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1 In format provided by Swinney et al. (JULY 2011) Box S2 Discovery of first-in-class medicines Here, a brief background of the discovery of the 50 first-in class smallmolecule new molecular entities and 25 biologics, categorized as in Figure 1, is provided. References not in the print version of the article are provided at the end of this document. Phenotypic screening: serendipitous discoveries Memantine. Memantine was first synthesized by Eli Lilly in the early 1960s as a potential antidiabetic agent, but was ineffective at lowering elevated blood sugar. It was later found to have central nervous system (CNS) activity, and entered clinical trials for dementia in Germany in 1986, but NMDA receptor antagonism was not identified as the mechanism of its therapeutic action until 1989, the year when it was launched in Germany for dementia by Merz. Memantine is an uncompetitive and low moderate-affinity NMDA receptor antagonist, but seems to lack the CNS side effects associated with other NMDA antagonists. It has been proposed that this could be due to the strong voltage dependency and rapid unblocking kinetics of memantine, resulting in memantine blocking the pathological, but not the physiological, activation of NMDA receptors. Lipton has term this type of target as a pathophysiologically activated target 22,23, Sinecatechins. The mode of action of sincatechins in the clearance of genital and perianal warts is unknown. In vitro, they have antioxidative activity; however, the clinical significance of this finding is unknown. Archaeological evidence suggests that tea plant culture is likely to have originated in China more than 5,000 years ago, from where it was brought to India, Japan, Thailand, Korea and Sri Lanka. The medicinal properties of tea leaves first appeared in a Chinese book on pharmaceutical plants (~200 BC). Later, in the KissaYojoki (Book of Tea, ~1191), tea was listed as a remedy to control bleeding, help wounds heal, regulate body temperature, control blood sugar and promote digestion 125. Vorinostat. Charlotte Friend was studying murine erythroleukaemia cells (MELC) and in an effort to soften them for transfection, she cultured the cells with 280 mm DMSO in aqueous buffer. Approximately two-thirds of these cancer cells turned red suggesting the presence of haemoglobin. Subsequently, other polar, small-molecule solvent species were observed to also induce the cytodifferentiation and growth arrest of MELCs and that simple amides were in fact somewhat more potent than DMSO. The molecular mechanism of action (MMOA) was identified using cell-based screens. It was subsequently discovered that suberoylanilidehydroxamic acid (SAHA, vorinostat) inhibits the activity of histone deacetylases (HDACs), including all 11 known human class I and class II HDACs 127,128. Phenotypic screening of random compound library Daptomycin. Daptomycin is a fermentation product of Streptomyces roseosporus. It was discovered in the early 1980s, at Eli Lilly and Company, and in 1997, Cubist Pharmaceuticals licensed worldwide rights from Lilly. The mechanism of action of daptomycin is distinct from that of any other antibiotic. Daptomycin binds to bacterial membranes and causes a rapid depolarization of membrane potential. This loss of membrane potential causes inhibition of DNA, RNA, and protein synthesis, which results in bacterial cell death 133. Ezetimibe. Ezetimibe reduces levels of blood cholesterol by inhibiting the absorption of cholesterol by the small intestine. Ezetimibe has a mechanism of action that differs from those of other classes of cholesterol-reducing compounds (statins, bile acid sequestrants, fibric acid derivatives and plant stanols). The molecular target of ezetimibe has been shown to be the sterol transporter, Niemann-Pick C1-Like 1 (NPC1L1), which is involved in the intestinal uptake of cholesterol and phytosterols. The predecessors to ezetimibe were discovered by using animal models for cholesterol lowering with no idea of the MMOA 30,163. Linezolid. A new class of antibiotics known as oxazolidinones were discovered by scientists from Dupont. They inhibited Gram-positive bacteria associated with serious infections without demonstrating cross resistance with any known antibiotics; Dupont scientists also were unable to induce resistance in the laboratory. A team of scientists lead by Steven Brickner at Upjohn optimized these compounds to eventually identify linezolid as a safe effective antibiotic. Oxazolidinones are protein synthesis inhibitors that target an early step involving the binding of N-formylmethionyl-tRNA to the ribosome. Binding studies demonstrated that these agents interact with the 50S subunit and impart their inhibitory effect by perturbing the correct positioning of trnas on the ribosome 28,96,97. Nateglinide. Nateglinide was discovered by Ajinomoto Co. in their efforts to find another type of antidiabetic drug. In the course of screening in 18-hour-fasted normal mice for hypoglycemic effects, Shinkai and coworkers found that N-benzoyl-DL-phenylalanine exhibited a slight blood glucose lowering activity at an oral dose of 500 mg per kg. The component parts of the molecule were systematically varied leading to the identification of nateglinide Pemirolast. Pemirolast is a mast-cell stabilizer that is approved as an ophthalmic solution for the prevention of itching of the eye due to allergic conjunctivitis. It was initially identified in animal studies to strongly inhibit both rat and guinea pig passive cutaneous anaphylaxis mediated by homologous IgE or IgG antibody 117. Rufinamide. Rufinamide was discovered by Novartis and initially evaluated within the framework of NIH-sponsored anticonvulsant drug screening program. Rufinamide has a distinctive profile in animal testing activity in both MES and PTZ models and a very high protective index that differentiates it from other anti-epileptic drugs 123,124. Phenotypic screening of compound-specific libraries based on significant prior knowledge of compound properties Azacitidine. Azacitidine is a nucleoside analogue of cytidine that specifically inhibits DNA methylation by trapping DNA methyltransferases. It was originally developed as a cytotoxic agent, and an application to the FDA requesting its approval as such was turned down more than 25 years ago. The discovery in the early 1980s that it was a hypomethylating agent, and the elucidation of the role of DNA hypermethylation in cancer, prompted its re-evaluation and eventually led to its approval 69,85. Caspofungin. Caspofungin, an echinocandin, inhibits the synthesis of β(1,3)-d-glucan, an essential component of the cell wall of susceptible Aspergillus species and Candida species. β (1,3)-D-glucan is not present in mammalian cells. The inhibition is reported to be non-competitive. Echinocandins produce a rapid lysis of growing cells and thinning of newly formed yeast cell wall. The disruption of the cell wall was attributed to inhibition of 1,3-b-glucan synthase activity 71,86.

