The challenge of developing robust drugs to overcome resistance

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feature The challenge of developing robust drugs to overcome resistance

Drug resistance is problematic in microbial disease, viral disease and cancer. Understanding at the outset that resistance will impact the effectiveness of any new drug that is developed for these disease categories is imperative. In this Feature, we detail approaches that have been taken with selected drug targets to reduce the susceptibility of new drugs to resistance mechanisms. We will also define the concepts of robust drugs and resilient targets, and discuss how the design of robust drugs and the selection of resilient targets can lead to successful strategies for combating resistance. Introduction Drug resistance is defined, in a clinical setting, as the point at which administration of the drug can no longer safely treat the disease state owing to an induced change in the drug target or an inability of the drug to reach the target. With an antimicrobial agent, clinical resistance occurs when the minimum inhibitory concentration (MIC) of the drug, for a given microbial strain, exceeds the concentration of drug that can safely be administered. Resistance to a drug can arise by: (i) mutation of the gene (or gene cluster); (ii) acquisition of extrachromosomal DNA, or a transposable plasmid, that carries the resistance gene or genes; (iii) upregulation of the target or; (iv) upregulation of an efflux mechanism. We have recently suggested [1] an approach for combating drug resistance which involves the selection of resilient drug targets [2] that are evolutionally constrained and the development of robust drugs [3] that are less susceptible to the development of resistance. A drug target is likely to develop resistance if it cannot easily tolerate change and maintain

function. Many drug targets are enzymes. Because these enzymes perform highly constrained and crucial chemical reactions, they can represent resilient targets that are less susceptible to drug resistance. However, disrupting the therapeutic target activity is necessary but not sufficient for developing a drug that avoids resistance. A robust inhibitor is one that successfully inhibits a resilient target, and one that does not lose effectiveness quickly owing to resistance. Such an inhibitor could only bind to crucial regions within the target that would be essential for function and, thus, intolerant to change. The use of high-resolution structures and evolutionary constraints aids the design of robust inhibitors. By choosing resilient targets and designing robust inhibitors the Institute for Drug Resistance (IDR) [1] proposes to focus on drug resistance in drug design strategies, and develop a new generation of more-effective therapeutics. This Feature will focus on selected, potentially resilient drug targets and describe efforts to produce drugs with various degrees of robust-

1359-6446/06/$ - see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.drudis.2011.07.001

ness for these targets in the disease areas of cancer and bacterial and viral infections. The highlighted drug targets include: BCR-ABL kinase; epithelial growth factor receptor (EGFR) kinase and platelet-derived growth factor (PDGFR) kinase in cancer; dihydrofolate reductase (DHFR) and DNA gyrase/topoisomerase IV in bacteria; and human immunodeficiency virus (HIV) protease and hepatitis C (NS3/4A) protease in viruses. Because enzymes are evolutionally constrained to catalyze a chemical reaction, they have the potential of being resilient targets; however, properly identifying robust inhibitors remains a challenge for the field.

Drug resistance in enzyme targets of cancer Drug resistance is now a widespread problem in cancer and is particularly problematic with kinase inhibitors, proteasome inhibitors [4,5] and monoclonal antibodies [6]. Of these, kinase inhibitors comprise the largest class of anticancer agents where drug resistance has significantly limited treatment. In this article the focus will be www.drugdiscoverytoday.com

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Amy C. Anderson1,2, Michael P. Pollastri1,3, Celia A. Schiffer1,4,*, [email protected] and Norton P. Peet1,5, [email protected]

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on the challenges relating to developing robust drugs that overcome resistance to inhibitors of the kinase domains of BCR-ABL, EGFR and PDGF.

