Avenues For Drug Discovery Research Articles

Targeting selenium metabolism and selenoproteins: novel avenues for drug discovery.

Selenoproteins play a wide range of roles in metabolism and oxidative stress defense and are produced by organisms in all three domains of life. Recent evidence has been presented that metal based cancer drugs target the selenol nucleophile of the active site selenocysteine in thioredoxin reductase isoenzymes. Other metals and metalloids, such as tin, arsenic and gold, have also recently been shown to form stable complexes with hydrogen selenide, a required precursor for the synthesis of selenoproteins in all biological organisms. Moreover these metal based compounds have been shown to inhibit growth of pathogens such as Clostridium difficile and Treponema denticola due to their reactivity with this highly reactive metabolic precursor. This review summarizes the recent finding on these two avenues for drug discovery, and puts this work in context with the larger field of selenium biology.

The SCHOOL of nature: I. Transmembrane signaling.

Receptor-mediated transmembrane signaling plays an important role in health and disease. Recent significant advances in our understanding of the molecular mechanisms linking ligand binding to receptor activation revealed previously unrecognized striking similarities in the basic structural principles of function of numerous cell surface receptors. In this work, I demonstrate that the Signaling Chain Homooligomerization (SCHOOL)-based mechanism represents a general biological mechanism of transmembrane signal transduction mediated by a variety of functionally unrelated single- and multichain activating receptors. within the SCHOOL platform, ligand binding-induced receptor clustering is translated across the membrane into protein oligomerization in cytoplasmic milieu. This platform resolves a long-standing puzzle in transmembrane signal transduction and reveals the major driving forces coupling recognition and activation functions at the level of protein-protein interactions-biochemical processes that can be influenced and controlled. The basic principles of transmembrane signaling learned from the SCHOOL model can be used in different fields of immunology, virology, molecular and cell biology and others to describe, explain and predict various phenomena and processes mediated by a variety of functionally diverse and unrelated receptors. Beyond providing novel perspectives for fundamental research, the platform opens new avenues for drug discovery and development.

Novel Intermolecular Iterative Mechanism for Biosynthesis of Mycoketide Catalyzed by a Bimodular Polyketide Synthase

In recent years, remarkable versatility of polyketide synthases (PKSs) has been recognized; both in terms of their structural and functional organization as well as their ability to produce compounds other than typical secondary metabolites. Multifunctional Type I PKSs catalyze the biosynthesis of polyketide products by either using the same active sites repetitively (iterative) or by using these catalytic domains only once (modular) during the entire biosynthetic process. The largest open reading frame in Mycobacterium tuberculosis, pks12, was recently proposed to be involved in the biosynthesis of mannosyl-β-1-phosphomycoketide (MPM). The PKS12 protein contains two complete sets of modules and has been suggested to synthesize mycoketide by five alternating condensations of methylmalonyl and malonyl units by using an iterative mode of catalysis. The bimodular iterative catalysis would require transfer of intermediate chains from acyl carrier protein domain of module 2 to ketosynthase domain of module 1. Such bimodular iterations during PKS biosynthesis have not been characterized and appear unlikely based on recent understanding of the three-dimensional organization of these proteins. Moreover, all known examples of iterative PKSs so far characterized involve unimodular iterations. Based on cell-free reconstitution of PKS12 enzymatic machinery, in this study, we provide the first evidence for a novel “modularly iterative” mechanism of biosynthesis. By combination of biochemical, computational, mutagenic, analytical ultracentrifugation and atomic force microscopy studies, we propose that PKS12 protein is organized as a large supramolecular assembly mediated through specific interactions between the C- and N-terminus linkers. PKS12 protein thus forms a modular assembly to perform repetitive condensations analogous to iterative proteins. This novel intermolecular iterative biosynthetic mechanism provides new perspective to our understanding of polyketide biosynthetic machinery and also suggests new ways to engineer polyketide metabolites. The characterization of novel molecular mechanisms involved in biosynthesis of mycobacterial virulent lipids has opened new avenues for drug discovery.

