Small molecules enhance autophagy and reduce toxicity in Huntington's disease models

The target of rapamycin proteins regulate various cellular processes including autophagy, which may play a protective role in certain neurodegenerative and infectious diseases. Here we show that a primary small-molecule screen in yeast yields novel small-molecule modulators of mammalian autophagy. We first identified new small-molecule enhancers (SMER) and inhibitors (SMIR) of the cytostatic effects of rapamycin in Saccharomyces cerevisiae. Three SMERs induced autophagy independently of rapamycin in mammalian cells, enhancing the clearance of autophagy substrates such as mutant huntingtin and A53T alpha-synuclein, which are associated with Huntington's disease and familial Parkinson's disease, respectively. These SMERs, which seem to act either independently or downstream of the target of rapamycin, attenuated mutant huntingtin-fragment toxicity in Huntington's disease cell and Drosophila melanogaster models, which suggests therapeutic potential. We also screened structural analogs of these SMERs and identified additional candidate drugs that enhanced autophagy substrate clearance. Thus, we have demonstrated proof of principle for a new approach for discovery of small-molecule modulators of mammalian autophagy.     

Emerging roles of epigenetic mechanisms in Parkinson’s disease

Epigenetic mechanisms have emerged as important components of a variety of human diseases, including cancer and central nervous system disorders. Despite recent studies highlighting the role of epigenetic mechanisms in several neurodegenerative and neuropsychiatric disorders, to date, there has been a paucity of studies exploring the role of epigenetic factors in Parkinson’s disease (PD). PD is a progressive neurological disorder with characteristic motor and non-motor symptoms, including a range of neuropsychiatric features, for which neither preventative nor effective long-term treatment strategies are available. It is one of the most common neurodegenerative disorders and the second most prevalent after Alzheimer’s disease. In this review, we present several lines of evidence suggesting that epigenetic factors may play an important role in the pathogenesis of PD and propose on this basis a framework to guide future investigations into epigenetic mechanisms and systems biology of PD. These notions, together with technical advances in the ability to perform genome-wide analysis of epigenomic states, and newly available small-molecule probes targeting chromatin-modifying enzymes, may help design new treatment strategies for PD and other human diseases involving epigenetic dysregulation.     

Discovery and characterization of orthosteric and allosteric muscarinic M2 acetylcholine receptor ligands by affinity selection-mass spectrometry

Screening assays using target-based affinity selection coupled with high-sensitivity detection technologies to identify small-molecule hits from chemical libraries can provide a useful discovery approach that complements traditional assay systems. Affinity selection-mass spectrometry (AS-MS) is one such methodology that holds promise for providing selective and sensitive high-throughput screening platforms. Although AS-MS screening platforms have been used to discover small-molecule ligands of proteins from many target families, they have not yet been used routinely to screen integral membrane proteins. The authors present a proof-of-concept study using size exclusion chromatography coupled to AS-MS to perform a primary screen for small-molecule ligands of the purified muscarinic M2 acetylcholine receptor, a G-protein-coupled receptor. AS-MS is used to characterize the binding mechanisms of 2 newly discovered ligands. NGD-3350 is a novel M2-specific orthosteric antagonist of M2 function. NGD-3366 is an allosteric ligand with binding properties similar to the allosteric antagonist W-84, which decreases the dissociation rate of N-methyl-scopolamine from the M2 receptor. Binding properties of the ligands discerned from AS-MS assays agree with those from in vitro biochemical assays. The authors conclude that when used with appropriate small-molecule libraries, AS-MS may provide a useful high-throughput assay system for the discovery and characterization of all classes of integral membrane protein ligands, including allosteric modulators.     

Novel symmetrical ureas as modulators of protein arginine methyl transferases

Methylation of histone arginine residues is an epigenetic mark related to gene expression that is implicated in a variety of biological processes and can be reversed by small-molecule modulators of protein arginine methyltransferases (PRMTs). A series of symmetrical ureas, designed as analogues of the known PRMT1 inhibitor AMI-1 have been synthesized using Pd-catalyzed Ar–N amide bond formation processes or carbonylation reactions as key steps. Their inhibitory profile has been characterized. The enzymatic assays showed a weak effect on PRMT1 and PRMT5 activity for most of the compounds. The acyclic urea that exhibited the strongest effect on the inhibition of the PRMT1 activity also showed the greatest effect on the expression of some androgen receptor target genes (TMPRSS2 and FKBP5), which may be related with its enzymatic activity. Surprisingly, AMI-1 behaved as an activator of PRMT5 activity, a result not reported so far.     