BCR-ABL inhibitors

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Gleevec1 (imatinib) shows remarkable efficacy by achieving clinical remission in chronic myeloid leukemia (CML). Imatinib inhibits the constitutively active kinase domain, c-Abl, of the fusion protein BCR-ABL [7] existing in CML patients. Evidence from biochemical and crystallographic studies [8] shows that imatinib selectively binds a unique conformation of the activation loop in which a conserved phenylalanine, a member of the Asp-Phe-Gly (DFG) trio ˚ of amino acids, undergoes an extensive 10 A conformational change, into what is called the DFG-out conformation [9,10]. Imatinib binding maintains the kinase in the inactivated state. Interestingly, many kinases cannot adopt an inactive conformation [11]; therefore, targeting this state yields inhibitors with selectivity over other kinases. Crystal structures of other kinases in the DGF-out conformation include: KIT (mast/ stem cell growth factor receptor); Aurora A; EGFR (covered in this article); p38 or mitogen-activated protein kinase (MAPK)14; kinase insert domain receptor (KDR); and BRAF [9]. However, the success of imatinib was limited because of the selection of resistant mutants. Specifically, the BCR-ABL variant with a T315I gatekeeper mutation in the ATP-binding site was observed in 10–20% of CML patients after failure of imatinib [12,13]. Consequently, there are 200 follow-on inhibitors of BCR-ABL kinase [14–16]. Unfortunately, it has been difficult to develop robust inhibitors against the mutant variant of BCR-ABL. Two recently approved agents, dasatinib and nilotinib (Fig. 1a), have shown success against imatinib-resistant CML in clinical trials. However, the BCR-ABL1 T315I variant also confers resistance to nilotinib and dasatanib [17–21]. Thus, the problem of resistance to this class of inhibitors is still unsolved. Approaches that are being taken to develop robust drugs that are effective against resistant BCR-ABL include: (i) the design of compounds to inhibit the mutated enzyme; (ii) allosteric inhibitors that bind at a site different from the ATP-binding site; (iii) inhibitors that target other tyrosine kinases and simultaneously inhibit the T315I variant of BCR-ABL; and (iv) development of other dual or multikinase inhibitors that simultaneously inhibit the ABL and SFK (SRC family of kinases) families [15]. The SFKs are thought to be involved in the proliferation of BCR-ABL1-expressing cell lines [15,16,22]. The outcomes of these approaches will be crucial to the successful management of CML. 756

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EGFR inhibitors Because EGFR, a receptor tyrosine kinase, plays a crucial part in cellular signaling leading to growth, proliferation and metastasis [23–30], some investigators believed that inhibitors of its function would serve as leads for antineoplastic agents. Gefitinib (Fig. 1b) was approved in 2003 for the treatment of non-small-cell lung cancer (NSCLC) after failure of platinum-based and docetaxel therapies, but did not significantly improve survival [31]. In 2004 erlotinib (Fig. 1b) was also approved for NSCLC (after failure of one other agent) and approved in 2005 for pancreatic cancer in combination with gemcitabine. Both compounds are anilinoquinazolines that compete with ATP to bind the active site of the EGFR tyrosine kinase; a cocrystal structure of erlotinib bound to the EGFR tyrosine kinase [32] reveals the details of that interaction (Fig. 2a). Genetic evidence shows that resistance occurs in response to gefinitib and erlotinib in EGFR through mutation of the ‘gatekeeper’ residue T790M [33,34], similar to mechanisms of resistance that occur with mutants T315I (BCR-ABL/ imatinib [35]), T6741 (PDGFR/imatinib [36]) and T670I (c-KIT/imatinib [37]). Interestingly, there is evidence that the T790M mutation exists before the initiation of therapy [34] and that cells with the mutation are then selected during therapy. The T790M mutation was thought to introduce steric bulk that interfered with the binding of the inhibitors [38]. However, crystal structures and biochemical experiments with the EGFR T790M and T790M/L858R variants bound to inhibitors reveal that the T790M mutation increases ATP affinity, especially in the context of the L858R mutation that activates the kinase [39]. By increasing affinity for ATP, the T790M mutation is considered to be a multi-drug resistant mutation that will reduce the potency of any ATP-competitive inhibitor. The development of irreversible inhibitors such as HKI272 (Figs 1b and 2b), which is now in Phase II trials [33,40,41], can overcome the effects of the T790M mutation. The irreversible inhibitors have a Michael acceptor that forms a covalent bond with a cysteine thiol in the active site pocket, thus eliminating the equilibrium condition with ATP. HKI272 has been shown to inhibit EGFR function even in the presence of the T790M mutation, but could be subject to resistance via a C797 mutation [40].