2 Cilostazol. The mechanism ofcilostazol on the symptoms of intermittent claudication is not fully understood. Cilostazol and several of its metabolites are cyclic AMP (camp) phosphodiesterase 3 inhibitors, inhibiting phosphodiesterase activity and suppressing camp degradation with a resultant increase in camp in platelets and blood vessels, leading to inhibition of platelet aggregation and vasodilation, respectively. The predecessors of cilostazol were identified by screening new synthetic compounds in blood platelet aggregation assays 87. Cinacalcet. Nemeth and coworkers used a library of phenylalkylamines in a phenotypic assay to discover cinacalcet. The following narrative highlights the role of a functional (phenotypic) assay as the pivotal point for the discovery. To screen for agonist-like activity on the Ca 2+ receptor, we assessed the ability of test substances to evoke an increase in [Ca 2+ ] i in bovine parathyroid cells. It was a functional assay, with a physiologically meaningful readout, and this, it turns out, was pivotal to the discovery of type II calcimimetics the allosteric modulators. In 1990, when the calcimimetic drug discovery project began in earnest, there were 50 years of physiology dictating what the drug must do it had to do what extracellular Ca 2+ does because that is the primary physiological stimulus regulating the secretion of PTH. It was a physiological phenomenon searching for a mechanism. The calcium receptor provided that mechanism, and thereby a target with an intrinsically high probability of success. Most drug discovery programs today start with a molecular mechanism in the hope that it will explain some physiological phenomenon 29. Docosanol. The antiviral activity of a long-chain, saturated fatty acid was initially reported 70 years ago by Stock and Francis. Their work was based on previous studies showing the anti-infective properties of soaps. Katz and coworkers reported in 1991 that 1-docosanol, a 22-carbon-long saturated alcohol inhibited viral replication The precise MMOA of this fatty acid, which is approved to treat genital warts, is unknown. Levetiracetam. In 1992, Alma Gower observed in routine screening that the (S)-enantiomer of the ethyl analogue of piracetam provided more potent protection against sound-induced convulsions in audiogenic seizure susceptible mice than piracetam. This compound now known as levetiracetam had activity in a variety of other seizure models, although its spectrum of activity was distinctly different from other antiepileptic drugs 95. The precise MMOA is unknown. Lubiprostone. Lubiprostone is a derivative of prostaglandin E1 (PGE1). The first evidence that lubiprostone increased intestinal water secretion and intestinal fluid chloride concentrations was reported in The exact MMOA is still unclear although some controversial evidence suggests a role for chloride channels. We could not identify a report that explicitly described the method of discovery. However, we deduce for the literature that it was discovered using phenotypic assays It is marketed for the treatment of chronic constipation. Miglustat. Miglustat is an inhibitor of glucosylceramide synthase that is approved for the glycosphingolipid storage disorder Gaucher s disease. It was originally developed as an antiviral agent because most enveloped viruses use the same pathway for glycoprotein synthesis in infected cells 104,105. Nelarabine. Nucleoside analogues have proven to be a highly successful class of cytotoxic drugs for the treatment of haematological cancers. These compounds, such as cytarabine, work primarily by incorporation of their triphosphate form into DNA, resulting in apoptosis. The deoxyguanosine analogue 9-b-D-arabinofuranosylguanine (ara-g), was discovered in the 1960s, but was not used clinically because of its poor solubility characteristics. However, studies starting in the 1970s on a naturally occurring disease, purine nucleoside phosphorylase deficiency (PNP), provided a rationale for developing deoxyguanosine analogues. Ara-G was found to be selectively toxic to T cells compared with B cells, and a water-soluble prodrug of ara-g, nelarabine (also known as compound 506U78), was subsequently developed Nitazoxanide. Nitazoanide was first described in 1975 and was initially developed as a veterinary antihelminthic agent that targets intestinal cestodes. The antiprotozoal activity of nitazoxanide is believed to be due to interference with the pyruvate:ferredoxinoxidoreduct ase (PFOR) enzyme-dependent electron transfer reaction, which is essential to anaerobic energy metabolism 78,79. Nitisinone. Nitisinone is a triketone herbicide that is an irreversible inhibitor of 4-hydroxyphenylpyruvate dioxygenase in plants. It is now approved in humans to treat tyrosinaemia type 1 in order to block or reduce the formation of toxic metabolites in the liver. The discovery of both molecules originated with phenotypic assays, then using chemical biology the molecules were used to test hypothesis that led to the approved indication Ranolazine. Ranolazine was found to have anti-anginal and antiischaemic effects that do not depend on reductions in heart rate or blood pressure, leading to its development as an anti-anginal drug. The mechanisms underlying the anti-anginal and anti-ischaemic effects of ranolazine are not clear. Ranolazine (RS-43285) was first identified