Good Drug Target Research Articles

Lipid A as a Drug Target and Therapeutic Molecule.

In this review, lipid A, from its discovery to recent findings, is presented as a drug target and therapeutic molecule. First, the biosynthetic pathway for lipid A, the Raetz pathway, serves as a good drug target for antibiotic development. Several assay methods used to screen for inhibitors of lipid A synthesis will be presented, and some of the promising lead compounds will be described. Second, utilization of lipid A biosynthetic pathways by various bacterial species can generate modified lipid A molecules with therapeutic value.

Advances in Bacterial Methionine Aminopeptidase Inhibition.

Methionine aminopeptidases (MetAPs) are metalloenzymes that cleave the N-terminal methionine from newly synthesized peptides and proteins. These MetAP enzymes are present in bacteria, and knockout experiments have shown that MetAP activity is essential for cell life, suggesting that MetAPs are good antibacterial drug targets. MetAP enzymes are also present in the human host and selectivity is essential. There have been significant structural biology efforts and over 65 protein crystal structures of bacterial MetAPs are deposited into the PDB. This review highlights the available crystallographic data for bacterial MetAPs. Structural comparison of bacterial MetAPs with human MetAPs highlights differences that can lead to selectivity. In addition, this review includes the chemical diversity of molecules that bind and inhibit the bacterial MetAP enzymes. Analysis of the structural biology and chemical space of known bacterial MetAP inhibitors leads to a greater understanding of this antibacterial target and the likely development of potential antibacterial agents.

Does epigenetic dysregulation of pancreatic islets contribute to impaired insulin secretion and type 2 diabetes?

β cell dysfunction is central to the development and progression of type 2 diabetes (T2D). T2D develops when β cells are not able to compensate for the increasing demand for insulin caused by insulin resistance. Epigenetic modifications play an important role in establishing and maintaining β cell identity and function in physiological conditions. On the other hand, epigenetic dysregulation can cause a loss of β cell identity, which is characterized by reduced expression of genes that are important for β cell function, ectopic expression of genes that are not supposed to be expressed in β cells, and loss of genetic imprinting. Consequently, this may lead to β cell dysfunction and impaired insulin secretion. Risk factors that can cause epigenetic dysregulation include parental obesity, an adverse intrauterine environment, hyperglycemia, lipotoxicity, aging, physical inactivity, and mitochondrial dysfunction. These risk factors can affect the epigenome at different time points throughout the lifetime of an individual and even before an individual is conceived. The plasticity of the epigenome enables it to change in response to environmental factors such as diet and exercise, and also makes the epigenome a good target for epigenetic drugs that may be used to enhance insulin secretion and potentially treat diabetes.

Heterocyclic chalcone activators of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) with improved in vivo efficacy

Nrf2 activators represent a good drug target for designing agents to treat diseases associated with oxidative stress. Building upon previous work, we designed and prepared a series of heterocyclic chalcone-based Nrf2 activators with reduced lipophilicity and, in some cases, greater in vitro potency compared to the respective carbocyclic scaffold. These changes resulted in enhanced oral bioavailability and a superior pharmacodynamic effect in vivo.

The YPEL5–PPP1CB fusion transcript is detected in different hematological malignancies and in normal samples

Chronic lymphocytic leukemia (CLL) is the most frequent leukemia in Western adults. It was suggested that transcripts from a reciprocal trans-splicing event between YPEL5 and PPP1CB were present exclusively in CLL patients (more than 90%). Here we show that the YPEL5–PPP1CB fusion is not specific for CLL but is also detected in other hematological malignancies such as chronic myeloid leukemia, monoclonal B cell lymphocytosis or acute leukemia and also in normal samples. As such, it is unlikely that the YPEL5–PPP1CB fusion is a good drug target in CLL or a suitable target to monitor disease.