Acute coagulopathy of trauma: mechanism, identification and effect

Acute coagulopathy of trauma has only been described relatively recently. Developing early in the postinjury phase, it is associated with increased transfusion requirements and poor outcomes. This review examines the possible initiators, mechanism and clinical importance of acute coagulopathy. Acute coagulopathy of trauma occurs in patients with shock and is characterized by a systemic anticoagulation and hyperfibrinolysis. Dilution, acidemia and consumption of coagulation proteases do not appear to be significant factors at this stage. There is evidence to implicate activation of the protein C pathway in this process. Diagnosis of acute coagulopathy currently relies on laboratory assessment of clotting times. These tests do not fully characterize the coagulopathy and have significant limitations, which reduce their clinical utility. Acute coagulopathy results in increased transfusion requirements, incidence of organ dysfunction, critical care stay and mortality. Recognition of an early coagulopathic state has implications for the care of shocked patients and the management of massive transfusion. Identification of novel mechanisms for traumatic coagulopathy may lead to new avenues for drug discovery and therapeutic intervention.

The PharmD Investigator in Clinical Pharmacology: Supply and Demand

The article by Douglas Figg in this issue1 addresses two important questions in clinical medicine and clinical research. First, how should the United States respond to the growing shortage of clinical pharmacologists who have traditionally fulfilled key roles in patient care, drug development research, and drug policy? Second, how can doctor of pharmacy (PharmD) graduates be prepared for postgraduate education in a manner that allows them to effectively contribute to these three critical areas as health-care practice and research become increasingly interdisciplinary? Clinical Pharmacology & Therapeutics (2008); 84, 4, 445–447 doi:10.1038/sj.clpt.6100475

GPCR functional selectivity has therapeutic impact

Many in vitro data show that some ligands can cause the differential activation of signaling pathways mediated by a single receptor (termed 'functional selectivity'). It remains unclear, however, whether functionally selective properties are meaningful in vivo. Data obtained with experimental compounds that are functionally selective at the dopamine D2L receptor in vitro suggest that these properties might predict atypical behavioral actions. Moreover, the antipsychotic drug aripiprazole is commonly thought to be a D2 partial agonist, but data clearly show that aripiprazole is functionally selective in vitro. It is proposed that the effects of aripiprazole in animal models and humans can be reconciled only with its functionally selective D2 properties, not its partial D2 agonism. Together, these data provide support for the hypothesis that compounds with functionally selective properties in vitro are likely to have novel actions in vivo, opening doors to new avenues of drug discovery.

Characterizing the neuroprotective effects of alkaline extract of Lycium barbarum on β-amyloid peptide neurotoxicity

Lycium barbarum is an oriental medicinal herb that has long been used for its anti-aging and cell-protective properties. Previous studies have shown that aqueous extracts from L. barbarum exhibit neuroprotection via inhibiting pro-apoptotic signaling pathways. Other active components can also be accomplished by novel alkaline extraction method, which may give different profiles of water-soluble components. We hypothesize that another active component obtained by alkaline extraction method exerts different biological mechanisms to protect neurons. In this study, we aim to examine the neuroprotective effects from the alkaline extract of L. barbarum, namely LBB, to attenuate β-amyloid (Aβ) peptide neurotoxicity. Primary cortical neurons were exposed to Aβ-peptides inducing apoptosis and neuronal cell death. Pretreatment of LBB significantly reduced the level of lactate dehydrogenase (LDH) release and the activity of caspase-3 triggered by Aβ. “Wash-out” procedures did not reduce its neuroprotective effects, suggesting that LBB may not bind directly to Aβ. We have further isolated three subfractions from LBB, namely LBB-0, LBB-I and LBB-II. LBB-I and LBB-II showed differential neuroprotective effects. Western blot analysis demonstrated that LBB-I and LBB-II markedly enhanced the phosphorylation of Akt. Taken together, our results suggested that the glycoconjugate isolated from novel alkaline extraction method can open up a new avenue for drug discovery in neurodegenerative diseases.