by screening potential channel blockers in a canine model of myocardial ischaemia Retapamulin. Retapamulin is a semisynthetic derivative of the compound pleuromutilin, which is isolated through fermentation from Clitopiluspasseckerianus (formerly Pleurotuspasseckerianus). In vitro activity of retapamulin against isolates of Staphylococcus aureus as well as Streptococcus pyogenes has been demonstrated. Pleuromutilin was first isolated in 1951 from two basidiomycetes species and was characterized as a crystalline antibiotic with modest activity against Gram-positive organisms in vitro and weak activity in vivo. Retapamulin selectively inhibits bacterial protein synthesis by interacting at a site on the 50S subunit of the bacterial ribosome through an interaction that is different from that of other antibiotics. This binding site involves ribosomal protein L3 and is in the region of the ribosomal P site and peptidyltransferase center. By virtue of binding to this site, pleuromutilins inhibit peptidyl transfer, block P-site interactions, and prevent the normal formation of active 50S ribosomal subunits 122. Sirolimus. The discovery of sirolimus (rapamycin) was first reported in It was isolated from an Easter Island soil sample and found to inhibit Candida albicans, Microsporum gypseum and Trichophyton granulosum. Rapamycin is the third addition to a lineage of antifungal antibiotics that has been exploited to prolong the survival of transplanted grafts. However, its mechanism of action is distinct from cyclosporine and FK-506 (tacrolimus). The sirolimus FKBP12 complex binds to and inhibits the activation of the mammalian target of rapamycin (mtor), a key regulatory kinase. This inhibition suppresses cytokine-driven T-cell proliferation, inhibiting the progression from the G1 to the S phase of the cell cycle 70,126.

3 Ziconitide. Ziconitide is derived from a toxin iolated from Conus geogruphus, a marine snail that captures fish by injecting a potent venom through a disposable harpoon-like tooth. During routine bioassays of chromatographic fractions, some fractions caused a persistent shaking in mice that had been injected intracerebrally. The isolated c-conopeptide GVIA was eventually found to bind to N-type calcium channels located on the primary nociceptive (A-δ and C) afferent nerves in the superficial layers (Rexed laminae I and II) of the dorsal horn in the spinal cord. The N-type channel has been distinguished from the other classes of calcium channels by its sensitivity to GVIA. However, the propensity of GVIA to bind irreversibly to N-type channels has limited its use. Valentino and coworkers investigated ω-conopeptides from several species of Conus and characterized peptides with high affinity for N-type channels that bind in a reversible manner. They synthesized relatively large quantities of peptides corresponding to nine naturally occurring ω-conopeptides derived from five different species of Conus and examined their biochemical, electrophysiological, and pharmacological properties. The synthetic peptide SNX-111 (ziconotide), corresponding to the structure of the ω-conopeptide MVIIA from Conus magus, was found to be a highly effective neuroprotective agent in animal models of transient global ischemia 31. Zonisamide. The anticonvulsant properties of zonisamide were discovered through the testing of numerous 3-substituted 1,2-benzisoxazole compounds in animal models. The exact MMOA is unknown 129. Phenotypic screening: intentional/known target seeking improved MMOA Aripiprazole. In some cases, the target has been known for some time; however, a novel MMOA provides an advantage. This is the case with aripiprazole, a partial agonist of the dopamine D 2 receptor. The identification of aripiprazole and its MMOA was a logical progression in the discovery and development of improved antipsychotics. Aripiprazole was discovered through investigating dopamine antagonist activity in animal models. The mechanism of partial agonism was demonstrated later, and the therapeutic advantage for partial agonism described by Pulvirenti and Koob 75,76, Fulvestrant. In 1991, Wakeling and coworkers reported the properties of a new pure antiestrogen, ICI 182,780 (fulvestrant). The rationale for the search for a pure estrogen antagonist was driven by the observations that antiestrogens compete with endogenous estrogens for binding to estrogen receptor, but have a diversity of biological actions that range between full agonist, with estrogen-like trophic effects, through partial agonism to complete blockade of estrogen action. Subsequently, if was revealed that fulvestrant is a pure estrogen receptor antagonist due to its ability to induce degradation of the estrogen receptors. The mechanism of antagonism was unexpected 47,93,94. Varenicline. The discovery of varenicline is an excellent example of a MMOA-driven approach based on pharmacology and the requirement to minimize mechanism-based toxicity. It was anticipated that a selective partial agonist of nicotinic acetylcholine receptors (nachrs) could have potential as a smoking cessation aid. It was hypothesized that a partial agonist of nachrs would lead to a moderate and sustained increase of mesolimbic dopamine levels. This might counteract the low dopamine levels that result from a lack of nicotine during attempts to quit smoking, which seem to be important in craving and