AlphaScreen HTS and Live-Cell Bioluminescence Resonance Energy Transfer (BRET) Assays for Identification of Tau–Fyn SH3 Interaction Inhibitors for Alzheimer Disease

Alzheimer disease (AD) is the most common neurodegenerative disease, and with Americans’ increasing longevity, it is becoming an epidemic. There are currently no effective treatments for this disorder. Abnormalities of Tau track more closely with cognitive decline than the most studied therapeutic target in AD, amyloid-β, but the optimal strategy for targeting Tau has not yet been identified. On the basis of considerable preclinical data from AD models, we hypothesize that interactions between Tau and the Src-family tyrosine kinase, Fyn, are pathogenic in AD. Genetically reducing either Tau or Fyn is protective in AD mouse models, and a dominant negative fragment of Tau that alters Fyn localization is also protective. Here, we describe a new AlphaScreen assay and a live-cell bioluminescence resonance energy transfer (BRET) assay using a novel BRET pair for quantifying the Tau–Fyn interaction. We used these assays to map the binding site on Tau for Fyn to the fifth and sixth PXXP motifs to show that AD-associated phosphorylation at microtubule affinity regulating kinase sites increases the affinity of the Tau–Fyn interaction and to identify Tau–Fyn interaction inhibitors by high-throughput screening. This screen has identified a variety of chemically tractable hits, suggesting that the Tau–Fyn interaction may represent a good drug target for AD.

Abstract A47: Targeting Ras - is there Ras-specific palmitoylation in human cells that can be targeted for cancer treatment?

Abstract Ras GTPases act on the plasma membrane (PM) to mediate extracellular signaling and tumorigenesis. PM association for many Ras proteins depends on palmitoylation, which in budding yeast is catalyzed by the Erf2-Erf4 palmitoyl transferase. Despite this, there was no evidence that its human ortholog DHHC9 can similarly control Ras in vivo, and some studies even proclaimed that there was no Ras-specific palmitoylation. We performed an unbiased screen to seek fission yeast (Schizosaccharomyces pombe, sp) mutants with reduced PM Ras. Mutations affecting sp-erf2 to varying degrees were isolated 5 times; in contrast, deleting several other S. pombe palmitoyl transferases did not affect Ras. Sp-Erf2 localizes to the trans-Golgi, mediated by its third transmembrane domain and the Erf4 cofactor. In fission yeast, the human ortholog zDHHC9 fully rescues the phenotypes of sp-erf2 null cells, while expressing zDHHC14, another sp-Erf2-like human protein, did not rescue Ras1 mislocalization in these cells. Importantly, ZDHHC9 is widely overexpressed in human cancers, including several types that do not contain frequent oncogenic RAS mutations. Overexpressing ZDHHC9 in mammalian cells promotes, while repressing it diminishes, Ras PM localization, as well as transformation. These data strongly demonstrate that sp-Erf2/zDHHC9 palmitoylates Ras proteins in a highly selective manner in the trans-Golgi to facilitate PM targeting via the trans Golgi network. zDHHC9 overexpression can apparently increase Ras signaling outputs at the PM, such that tumor formation can occur in many tissues with no oncogenic mutations in RAS genes themselves. zDHHC9 may thus be a good drug target for the treatment of a wide range of cancers. Citation Format: Evelin C. Young, Ze-Yi Zheng, Angela D. Wilkins, Hee-Tae Jeong, Min Li, Lichtarge Olivier, Eric C. Chang. Targeting Ras - is there Ras-specific palmitoylation in human cells that can be targeted for cancer treatment? [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A47. doi: 10.1158/1557-3125.RASONC14-A47

Abstract SY37-03: Lifting the metabolic break on caspase 2 to enhance chemotherapy