Receptor-Mediated Activation of Heterotrimeric G-Proteins: Current Structural Insights

G-protein-coupled receptors (GPCRs) serve as catalytic activators of heterotrimeric G-proteins (Galphabetagamma) by exchanging GTP for the bound GDP on the Galpha subunit. This guanine nucleotide exchange factor activity of GPCRs is the initial step in the G-protein cycle and determines the onset of various intracellular signaling pathways that govern critical physiological responses to extracellular cues. Although the structural basis for many steps in the G-protein nucleotide cycle have been made clear over the past decade, the precise mechanism for receptor-mediated G-protein activation remains incompletely defined. Given that these receptors have historically represented a set of rich drug targets, a more complete understanding of their mechanism of action should provide further avenues for drug discovery. Several models have been proposed to explain the communication between activated GPCRs and Galphabetagamma leading to the structural changes required for guanine nucleotide exchange. This review is focused on the structural biology of G-protein signal transduction with an emphasis on the current hypotheses regarding Galphabetagamma activation. We highlight several recent results shedding new light on the structural changes in Galpha that may underlie GDP release.

PET and SPECT Imaging of the Opioid System: Receptors, Radioligands and Avenues for Drug Discovery and Development

As we celebrate the bicentennial of the isolation of morphine by Sertürner, opioids continue to dominate major sectors of the analgesic market worldwide. The pharmaceutical industry stands to benefit greatly from molecular imaging in preclinical and early clinical trials of new or improved opioid drugs. At this juncture, it seems fitting to summarize the past twenty or so years of research on molecular imaging of the opioid system from the viewpoint of drug discovery and development. Opioid receptors were first imaged in human volunteers by positron emission tomography (PET) in 1984. Now, quantitative PET imaging of the major opioid receptor types (micro, delta , kappa) is possible in the brain and peripheral organs of healthy persons and patient populations. Radioligands are under development for single photon emission computed tomography (SPECT) of opioid receptors as well. These functional, nuclear imaging techniques can trace the fate of radiolabeled molecules directly, but non-invasively, and allow precise pharmacokinetic and pharmacodynamic measurements. Molecular imaging provides unique data that can aid in selecting the best drug candidates, determining optimal dosing regimens, clearing regulatory hurdles and lowering risks of failure. Using a historical perspective, this review touches on opioid receptors as drug targets, and focuses on the status and use of radiotracers for opioid receptor PET and SPECT. Selected studies are discussed to illustrate the power of molecular imaging for facilitating opioid drug discovery and development.

Aspirin and steroids: new mechanistic findings and avenues for drug discovery

The inflammatory response is a life-saving protective process mounted by the body to overcome pathogen infection and injury; however, in chronic inflammatory pathologies, this response can become deregulated. Aspirin and glucocorticoids are two examples of drugs developed over the years to rectify deregulated inflammatory reactions. Interestingly, both these prototypes of anti-inflammatory therapeutics have been 'borrowed' from Mother Nature, identified from the plant and animal world, respectively. In the past century, systematic organic chemistry has been the major approach for producing new drugs, and vast quantities of aspirin and prednisolone have been synthesized, packaged and sold. However, the fascination provoked by these often life-saving drugs has not subsided, and recent work into the endogenous control of the host inflammatory response has revitalized these compounds. Thus, epi-lipoxins, produced after aspirin acetylation of inducible cyclooxygenase-2, and glucocorticoid-regulated annexin 1 appear to be important endogenous mediators of their respective anti-inflammatory effects. In addition, aspirin-triggered epi-lipoxins and glucocorticoid-regulated annexin 1 might act on the same G-protein-coupled receptor, thus rendering this shared receptor a more likely and worthwhile target for fruitful drug discovery.

G-protein-coupled receptor accessory proteins: their potential role in future drug discovery

Historically, the activation and inhibition of GPCR (G-protein-coupled receptor) function have been a very successful avenue for drug discovery and development. However, it is clear that receptors do not function in isolation but are impacted by other proteins. These proteins may alter either binding or functional responses. Identification and study of these interactions have grown rapidly in recent years and continue to do so, resulting in a plethora of potential receptor-protein connections. These associations can be regarded as alternative intervention points to modulate GPCR function and may not only provide alternative ways to modify receptor activity but also to exploit new chemical space for drug-like molecules. Such interactions may account for side-effects or undesirable properties associated with otherwise well-validated GPCR targets. Understanding and/or intervening in these interactions may allow scientists to progress those targets that may have been deemed unsuitable for therapeutic intervention. The present study reviews the opportunities for utilizing receptor interacting proteins as potential drug targets and the issues associated with them.