Targeted therapies in epithelial ovarian cancer: Molecular mechanisms of action

Ovarian cancer is the leading cause of death in women with gynecological cancer. Most patients are diagnosed at an advanced stage and have a poor prognosis. Currently, surgical tumor debulking, followed by platinum- and taxane-based chemotherapy is the standard treatment for advanced ovarian cancer. However, these patients are at great risk of recurrence and emerging drug resistance. Therefore, novel treatment strategies are required to improve outcomes for women with advanced ovarian cancer. A variety of molecular targeted agents, the majority of which are monoclonal antibodies and small-molecule protein-kinase inhibitors, have been explored in the management of ovarian cancer. The targets of these agents include angiogenesis, the human epidermal growth factor receptor family, ubiquitin-proteasome pathway, epigenetic modulators, poly(ADP-ribose) polymerase (PARP), and mammalian target of rapamycin (mTOR) signaling pathway, which are aberrant in tumor tissue. The antiangiogenic agent, bevacizumab, has been reported as the most effective targeted agent and should be included in the standard chemotherapeutic regimen for advanced ovarian cancer. PARP inhibitors, which are mainly used in breast and ovarian cancer susceptibility gene-mutated patients, and mTOR inhibitors are also attractive treatment strategies, either alone or combination with chemotherapy, for ovarian cancer. Understanding the tumor molecular biology and identification of predictive biomarkers are essential steps for selection of the best treatment strategies. This article reviews the molecular mechanisms of the most promising targeted agents that are under early phase clinical evaluation for ovarian cancer.     

Surface plasmon resonance biosensor assay for the analysis of small-molecule inhibitor binding to human and parasitic phosphodiesterases

In the past decade, surface plasmon resonance (SPR) biosensor-based technology has been exploited more and more to characterize the interaction between drug targets and small-molecule modulators. Here, we report the successful application of SPR methodology for the analysis of small-molecule binding to two therapeutically relevant cAMP phosphodiesterases (PDEs), Trypanosoma brucei PDEB1 which is implicated in African sleeping sickness and human PDE4D which is implicated in a plethora of disease conditions including inflammatory pulmonary disorders such as asthma, chronic obstructive pulmonary disease and central nervous system (CNS) disorders. A protocol combining the use of directed capture using His-tagged PDE_CDs with covalent attachment to the SPR surface was developed. This methodology allows the determination of the binding kinetics of small-molecule PDE inhibitors and also allows testing their specificity for the two PDEs. The SPR-based assay could serve as a technology platform for the development of highly specific and high-affinity PDE inhibitors, accelerating drug discovery processes.     

High-throughput screening for small-molecule adiponectin secretion modulators

Adiponectin is an adipokine secreted by adipocytes and plays a role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. Several studies have shown that upregulation of adiponectin has a number of therapeutic benefits. Although peroxisome proliferator-activated receptor γ (PPARγ) agonists are known to increase adiponectin secretion both in cultured adipocytes and humans, they have several side effects, such as weight gain, congestive heart failure, and edema. Therefore, adiponectin secretion modulators that do not possess PPARγ agonistic activity seem to promising for a number of conditions. Here, the authors report on the development of a reporter-based high-throughput screening (HTS) assay using insulin-resistant-mimic 3T3-L1 adipocytes for discovery of adiponectin secretion modulators. They screened a library of approximately 100 000 small-molecule compounds using this model, performed several follow-up screens, and identified six hit compounds that increase adiponectin secretion without having PPARγ agonistic activity. These compounds may be useful drug candidates for diabetes, obesity, atherosclerosis, and other metabolic syndromes. This HTS assay might be applicable to screening for other adipokine modulators that can be useful for the treatment of other conditions.     

发现靶向蛋白质间相互作用的小分子药物研究进展

蛋白质-蛋白质相互作用在多种细胞功能中具有重要的作用。靶向蛋白质-蛋白质相互作用已经成为新药发现的重要策略,但发现能阻断蛋白质-蛋白质相互作用的小分子药物是一个巨大的挑战。尽管如此,近年来人们还是发现了许多能调控蛋白质-蛋白质相互作用的小分子。该文主要总结了在病毒进入、细胞凋亡通路和神经退行性疾病等方面的蛋白质-蛋白质相互作用小分子抑制剂的研究进展。     

Adenylyl cyclase isoforms as novel therapeutic targets: an exciting example of excitotoxicity neuroprotection

The use of transgenic animals lacking one or multiple adenylyl cyclase (AC) isoforms has provided significant information on the roles of AC-dependent signaling in the central nervous system. A recent study provides evidence that AC type 1 (AC1) might be important in glutamate-induced neuronal toxicity. However, the absolute AC specificity revealed in this study contrasts earlier work examining other forms of neurodegeneration. Nonetheless, these observations suggest that specific AC isoforms may represent novel targets for the treatment of central nervous system disorders. It is anticipated that such findings will help catalyze new drug discovery efforts to identify small-molecule modulators of individual AC isoforms.     