Abstract Chemotherapeutic elimination of many tumors depends on the induction of programmed cell death (apoptosis) in tumor cells. We and others have found that DNA damaging chemotherapeutics (e.g., cisplatin) and cytotoxic agents (e.g., paclitaxel) commonly used in cancer therapy (for eg. ovarian cancer, breast cancer and others) requires the initiator caspase, caspase 2, for robust induction of apoptosis. However, resistance to chemotherapy-induced apoptosis remains a significant obstacle in the treatment of these cancers. Recently, it has been recognized that the increased metabolic rate found in many cancer cells is a contributing factor in chemoresistance. While the mechanism through which metabolism regulates chemoresistance is not fully elucidated, we have found that caspase 2 activity is subject to metabolic control. Specifically, a highly active pentose phosphate pathway (PPP) leads to high levels of NADPH. This promotes suppressive phosphorylation of caspase 2, thereby inhibiting cancer cell death. Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme for entry of G6P into the PPP. G6PD has been reported to promote oncogenic transformation in vitro and in vivo and G6PD activity is elevated in a variety of cancers (1). Moreover, loss of G6PD in both Chinese hamster ovary cells and human fibroblasts has been reported to enhance radiosensitivity (2, 3). PPP flux is enhanced in cancer cells through a variety of mechanisms: it has been reported that mutation of the tumor suppressor p53 prevents its suppression of G6PD and overexpression of TAp73 increases G6PD expression in tumors (4). Our data suggest that a highly significant consequence of increased G6PD levels is the suppression of caspase 2. This is independent of any effects G6PD may have on redox potential in tumors. We now show that lifting this suppression of caspase 2, through inhibition of G6PD, will markedly sensitize ovarian, breast and pancreatic cancer cells to chemotherapeutics. G6PD is likely to be a good drug target for cancer therapy: loss of G6PD is known to have minimal adverse consequences for humans, with nearly 7.5% of the world's population exhibiting some degree of G6PD enzyme deficiency (5). Thus, inhibition of G6PD in normal cells (particularly in the temporally restricted setting of cancer therapy) is likely to cause little toxicity. On a mechanistic level, we have reported that the metabolic control of caspase 2 is exerted via its inhibitory phosphorylation by activated CaMKII (6). We now demonstrate a novel form of control of CaMKII activation by metabolism that depends, at least in part, on dephosphorylation of CaMKII at previously uncharacterized sites (T393/S395). We show that this dephosphorylation is mediated by metabolic activation of protein phosphatase 2A (PP2A) in complex with the B55β targeting subunit and that metabolic activity controls the association of the B55β/PP2A complex . This represents a novel locus of CaMKII control and also provides a mechanism contributing to metabolic control of apoptosis. These findings may also have implications for metabolic control of the many CaMKII-controlled and PP2A-regulated physiological processes, as both enzymes appear to be responsive to alterations in glucose metabolized via the PPP. Finally, we will discuss our recent findings involving a role for caspase 2 in the induction of lipoapoptosis in the setting of fatty liver disease, the most severe form of which (nonalcoholic steatohepatitis) may predispose patients to development of liver cancer. We have found that fatty liver can promote a dramatic induction of caspase 2 in both mouse models of disease and in human patients and will present data to suggest that this is a critical component of disease etiology.

Dozens of Schizophrenia Risk Loci Identified

The genetics of schizophrenia enters the modern era, as a landmark, genomewide analysis identifies 108 risk loci for this disorder—most of them new.

Plasmepsin V, a Secret Weapon Against Malaria

Plasmepsin V, a Secret Weapon Against Malaria

Metabolic perturbation of an Hsp90 C-domain inhibitor in a lung cancer cell line, A549 studied by NMR-based chemometric analysis

Hsp90 is a good drug target molecule that is involved in regulating various signaling pathway in normal cell and the role of Hsp90 is highly emphasized especially in cancer cells. Thus, much efforts for discovery and development of Hsp90 inhibitor have been continued and a few Hsp90 inhibitors targeting the N-terminal ATP binding site are being tested in the clinical trials. There are no metabolic signature molecules that can be used to evaluate the effect of Hsp90 inhibition. We previously found a potential C-domain binder named PPC1 that is a synthetic small molecule. Here we report the metabolomics study to find signature metabolites upon treatment of PPC1 compound in lung cancer cell line, A549 and discuss the potentiality of metabolomic approach for evaluation of hit compounds.

