary medicinal-chemistry strategies for the discovery of selective butyrylcholinesterase inhibitors

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New methods for the discovery and synthesis of PDE7 inhibitors as new drugs for neurological and inflammatory disorders

Introduction: Phosphodiesterase 7 (PDE7) is a high-affinity cyclic AMP (cAMP)-specific PDE that is expressed in immune and proinflammatory cells. The PDE7 recently emerged a pharmacological target in the context of the immune and neurological responses to alleviate chronic inflammation and neurodegenerative disorders.

Areas covered: This review explains PDE7 as a therapeutic target for neurological and inflammation disorders. Additionally, specification of PDE7 inhibitors, functional diversity between PDE7A and 7B, inhibitor selectivity to PDE7 isoforms, and new discovery methods for PDE7 inhibitors such as synthesis, quantitative structure activity relationship (QSAR) studies, ligand-based virtual screening, and structure-based screening (docking) are discussed.

Expert opinion: There are only a few selective PDE7 inhibitors that can discriminate between PDE7A and PDE7B, but due to different tissue distribution and physiological functions in the body, the hope is to develop selective PDE7A and PDE7B inhibitors to target more specific functions and pathological conditions without a high likelihood of causing nonspecific side effects. Conventional approaches such as synthesis and QSARs are currently used to find specific PDE7 inhibitors. The development of computational-based screening of databases has provided many opportunities to discover new ligands based on biological activity in a very short period.     

Progress in the discovery of BACE inhibitors

Dementia caused by Alzheimer's disease is a large medical burden on society in the developed world. Current treatments are largely symptomatic, and there is an urgent need for therapies which can interrupt or reverse the progression of disease. A number of strategies for intervention are being actively pursued; among the most promising is the inhibition of beta-secretase, or BACE. BACE is the enzyme responsible for N-terminal cleavage of the Alzheimer's precursor protein leading to the production of the beta-amyloid peptide. This cascade ultimately leads to the formation of amyloid plaques, one of the hallmark lesions of the disease. It is expected that inhibitors of BACE may therefore serve as an effective disease-modifing therapy for the treatment of AD. This concept has received significant attention by both academics and the pharmaceutical industry. This review focuses on a discussion of the reported structure-activity relationships for inhibitors of this important therapeutic target.