withdrawal, and could therefore be crucial in leading to relapse to smoking. Furthermore, by competitively binding to nachrs, a partial agonist might shield a smoker from nicotineinduced increases in dopamine levels and the associated rewarding properties in the event of relapse. Varenicline was discovered through the synthesis of a series of compounds inspired by the natural product ( )-cytisine, which was previously known to have partial agonist activity at nachrs 77. Target-based screening: optimized MMOA subsequently identified Gefitinib. Gefitinib was discovered on the basis of assays for inhibition of the intracellular phosphorylation of the tyrosine kinase associated with the epidermal growth factor receptor (EGFR), which is overexpressed in non-small-cell lung cancer cells. Gefitinib and related quinazolines were later found to sequester EGFR plus the ligand into inactive receptor ligand complexes. This novel MMOA of quinazoline tyrosine kinase inhibitors may be the basis for their high potency, especially in conditions when the ligand is present in limiting amounts 41,42. Imatinib. The discovery of imatinib was based on the understanding that chronic myelogenous leukemia (CML) was caused by a chromosomal translocation that creates the oncogenic BCR ABL gene. As the tyrosine kinase activity of BCR ABL is crucial for its transforming activity, the enzymatic activity of this deregulated gene was targeted for addressing BCR ABL-positive leukaemias. Cell lines and enzyme assays were developed to screen for inhibitors of this activity. Molecules with good drug-like properties were identified in screens against protein kinase C. Medicinal chemistry efforts ultimately developed these leads into imatinib. Subsequent analysis of the crystal structure showed that imatinib inhibits the ABL kinase by binding with high specificity to an inactive form of the kinase. The need for the kinase to adopt this unusual conformation, which favours binding, might contribute to the high selectivity of the compound 49. Maraviroc. Maraviroc, a selective C C chemokine receptor 5 (CCR5) antagonist, prevents the interaction of HIV1 gp120 and CCR5 that is necessary for CCR5-using HIV1 strains to enter cells. The CCR5 co-receptor had attracted considerable interest as a therapeutic target, as individuals with a natural mutation (CCR5 32) that results in an absence of surface-expressed CCR5 have a high degree of resistance to HIV1 infection, with no apparent significant adverse consequences. Maraviroc was discovered through the medicinal chemistry optimization of a hit compound identified from high-throughput screening. Maraviroc is an allosteric antagonist that induces a unique conformation of the receptor leading to a block in HIV1 entry and infection Raltegravir. HIV1 integrase is a virally encoded enzyme that integrates the viral DNA into the genome of the host cell, which is essential for viral replication. Integration involves two steps catalyzed by HIV1 integrase: endonucleolytic processing of the viral DNA to remove the terminal dinucleotide from each 3 end, and strand transfer, in which the viral DNA 3 ends are covalently linked to the cellular DNA. The discovery of diketo acids that selectively inhibit the strand transfer function of HIV1 integrase and thereby prevent integration and inhibit HIV1 replication provided proof of concept for this enzyme as a therapeutic target for HIV1 infection. The diketo acid inhibitors can only bind to the acceptor site after 3 -processing and thereby trap the intermediate state. Use of the strand transfer assay and medicinal chemistry efforts based on an understanding of the MMOA of diketo acids and the required pharmacophore led to the identification of the HIV1 integrase inhibitor raltegravir. Raltegravir potently binds

4 only when integrase is in its binary complex with donor viral DNA, possibly binding to a transient intermediate along the integration pathway. Integrase inhibitors that disrupt earlier steps in integration (for example, assembly) have so far not proved to be effective inhibitors of preintegration complexes or of viral replication. The diketo acids are the first inhibitors of HIV1 integrase whose antiviral activity clearly results from inhibition of integration 39,40,143,144. Sorafenib. Considerable biological data supported the choice of targeting Raf kinase as an anticancer target. Additionally, drug discovery assays were available to allow initiation of a high-throughput screening (HTS) approach, followed by medicinal chemistry optimization. These efforts lead to the discovery of sorafenib. The net effect of sorafenib s interactions with Raf kinase is to stabilize the DFG motif in an inactive conformation. As well as inhibiting Raf kinase, sorafenib is a potent inhibitor of vascular endothelial growth factor receptor (VEGFR) kinases. Its efficacy in renal cell carcinoma, which was the first indication for which it was approved, is thought to be primarily due to inhibition of VEGFR kinases 44. Sunitinib. Sunitinib (SU11248) was identified as a potent inhibitor of the VEGFR and platelet-derived growth factor receptor (PDGFR) kinases. VEGFR2 and PDGFR are important in cancer-cell proliferation and survival, and also in tumour angiogenesis. Data from preclinical and animal models suggested that simultaneous inhibition of VEGFR2 and PDGFR might produce greater antitumour