ERK5 activation in macrophages promotes efferocytosis and inhibits atherosclerosis.

Efferocytosis is a process by which dead and dying cells are removed by phagocytic cells. Efferocytosis by macrophages is thought to curb the progression of atherosclerosis, but the mechanistic insight of this process is lacking. When macrophages were fed apoptotic cells or treated with pitavastatin in vitro, efferocytosis-related signaling and phagocytic capacity were upregulated in an ERK5 activity-dependent manner. Macrophages isolated from macrophage-specific ERK5-null mice exhibited reduced efferocytosis and levels of gene and protein expression of efferocytosis-related molecules. When these mice were crossed with low-density lipoprotein receptor(-/-) mice and fed a high-cholesterol diet, atherosclerotic plaque formation was accelerated, and the plaques had more advanced and vulnerable morphology. Our results demonstrate that ERK5, which is robustly activated by statins, is a hub molecule that upregulates macrophage efferocytosis, thereby suppressing atherosclerotic plaque formation. Molecules that upregulate ERK5 and its signaling in macrophages may be good drug targets for suppressing cardiovascular diseases.

Voltage-Gated Proton Channels as Novel Drug Targets: From NADPH Oxidase Regulation to Sperm Biology

Voltage-gated proton channels are increasingly implicated in cellular proton homeostasis. Proton currents were originally identified in snail neurons less than 40 years ago, and subsequently shown to play an important auxiliary role in the functioning of reactive oxygen species (ROS)-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. Molecular identification of voltage-gated proton channels was achieved less than 10 years ago. Interestingly, so far, only one gene coding for voltage-gated proton channels has been identified, namely hydrogen voltage-gated channel 1 (HVCN1), which codes for the HV1 proton channel protein. Over the last years, the first picture of putative physiological functions of HV1 has been emerging. The best-studied role remains charge and pH compensation during the respiratory burst of the phagocyte NADPH oxidase (NOX). Strong evidence for a role of HV1 is also emerging in sperm biology, but the relationship with the sperm NOX5 remains unclear. Probably in many instances, HV1 functions independently of NOX: for example in snail neurons, basophils, osteoclasts, and cancer cells. Generally, ion channels are good drug targets; however, this feature has so far not been exploited for HV1, and hitherto no inhibitors compatible with clinical use exist. However, there are emerging indications for HV1 inhibitors, ranging from diseases with a strong activation of the phagocyte NOX (e.g., stroke) to infertility, osteoporosis, and cancer. Clinically useful HV1-active drugs should be developed and might become interesting drugs of the future.

Solution structure of a putative FKBP-type peptidyl-propyl cis–trans isomerase from Giardia lamblia

The FK506 Binding Protein (FKBP) super-family was named for the ability of these proteins to bind to the immunosuppressive drug FK506, a 23-member macrolide lactone. Immunosuppression is achieved when the proteinFK506 complex binds to calcineurin to form a larger complex that blocks signal transduction in the T-lymphocyte transduction pathway (Gothel and Marahiel 1999). FKBP proteins also have peptidyl-proline cis–trans isomerases (PPIase) activity that catalyzes the interconversion of the peptidyl-prolyl imide bond between the cis and trans configuration in peptide and protein substrates (Gothel and Marahiel 1999). The isomerase activity is unrelated to the immunosuppressive effects when bound to FK506, but, is essential for the proper folding of many proteins and a number of PPIases act as chaperones. Because proper protein folding is often necessary for proper biochemical function, FKBP proteins are indirectly associated with many diverse biologically processes including trafficking, cell-cycle regulation, development, and signal transduction (Gothel and Marahiel 1999). It has been observed that the parasite Trypanosoma cruzi secretes a FKBP-type protein upon infecting its host, leading to the suggestion that some parasites may use these proteins as virulence factors (Moro et al. 1995). Cultures of the parasite responsible for malaria, Plasmodium falciparum, are sensitive to FK506 (Bell et al. 1994). Regardless of the potential role of FKBP-type proteins as parasite virulence factors, the many critical biologically functions dependent on FKBP-type proteins makes this super-family of proteins good drug targets (Moro et al. 1995; Alag et al. 2010). The most common parasitic protist in the United States is Giardia lamblia (Krappus et al. 1994): the etiological agent responsible for giardiasis, an infection of the small intestines that often causes diarrhea. While giardiasis is generally not life-threatening, in developing countries this enteric protozoan parasite contributes to malnutrition and underperformance in school, and consequently, has been added to the ‘‘Neglected Disease Initiative’’ list by the World Health Organization (Savioli et al. 2006). Recently, the G. lamblia genome was sequenced (Morrison et al. 2007) and among the 6,470 genes encoded in the five chromosomes is a putative, 109-residue FKBP-type PPIase, Gl-FKBP. To assist structure-based design of a more palatable, single-dose treatment for giardiasis targeting GlFKBP, we used NMR-based methods to determine its structure in solution. The structure of Gl-FKBP and the G. W. Buchko S. N. Hewitt W. C. Van Voorhis P. J. Myler Seattle Structural Genomics Center for Infectious Disease, Seattle, WA, USA