Resorufin butyrate as a soluble and monomeric high-throughput substrate for a triglyceride lipase

Triglyceride lipases such as lipoprotein lipase, endothelial lipase, and hepatic lipase play key roles in controlling the levels of plasma lipoprotein. Accordingly, small-molecule modulation of these species could alter patient lipid profiles with corresponding health effects. Screening of these enzymes for small-molecule therapeutics has historically involved the use of lipid-based particles to mimic native substrates. However, particle-based artifacts can complicate the discovery of therapeutic molecules. As a simplifying solution, the authors sought to develop an approach involving a soluble and monomeric lipase substrate. Using purified bovine lipoprotein lipase as a model system, they show that the hydrolysis of resorufin butyrate can be fluorescently monitored to give a robust assay (Z' > 0.8). Critically, using parallel approaches, they show that resorufin butyrate is soluble and monomeric under assay conditions. The presented assay should be useful as a simple and inexpensive primary or secondary screen for the discovery of therapeutic lipase modulators.     

The Multifaceted Roles of USP7: New Therapeutic Opportunities

The deubiquitylating enzyme USP7 (HAUSP) sits at a critical node regulating the activities of numerous proteins broadly characterized as tumor suppressors, DNA repair proteins, immune responders, viral proteins, and epigenetic modulators. Aberrant USP7 activity may promote oncogenesis and viral disease making it a compelling target for therapeutic intervention. Disclosed drug discovery programs have identified inhibitors of USP7 such as P005091 with cellular proof of concept and anti-proliferative activity in cancer models. Taken together, USP7 inhibitors hold promise as a new strategy for the treatment of disease.     

Drug discovery beyond the 'rule-of-five'

Although a very useful guideline for orally bioavailable small-molecule drug design, the 'rule-of-five' (also known as 'Lipinski's rule of drug-likeness') has to some extent been overemphasized. Firstly, only 51% of all FDA-approved small-molecule drugs are both used orally and comply with the 'rule-of-five'. This does not even include the increasing number of biologicals of which several have reached 'blockbuster' status. Secondly, it does not cover natural product and semisynthetic natural product drugs, which constitute over one-third of all marketed small-molecule drugs. A more balanced and programmatic approach to drug discovery should be more productive than to rely on an overemphasis of 'rule-of-five' compliance. Rather it should consider proactively the development of parenteral drugs in parallel to oral drugs and to consider the development of therapeutic antibodies in parallel to small-molecule drugs. These are particularly relevant for efforts against 'first-in-class' and/or particularly challenging targets such as proteases and those involving protein-protein interactions. In addition, more effort should be invested in natural product research. Emerging novel technologies such as synthetic biology (genetic engineering of living organisms to produce small-molecule therapeutics) may address several challenging issues of natural product-based drug discovery including synthetic feasibility and ligand efficiency.     

Pharmacology of voltage-gated and calcium-activated potassium channels.

Several important new findings have furthered the development of voltage-gated and calcium-activated potassium channel pharmacology. The molecular constituents of several members of these large ion channel families were identified. Small-molecule modulators of some of these channels were reported, including correolide, the first potent, small-molecule, natural product inhibitor of the Shaker family of voltage-gated potassium channels to be disclosed. The initial crystal structure of a bacterial potassium channel was determined; this work gives a physical basis for understanding the mechanisms of ion selectivity and ion conduction. With the recent molecular characterization of a potassium channel structure and the discovery of new templates for channel modulatory agents, the ability to rationally identify and develop potassium channel agonists and antagonists may become a reality in the near future.     