effects than inhibition of either receptor tyrosine kinase alone, leading to the development and prioritization of agents that inhibited both of these receptors Target-based screening Aliskiren. It took nearly 100 years following the discovery of the protease renin, which is a key regulator of blood pressure, for an orally active renin inhibitor, aliskiren to be approved for the treatment of hypertension. The breakthrough came in the 1980s when medicinal chemists identified inhibitors of renin that were more drug-like than substrate-like 38,130. Aprepitant. The isolation of substance P (SP) in 1931, and the later discovery of its preferred neurokinin (NK)1 receptor, led to an intense research effort aimed at elucidating the biological role of SP, particularly within the central nervous system. Large investments were made by multiple pharmaceutical companies in discovering (using HTS assays) and developing NK1 receptor antagonists such as aprepitant for indications including depression and pain. Although these trials were not successful, the role of the NK1 receptor in emesis provided the basis for the approval of aprepitant as an anti-emetic agent in patients receiving chemotherapy. It remains the only NK1 receptor antagonist on the market 46. Bortezomib. In 1992, Alfred Goldberg decided to use the growing basic knowledge on the proteasome to create a biotechnology company focused on using inhibitors to block the proteasome, with the aims of investigating the physiological roles of the proteasome and translating basic proteasome research into a therapeutic application 131,132. Bortezomib was discovered as proteasome inhibitor, and an initial aim was to harness this activity to treat muscle-wasting disorders. However, based on growing understanding of its biological activity in particular, its anticancer effects bortezomib was first approved for multiple myeloma. Bosentan. Various evidences suggested that endothelin 1 might have a role in the pathogenesis and progression of pulmonary arterial hypertension. In a program aimed at discovering nonpeptidic endothelin receptor antagonists, a class of substituted arylsulfonamidopyrimidines was identified in a chemical substance library. Chemical optimization of these compounds resulted in the discovery of bosentan 37. Conivaptan. Conivaptan was developed in a programme to identify orally active arginine vasopressin (AVP) antagonists for both the V1A and V2 receptors, based on the hypothesis that blocking the properties of both receptors vasoconstriction and water reabsorption, respectively would be beneficial in congestive heart failure 88. Eltrombopag. Eltrombopag is a thrombopoietin receptor (TPO) agonist approved for the treatment of thrombocytopenia in patients with chronic immune (idiopathic) thrombocytopenic purpura. It interacts with the transmembrane domain of the human TPO-receptor and initiates signalling cascades that induce proliferation and differentiation of megakaryocytes from bone marrow progenitor cells. Eltrombopag was identified through screening of small molecule libraries for the ability to activate a reporter molecule in TPO-dependent cell lines. Lead compounds were initially identified and then optimized for their biologic effects and pharmaceutical properties. It is suggested that eltrombopag binds the TPO receptor at a distance from the binding site for TPO and initiates signal transduction by a mechanism different from rhtpo, possibly by inducing dimerization 36. Orlistat. A target directed screening of microbial broths from soil organisms resulted in the discovery of a very potent, selective and irreversible inhibitor of pancreatic lipase, which was named lipstatin (orlistat). It exerts its therapeutic activity in the lumen of the stomach and small intestine by forming a covalent bond with the active serine residue site of gastric and pancreatic lipases 35,142. Sitagliptin. Advances in the understanding of the actions of endogenous glucoregulatory peptide hormones, known as incretins, identified new therapeutic targets for type 2 diabetes. Two incretins glucosedependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP1) potentiate glucose-dependent insulin secretion from islet b-cells by activating specific G-protein-coupled receptors. However, although native GLP1(7 36) amide effectively lowers blood glucose, it is rapidly degraded by the ubiquitous serine protease dipeptidyl peptidase-4 (DPP4). Sitagliptin, a DPP4 inhibitor, was discovered in an iterative discovery approach aimed at optimizing metabolic properties while retaining efficacy 33,146. Zanamivir. Influenza virus (both A and B) adopt a unique replication strategy by using one of its surface glycoproteins, haemagglutinin, to bind to the target-cell receptor, which contains a terminal sialic acid. The other surface glycoprotein, neuraminidase, cleaves off the terminal sialic acid of the host cell receptor, allowing virus particles to leave the cell after the viral replicative cycle has been completed. The viral neuraminidase is therefore needed for the elution of newly formed virus particles from the cells. Computer-assisted drug design, based on the crystal structure of the influenza viral neuraminidase, led to the identification of (GG167) as a specific and potent inhibitor Targeting the viral neuraminidase prevents the release of the influenza virus from the cells 34,151.