Inhibitory monoclonal antibodies to human cytochrome P450 enzymes: a new avenue for drug discovery

Monoclonal antibodies (mAbs)1,2 are reagents par excellence for the highly specific quantification of the amount and metabolic activity3 of the multiple isoforms of cytochrome P450 (P450), which collectively metabolize a multitude of drugs and non-drug xenobiotics including toxins, mutagens and carcinogens4–7. Drug metabolism in humans is catalysed by 12 major microsomal P450 enzymes heterogeneously distributed in liver and other tissues, and with different substrate and product specificities4,5. P450-directed metabolism is important with respect to the toxicity of a drug7, drug–drug interactions8 and a drug administered to an individual that is polymorphically defective in certain P450 isoforms, such as 2D6 (Ref.6). Metabolism of drugs by the same P450 can lead to toxic effects as a result of drug–drug competition for the same P450 isoform8. mAbs that are specific to individual P450 isoforms can be used to determine P450 protein content by immunoblot (IB)9 and can quantify the role of the target P450 isoform(s) in microsomes by their inhibition of substrate metabolism9–17. There are large interindividual variations in P450 phenotype18. Thus, mAb-identified drugs, metabolized by a single P450, could be used in vivo for screening individuals for their P450 isoform phenotype. A quantitative knowledge of the P450-specific metabolism of a drug and the individual’s phenotype could determine the clinical potential, efficacy, dosage and toxicity of the drug or partner drugs.

The pharmacologic basis of antiepileptic drug action.

The development of medications used in the treatment of epilepsy has accelerated over the past decade, and has benefited from a parallel growth in our knowledge of the basic mechanisms underlying neuronal excitability and synchronization. This understanding of the pharmacologic basis of antiepileptic drug (AED) action has, in large part, arisen from recent advances in cellular and molecular biology, coupled with avenues of drug discovery that have departed somewhat from the largely empiric approaches of the past. Physicians now have available to them an ever-growing armentarium of AEDs, necessitating a firmer appreciation of their mechanisms of action if more rational approaches toward both clinical application and research are to be adopted. An important example in this regard is the concept of rational polypharmacy for patients with epilepsy who are refractory to monotherapy. This review summarizes our current understanding of the molecular targets of clinically significant AEDs, comparing and contrasting their differing mechanisms of action.

Regulation of 10P2 murine mast cell proliferation and secretory function by stem cell factor or IL-9.

Mast cells are effectors of inflammatory responses. When triggered by immunological or nonimmunological mechanisms, mast cells release potent biological mediators from preformed stores and synthesize others de novo. In previous investigations from this laboratory, the signal transduction pathways of cloned 10P2 cytokine-independent mast cells were explored. Results suggested that 10P2 cells undergo activation-secretion coupling assessed as release of stored [14C]serotonin (5-HT) when challenged with IgE-specific antigen, influx of extracellular calcium, release of intracellular calcium stores, or by direct activation of protein kinase C isozymes. In the present investigations, cytokine proliferative effects and modulatory roles on release of stored [14C]5-HT have been explored. Following passive sensitization with anti-dinitrophenol (anti-DNP) IgE and challenge with DNP, mast cells released up to 32% of the stored [14C]5-HT. Pretreatment of cells with 10, 30, or 50 ng/ml stem cell factor (SCF) did not alter the response. SCF did not directly induce [14C]5-HT release. Pretreatment with 25 ng/ml interleukin-9 (IL-9) significantly potentiated the IgE-antigen release by 51.1%, 35.7%, or 31.6% when challenged with 3, 10 or 30 ng/ml DNP-HSA. Treatment of cells with 1-100 ng/ml SCF for 72 hr resulted in significantly enhanced proliferation whereas this did not occur when cells were treated with 1-100 ng/ml IL-9. Collectively, these results suggest that SCF alone has a proliferative effect, does not alter the IgE-specific antigen signal transduction pathway, and does not directly stimulate mast cell degranulation. In contrast, IL-9 potentiates the IgE-antigen signal transduction response but exerts no proliferative response. Reports of effects of orally administered cytokines are now beginning to emerge. This raises the possibility that cytokines may be a future therapeutic approach to treatment of allergic and nonallergic inflammatory diseases. The 10P2 cytokine-independent mast cell line may be a valuable adjunct to existing mast cell models as this avenue of drug discovery is explored.