New 7-Methylguanine Derivatives Targeting the Influenza Polymerase PB2 Cap-Binding Domain

The heterotrimeric influenza virus polymerase performs replication and transcription of viral RNA in the nucleus of infected cells. Transcription by "cap-snatching" requires that host-cell pre-mRNAs are bound via their 5' cap to the PB2 subunit. Thus, the PB2 cap-binding site is potentially a good target for new antiviral drugs that will directly inhibit viral replication. Docking studies using the structure of the PB2 cap-binding domain suggested that 7-alkylguanine derivatives substituted at position N-9 and N-2 could be good candidates. Four series of 7,9-di- and 2,7,9-trialkyl guanine derivatives were synthesized and evaluated by an AlphaScreen assay in competition with a biotinylated cap analogue. Three synthesized compounds display potent in vitro activity with IC50 values lower than 10 μM. High-resolution X-ray structures of three inhibitors in complex with the H5N1 PB2 cap-binding domain confirmed the binding mode and provide detailed information for further compound optimization.

Scleroderma models: skin in the game

North American researchers have mouse data showing that mutated fibrillin 1 recapitulates skin fibrosis and other symptoms seen in systemic scleroderma. The group also found downstream proteins whose expression was altered by the mutant glycoprotein that could be good drug targets.

Identification of Semicarbazones, Thiosemicarbazones and Triazine Nitriles as Inhibitors of Leishmania mexicana Cysteine Protease CPB

Cysteine proteases of the papain superfamily are present in nearly all eukaryotes. They play pivotal roles in the biology of parasites and inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas’ disease and leishmaniasis. Homology modeling of the mature Leishmania mexicana cysteine protease CPB2.8 suggested that it differs significantly from bovine cathepsin B and thus could be a good drug target. High throughput screening of a compound library against this enzyme and bovine cathepsin B in a counter assay identified four novel inhibitors, containing the warhead-types semicarbazone, thiosemicarbazone and triazine nitrile, that can be used as leads for antiparasite drug design. Covalent docking experiments confirmed the SARs of these lead compounds in an effort to understand the structural elements required for specific inhibition of CPB2.8. This study has provided starting points for the design of selective and highly potent inhibitors of L. mexicana cysteine protease CPB that may also have useful efficacy against other important cysteine proteases.