5 Target-based screen: modified natural target ligand Mifepristone. Antiprogestins were actively sought by Pincusin the early 1960s as a practical means for fertility control, with no success. The initial breakthrough came with the discovery of a general method providing access to 11b-substituted 19-norsteroids. This finding ultimately led scientists at Roussel-Uclaf to modify the 11b-position of norethindrone to produce the high affinity progesterone receptor antagonist RU (mifepristone) Ramelteon. Ramelteon, an insomnia drug, is a melatonin receptor agonist that has high affinity for the MT1 and MT2 receptors, both of which are thought to be involved in the maintenance of circadian rhythms. Ramelteon was synthesized as part of a program aimed at limiting the conformational flexibility of the methoxy group of melatonin, whose orientation is important for optimal binding to the MT1 receptor 72,145. Modified natural substance (non-biologics) Acamprosate. Acamprosate was discovered based on evidence for a role of GABA in the action of ethanol. The evidence led Boismare and coworkers to study the effects of homotaurine, a potent and stable GABA receptor ligand in rats. The acetylated form of homotaurine, acamprosate, was used to increase brain penetration 152. Aminolevulinic acid. Aminolevulinic acid (ALA) is a precursor of protoporphrin IX and is used in photodynamic therapy. In 1956, four volunteers who ingested doses of ALA developed photosensitization of the skin that persisted for approximately 24 hours. This suggested that at least some of the cells in the skin might synthesize and accumulate photosensitizing concentrations of protoporphrinix if provided with exogenous ALA 153,154. Fondaparinux. The mechanism of action of fondaparinux is related to the anticoagulant action of heparins. Unfractionated heparin, a glycosaminoglycan, exerts its anticoagulant effect via binding to antithrombin III, a natural anticoagulant. The structure of fondaparinux is identical to the heparin pentasaccharide sequence, the smallest fragment required for antithrombin III function. The pentasaccharide sequence was discovered by Choay and coworkers in their search for minimal molecular weight heparin fractions still retaining high anti-factor Xa potency in plasma Sapropterin. Tetrahydrobiopterin (BH 4 ) is a natural cofactor for phenylalanine hydroxylase. BH 4 -responsive phenylalanine-deficient patients, in whom BH 4 - deficiency was excluded, had been observed since 1985 by Neiderwieser and Curtius who suggested that the responses to BH 4 - may be caused by a K m mutant in the phenylalanine hydroxylase (PAH) gene. Several clinical studies showed that treatment with BH 4 can decrease blood phenylalanine levels in some patients with PAH deficiency. Sapropterin dihydrochloride is a synthetic preparation of the dihydrochloride salt of the biologically active 6R-(BH 4 ) stereoisomer Verteporfin. New uses for photodynamic therapy have led to the approval of verteporfin for macular degeneration. Verteporfin is a chemically modified version of protoporphrin IX. Verteporfin is activated by light in the presence of oxygen, generating highly reactive, short-lived singlet oxygen and reactive oxygen radicals. Light activation of verteporfin results in local damage to neovascular endothelium, resulting in vessel occlusion 74,162. Biologics Abatacept. Abatacept, a fusion protein composed of the extracellular domain of CTLA4 linked to a modified Fc portion of immunoglobulin G1, is a selective costimulation modulator that inhibits T lymphocyte activation by binding to CD80 and CD86, thereby blocking interaction with CD28. This interaction provides a costimulatory signal necessary for full activation of T lymphocytes. Activated T lymphocytes are implicated in the pathogenesis of rheumatoid arthritis and are found in the synovium of patients with rheumatoid arthritis 164, and it is approved for this indication. Agalsidase β. Agalsidase β is intended to provide an exogenous source of α-galactosidase A in patients with Fabry disease, an X-linked genetic disorder of glycosphingolipid metabolism. Deficiency of the lysosomal enzyme α-galactosidase A leads to progressive accumulation of glycosphingolipids, predominantly globotriaosylceramide (GL- 3), in many body tissues, starting early in life and continuing over decades. Clinical manifestations of Fabry disease include renal failure, cardiomyopathy, and cerebrovascular incidents. Accumulation of GL-3 in renal endothelial cells may play a role in renal failure. Agalsidase-b, a recombinant form of α-galactosidase A, reduces GL-3 deposition in the capillary endothelium of the kidney and certain other cell types 165. Alefacept. Alefacept is an immunosuppressive dimeric fusion protein that consists of the extracellular CD2-binding portion of the human leukocyte function antigen 3 (LFA3) linked to the Fc (hinge, CH2 and CH3 domains) portion of human IgG1. Alefacept interferes with lymphocyte activation by specifically binding to the lymphocyte antigen, CD2, and inhibiting LFA3/CD2 interaction. Activation of T lymphocytes involving the interaction between LFA3 on antigenpresenting cells and CD2 on T lymphocytes plays a role in the pathophysiology of chronic plaque psoriasis 166. Alemtuzumab. Alemtuzumab is a monoclonal antibody (mab) that binds to CD52, an antigen present on the surface of B and T lymphocytes, a majority of monocytes, macrophages, NK cells, and a subpopulation of granulocytes. It is approved for the treatment of chronic myeloid leukaemia (CLL). A proportion of bone marrow cells, including some CD34 + cells, express variable levels of CD52. The proposed mechanism of action in CLL is antibody-dependent cellularmediated lysis following cell surface binding of alemtuzumab to the leukaemic cells 167. Alglucosidase alfa. Alglucosidase alfa is an enzyme replacement therapy for treatment of