Development of dimeric modulators for anti-apoptotic Bcl-2 proteins

Bcl-2 family proteins can be classified into two subfamilies--anti-apoptotic members and pro-apoptotic members. Mechanistically, these two subfamilies can antagonize each other through heterodimerization while homodimerization has been proposed for each subfamily to carry out their corresponding anti-apoptotic or pro-apoptotic functions. To date, many small-molecule antagonists against anti-apoptotic Bcl-2 proteins have been developed, which are monomeric modulators. In this study, a series of BH3I-1 based dimeric modulators were developed with structure-activity relationship explored. Dimeric modulators compared to the monomeric antagonists have enhanced binding activity against anti-apoptotic Bcl-2 proteins. In addition, the acidic functional group was demonstrated to be critical for the binding interaction of the small-molecule antagonists with anti-apoptotic Bcl-2 proteins. Finally, the representative dimeric modulator revealed enhanced activity in inducing cytochrome c release from mitochondria compared to its monomeric counterpart. Taken together, dimerization of monomeric modulators is one practical approach to enhance the bioactivity of Bcl-2 antagonists.     

Membrane anchoring γ-secretase modulators with terpene-derived moieties

Modulation of γ-secretase activity is a promising therapeutic strategy for the treatment of Alzheimer’s disease. Herein we report on the synthesis of carprofen- and tocopherol-derived small-molecule modulators carrying terpene moieties as lipophilic membrane anchors. Additionally, these modulators are equipped with an acidic moiety, which contributes to the desired modulatory effect on the γ-secretase with decreased formation of Aβ₄₂ and increased Aβ₃₈ production.     

The Sirtuin family: therapeutic targets to treat diseases of aging

Sirtuins have emerged as therapeutic targets to treat age-related diseases. There are seven human Sirtuins (SIRT1-7) that display diversity in cellular localization and function. Growing evidence suggests that small-molecule activators of SIRT1 may counteract age-related afflictions such as type 2 diabetes. Alternatively, inhibitors of SIRT2 may be useful in the treatment of neurodegenerative diseases such as Parkinson's disease. Recent discoveries of small-molecule and protein modulators of Sirtuin deacetylation activity have provided enormous insight into the biological and molecular functions of Sirtuins and have validated their potential as therapeutics.     

Aptamers as reagents for high-throughput screening

The identification of new drug candidates from chemical libraries is a major component of discovery research in many pharmaceutical companies. Given the large size of many conventional and combinatorial libraries and the rapid increase in the number of possible therapeutic targets, the speed with which efficient high-throughput screening (HTS) assays can be developed can be a rate-limiting step in the discovery process. We show here that aptamers, nucleic acids that bind other molecules with high affinity, can be used as versatile reagents in competition binding HTS assays to identify and optimize small-molecule ligands to protein targets. To illustrate this application, we have used labeled aptamers to platelet-derived growth factor B-chain and wheat germ agglutinin to screen two sets of potential small-molecule ligands. In both cases, binding affinities of all ligands tested (small molecules and aptamers) were strongly correlated with their inhibitory potencies in functional assays. The major advantages of using aptamers in HTS assays are speed of aptamer identification, high affinity of aptamers for protein targets, relatively large aptamer-protein interaction surfaces, and compatibility with various labeling/detection strategies. Aptamers may be particularly useful in HTS assays with protein targets that have no known binding partners such as orphan receptors. Since aptamers that bind to proteins are often specific and potent antagonists of protein function, the use of aptamers for target validation can be coupled with their subsequent use in HTS.     

3D models of epithelial-mesenchymal transition in breast cancer metastasis: high-throughput screening assay development, validation, and pilot screen

Despite advancements in therapies developed for the treatment of cancer, patient prognosis and mortality rates have improved minimally, and metastasis remains the primary cause of cancer mortality worldwide. An underlying mechanism promoting metastasis in many types of cancer is epithelial-mesenchymal transition (EMT). Here the authors report a novel 3D model of EMT and metastatic breast cancer suitable for high-throughput screening (HTS) drug discovery. The primary assay incorporates the expression of the prognostic biomarker vimentin, as a luciferase reporter of EMT, in basil-like/triple-negative MDA-MB-231 breast carcinoma spheroids. Using this model, the authors developed a number of known antitumor agents as control modulators of EMT. U0126, PKC412, PF2341066, dasatinib, and axitinib downregulated vimentin expression by 70% to 90% as compared to untreated spheroids. Counterassays were developed to measure spheroid viability and the invasive potential of MDA-MB-231 spheroids after small-molecule treatment and used to confirm hits from primary screening. Finally, the authors conducted a pilot screen to validate this model for HTS using a purified library of marine secondary metabolites. From 230 compounds screened, they obtained a Z' score of 0.64, indicative of an excellent assay, and confirmed 4 hits, including isonaamidine B, papuamine, mycalolide E, and jaspamide. This HTS model demonstrates the potential to identify small-molecule modulators of EMT that could be used to discover novel antimetastatic agents for the treatment of cancer.