Discovery of a New Genetic Variant of Methionine Aminopeptidase from Streptococci with Possible Post-Translational Modifications: Biochemical and Structural Characterization

Protein N-terminal methionine excision is an essential co-translational process that occurs in the cytoplasm of all organisms. About 60-70% of the newly synthesized proteins undergo this modification. Enzyme responsible for the removal of initiator methionine is methionine aminopeptidase (MetAP), which is a dinuclear metalloprotease. This protein is conserved through all forms of life from bacteria to human except viruses. MetAP is classified into two isoforms, Type I and II. Removal of the map gene or chemical inhibition is lethal to bacteria and to human cell lines, suggesting that MetAP could be a good drug target. In the present study we describe the discovery of a new genetic variant of the Type I MetAP that is present predominantly in the streptococci bacteria. There are two inserts (insert one: 27 amino acids and insert two: four residues) within the catalytic domain. Possible glycosylation and phosphorylation posttranslational modification sites are identified in the ‘insert one’. Biochemical characterization suggests that this enzyme behaves similar to other MetAPs in terms of substrate specificity. Crystal structure Type Ia MetAP from Streptococcus pneumoniae (SpMetAP1a) revealed that it contains two molecules in the asymmetric unit and well ordered inserts with structural features that corroborate the possible posttranslational modification. Both the new inserts found in the SpMetAP1a structurally align with the P-X-X-P motif found in the M. tuberculosis and human Type I MetAPs as well as the 60 amino acid insert in the human Type II enzyme suggesting possible common function. In addition, one of the β-hairpins within in the catalytic domain undergoes a flip placing a residue which is essential for enzyme activity away from the active site and the β-hairpin loop of this secondary structure in the active site obstructing substrate binding. This is the first example of a MetAP crystallizing in the inactive form.

Exploration of N-(2-aminoethyl)piperidine-4-carboxamide as a potential scaffold for development of VEGFR-2, ERK-2 and Abl-1 multikinase inhibitor

VEGFR, ERK and Abl had been respectively identified as good drug targets, and their crosstalk also had been well elaborated. Multitarget drugs were more advantageous for cancer treatment, however, no inhibitors simultaneously acting on the three proteins were developed due to their structural diversities. Herein, N-(4-((2-(2-(naphthaen-1-yl)acetamido)ethyl)carbamoyl)piperidin-4-yl)-6-(trifluoromethyl)nicotinamide (NEPT, 6a) was discovered as an active scaffold against VEGFR-2, ERK-2 and Abl-1 kinases through the combination of support vector machine, similarity searching and molecular docking. NEPT and its derivatives were synthesized by convenient routine, their in vitro anti-proliferative abilities against human liver cancer cell line HepG2 were preliminarily evaluated. A representative compound 6b showed an IC50 value of 11.3μM and induced significant HepG2 cells apoptosis. Besides, these compounds displayed better anti-proliferative abilities against K562 cells (a cell line with typical hyperactivity of the above multikinases), for example compound 6b exhibited an IC50 value of 4.5μM. Based on hepatotoxicity case reports of Abl inhibitors, cytotoxicity of synthetic compounds against normal liver cell lines (QSG7701 and HL7702) was studied, 6b had a similar toxic effect with positive control imatinib, and most compounds showed less than 35% inhibition activities at 100μM. Molecular docking study disclosed interactions of 6b with VEGFR-2, ERK-2 and Abl-1 kinases, respectively. Our data suggested the biological activities of 6b may derived from collaborative effects of VEGFR-2, ERK-2 and Abl-1 inhibition.

Identification, Biochemical and Structural Evaluation of Species-Specific Inhibitors against Type I Methionine Aminopeptidases

Methionine aminopeptidases (MetAPs) are essential enzymes that make them good drug targets in cancer and microbial infections. MetAPs remove the initiator methionine from newly synthesized peptides in every living cell. MetAPs are broadly divided into type I and type II classes. Both prokaryotes and eukaryotes contain type I MetAPs, while eukaryotes have additional type II MetAP enzyme. Although several inhibitors have been reported against type I enzymes, subclass specificity is scarce. Here, using the fine differences in the entrance of the active sites of MetAPs from Mycobacterium tuberculosis , Enterococcus faecalis , and human, three hotspots have been identified and pyridinylpyrimidine-based molecules were selected from a commercial source to target these hotspots. In the biochemical evaluation, many of the 38 compounds displayed differential behavior against these three enzymes. Crystal structures of four selected inhibitors in complex with human MetAP1b and molecular modeling studies provided the basis for the binding specificity.