Pompe disease (glycogen storage disease type II), a rare lysosomal storage disorder. Pompe disease is an inherited disorder of glycogen metabolism cause by the absence or marker deficiency of the lysosomal enzyme alpha-glucosidase 168. Anakinra. Anakinra is a recombinant nonglycosylated form of the human interleukin receptor antagonist (IL- IRa) that was first approved for the treatment of rheumatoid arthritis. Anakinra differs from native human IL- IRa in that it has the addition of a single methionine residue at its amino terminus. Anakinra blocks the activity of IL-l by competitively inhibiting IL-l binding to the interleukin-l type I receptor (IL-IR1), which is expressed in a wide variety of tissues and organs. IL-l production is induced in response to inflammatory stimuli and mediates inflammatory and immunological responses. The levels of the naturally occurring IL-lRa in synovium and synovial fluid from rheumatoid arthritis patients are not sufficient to compete with the elevated amount of locally produced IL

6 Bevacizumab. The hypothesis that inhibiting angiogenesis might be an effective anticancer strategy was put forward more than 30 years ago by Judah Folkman. In the early 1990s, the demonstration that inhibition of VEGF-induced angiogenesis using a mab against VEGF markedly suppressed tumour growth in vivo led to the development of the humanized mab bevacizumab for various cancers, including colorectal cancer. Bevacizumab binds to VEGF and prevents the interaction of VEGF to its receptors (Flt-1 and KDR) on the surface of endothelial cells 48, Cetuximab Cetuximab is a chimeric mab that binds specifically to the extracellular domain of the human epidermal growth factor receptor (EGFR), and competitively inhibits the binding of epidermal growth factor and other ligands, such as transforming growth factor-a. In vitro assays and in vivo animal studies have shown that binding of cetuximab to the EGFR blocks phosphorylation and activation of receptor-associated kinases, resulting in inhibition of cell growth, induction of apoptosis, and decreased matrix metalloproteinase and VEGF production 173. It was first approved for colorectal cancer. Denileukin diftitox. Denileukin diftitox is a CD25-directed cytotoxin indicated for the treatment of patients with persistent or recurrent cutaneous T-cell lymphoma whose malignant cells express the CD25 component of the IL-2 receptor. Denileukin diftitox is a fusion protein designed to direct the cytocidal action of diphtheria toxin to cells that express the IL-2 receptor. Ex vivo studies report that after binding to the IL-2 receptor on the cell surface, denileukin diftitox is internalized by receptor-mediated endocytosis. The fusion protein is subsequently cleaved, releasing diphtheria toxin enzymatic and translocation domains from the IL-2 fragment, resulting in the inhibition of protein synthesis and ultimately, cell death 174. Drotrecogin a. Reduced levels of protein C, which inhibits coagulation and inflammation when activated, are found in most patients with sepsis and are associated with an increased risk of death. Drotrecogin a, a recombinant form of human activated protein C that exerts an antithrombotic effect by inhibiting Factors Va and VIIIa 175, is approved for the treatment of severe sepsis. Eculizumab. Eculizumab is a humanized mab that specifically binds to the complement protein C5 with high affinity, thereby inhibiting its cleavage to C5a and C5b and preventing the generation of the terminal complement complex C5b-9. Eculizumab inhibits terminal complement mediated intravascular hemolysis in patients with paroxysmal nocturnal haemoglobinuria (PNH). PNH results from the clonal expansion of haematopoietic stem cells that have somatic mutations in the phosphatidylinositol glycan complementation class A gene. C5 was considered to be a good therapeutic target for PNH because blockade at this point would prevent the creation of the membrane attack complex and the release of C5a Efalizumab, Efalizumab is a humanized mab that binds specifically to CD11a, was developed for the treatment of psoriasis. Efalizumab is immunosuppressive, blocking T-cell activation and migration, with increased chance for malignancy and risk of serious infection. During the clinical trials, there were no statistically significant increases in the risks of these outcomes prior to approval and atypical infections were uncommon. However, in 2009, after approximately 46,000 patients were exposed to efalizumab, the drug was withdrawn when three confirmed cases of progressive multifocal leukoencephalopathy (PML), and one additional suspected, but not confirmed, case were spontaneously reported in association with efalizumab use 180. Enfuvirtide. Enfuvirtide is a synthetic peptide derived from gp41 of HIV1. Initially, synthetic peptides derived from gp41 were not targeted as inhibitors of HIV1 fusion, but were investigated as part of epitope-mapping experiments aimed at evaluating strategies in vaccine development. It was observed that when these were incubated with human T cells, an antiviral effect was seen. An understanding of the fusion process, and how envelope glycoproteins interact, led to the appreciation of how these peptides inhibited fusion and thereby were able to interrupt infection 181. Exenatide. Exenatide is a synthetic derivative of 39-amino acid GLP1 agonist isolated from the salivary gland venom of the lizard Heloderma suspectum (Gila monster).this peptide has 53% amino acid similarity to mammalian GLP1, effectively binds to the GLP1 receptor, and is highly resistant to degradation by DPP Its enhancement of glucose-dependent insulin secretion and stability against degradation provide the basis for its use in the treatment of type 2 diabetes. Galsulfase. Galsulfase is used to treat mucopolysaccharidosis type VI, which is characterized by the absence or marked reduction in the lysosomal enzyme N-acetylgalactosamine 4-sulfatase. The deficiency results in the accumulation of its glycosaminoglycan (GAG) substrate, dermatan sulphate, in the lysosomes of many cell types, leading to widespread cellular, tissue and organ dysfunction. Galsulfase is intended to provide an exogenous enzyme that will be taken up into lysosomes and increase the catabolism of GAGs. Galsulfase uptake by cells into lysosomes is most likely mediated by the binding of mannose-6-phosphate-terminated oligosaccharide chains of galsulfase to specific mannose-6-phosphate receptors 185. Gemtuzumab. Gemtuzumab is a mab that binds specifically to CD33 coupled to the cytoxic drug calcheamicin. CD33 is expressed by acute myeloid leukeamia (AML) cells and thereby allows the targeting of the cytotoxic agent to the cancer cells. The use of potent cytotoxic agent such as calicheamicins was required due to the low concentration of CD33 on the AML cells. Gemtuzumab was recently withdrawn from the market 186. Idursulfase. Idursulfase is used to treat Hunter syndrome (mucopolysaccharidosis type II), an X-linked recessive disease caused by insufficient levels of the lysosomal enzyme iduronate- 2-sulfatase. This enzyme cleaves the terminal 2-O-sulfate moieties from the GAGs dermatan sulfate and heparan sulfate. Due to the missing or defective iduronate-2-sulfatase enzyme in patients with Hunter syndrome, GAGs progressively accumulate in the lysosomes of a variety of cells, leading to cellular engorgement, organomegaly, tissue destruction, and organ system dysfunction. Treatment of Hunter syndrome patients with idursulfase provides exogenous enzyme for uptake into cellular lysosomes. Mannose-6-phosphate residues on the oligosaccharide chains allow specific binding of the enzyme to the mannose-6-phosphate receptors on the cell surface, leading to cellular internalization of the enzyme, targeting to intracellular lysosomes and subsequent catabolism of accumulated GAGs 187. Laronidase. The rationale for laronidase therapy in mucopolysaccharidosis I (MPS type I) is to provide exogenous enzyme for uptake into lysosomes and to thereby increase the catabolism of GAGs. Laronidase uptake by cells into lysosomes is most likely mediated by the mannose-6-phosphate-terminated oligosaccharide chains of laronidase binding to specific mannose-6-phosphate receptors 188.

7 Natalizumab. In 1992, α4b1 integrin was identified as the key molecule involved in leukocyte homing to inflamed regions of the brain. An antibody to α4b1 integrin blocked paralysis in an animal model of multiple sclerosis, which led to the approval of the humanized anti-α4 mab natalizumab for the treatment of multiple sclerosis 189,190. Omalizumab. Omalizumab, a humanized mab that inhibits the binding of IgE to the high-affinity IgE receptor (FcεRI) on the surface of mast cells and basophils, is approved for the treatment of allergic asthma. Reduction in surface-bound IgE on FcεRI-bearing cells limits the degree of release of mediators of the allergic response. Treatment with omalizumab also reduces the number of FcεRI receptors on basophils in atopic patients 191. Palifermin. Palifermin, an N-terminally truncated form of fibroblast growth factor 7 with increased stability, is approved for the treatment of chemoradiation-induced oral mucositis in patients undergoing bone marrow transplantation 192. Pegvisomant. Pegivisomant is a protein growth hormone (GH) receptor antagonist that was designed to compete with native GH for the GH receptor and to prevent its proper functional dimerization process that is critical for the GH signal transduction and synthesis and secretion of insulin-like growth factor 1. Dimerization of the GH receptor occurs sequentially, such that a GH receptor molecule binds to one site on GH, followed by binding of a second molecule of GH receptor to a second site on the GH as well as to a site on the first GH receptor. Pegvisomant is a GH analogue that includes a single amino acid substitution at position 120 of glycine for arginine that generates the antagonism. The design idea was to retain a functional first site while the second site is sterically blocked by the glycine to arginine substitution. The substitution will result in antagonism of the response due to the inability of the GH receptors to dimerize Pramlintide. Amylin, a 37-aminoacid peptide that is cosecreted and localized with insulin within the b-cells of the pancreas, acts as a neuroendocrine hormone that complements the actions of insulin in postprandial glucose homeostasis. The synthetic analogue pramlintide acetate differs from amylin in that the amino acid residues at 25 (alanine), 28 (serine), and 29 (serine) have been replaced by proline residues. These substitutions increase the solubility of the drug and decrease its aggregation and adhesion properties, which have been noted with amylin 196,197. Rasburicase. Rasburicase is recombinant urate oxidase indicated for initial management of plasma uric acid levels in paediatric and adult patients with leukemia, lymphoma, and solid tumour malignancies who are receiving anti-cancer therapy expected to result in tumour lysis and subsequent elevation of plasma uric acid. In humans, uric acid is the final step in the catabolic pathway of purines. Rasburicase catalyses enzymatic oxidation of poorly soluble uric acid into an inactive and more soluble metabolite (allantoin) 198. Romiplostim. An innovative approach to mimic a natural ligand was used for the design of romiplostin, a thrombopoietin (TPO) receptor agonist for the treatment of thrombocytopenia in patients with chronic immune (idiopathic) thrombocytopenic purpura. Romiplostin is an Fc-peptide fusion protein (peptibody) that activates intracellular transcriptional pathways leading to increased platelet production via the TPO receptor. 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