disease in which the patient has a prolonged state of eosinophilia that can lead to organ dysfunction and death [36]. Some patients with this disease respond favorably to treatment with imatinib, because they harbor a fused Fip1-like 1 (FIP1L1)PDGFRa gene that generates an activated kinase. After initial treatment with imatinib, resistance is once again selected with the mutation of the gatekeeper residue T674I in the kinase domain of PDGFR. Interestingly, in an analogous fashion as EGFR, the gatekeeper mutation in many kinases, including PDGFR, does not exert a steric effect but has a role in favoring an activated kinase [42]. Several compounds in different stages of development are successful at overcoming the T674I variant (Fig. 1c). Ki11502 [43] is a potent multikinase inhibitor of PDGFRa/b and inhibits T674I as well as KIT, KDR and FLT3. Nilotinib [44] and EXEL0862 [45] also inhibit the growth of cells transfected with FIP1L1-PDGFRa harboring the T674I mutation. PKC412 (midostaurin) [36], a natural product isolated from Streptomyces, is reported to be an effective treatment for FIP1L1PDGFRa-induced disease and of imatinibinduced resistance. Although it is still ATP-competitive, PKC412 could have a different binding mode than imatinib, making it less susceptible to imatinib-specific resistance. Sorafenib is an inhibitor of the RAF-1, B-RAF, vascular endothelial growth factor receptor (VEGFR) and PDGFR tyrosine kinases and is also active against cells harboring the T674I mutation [46]. The inhibitor HG78501 is reported to bind kinases with the T674I mutation [47]. A cocrystal structure of HG78501 with Src kinase shows that the compound forms a H-bond with a neighboring methionine (Met 341 in Src), possibly explaining how HG78501 overcomes the effects of the mutation.

Drug resistance in enzyme targets of antibiotics Antibacterial agents, like antineoplastic agents, target fast-growing proliferative cells. Although the targets differ, the occurrence of resistance is common to both classes of drugs and the development of robust drugs to overcome resistant variants is an active area of research. In this article, we highlight efforts to develop robust drugs against DHFR and gyrase/topoisomerase IV.

PDGFR inhibitors

Dihydrofolate reductase and trimethoprim resistance

PDGFR tyrosine kinase inhibitors have been investigated as antineoplastic agents that inhibit angiogenesis. PDGFR inhibitors are also crucial in treating hypereosinophilic syndrome (HES), a

Trimethoprim (TMP) is a potent inhibitor of bacterial species with the essential enzyme DHFR, which plays a crucial part in the folate biosynthetic pathway. TMP, coadministered with

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[(Figure_1)TD$IG]

(a)

Imatinib (Gleevec)

Nilotinib

Dasitinib (Sprycel) (b)

Erlotinib

Gefitinib

HKl272 (c)

Nilotinib Ki11502 Features  PERSPECTIVE

PCK412 (Midostaurin)

HG78501

Sorafenib (d)

Nalidixic acid

Ciprofloxacin

Moxifloxacin

GSK299423

(e) Vertex

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FIGURE 1

(a) Structures of first- and second-generation BCR-ABL kinase inhibitors. (b) Structures of EGFR tyrosine kinase inhibitors. (c) Structures of PDGFR inhibitors. (d) Inhibitors that target the GyrA/ParA subunit of gyrase and/or TopoIV. (e) Benzimidazole GyrB/ParE inhibitors.

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[(Figure_2)TD$IG]

(a)

(b)

HKl272 T290M Cys 797

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FIGURE 2

Crystal structures of EGFR (green) with bound inhibitors (purple or magenta). (a) Erlotinib competes with ATP in the active site of EGFR; (b) HKI272 forms a covalent link with Cys 797 to eliminate the equilibrium between inhibitor and ATP.

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sulfamethoxazole (SMZ), is an effective therapeutic agent used to treat community-acquired methicillin-resistant Staphylococcus aureus (CAMRSA). However, epidemiological surveys show that 28% of MRSA strains are TMP-resistant [48–50]. A common resistance mechanism involves the acquisition of one of two sets of chromosomal mutations in the DHFR gene, either H30N/F98Y or F98Y/H149R. Structures of wild-type and mutant (F98Y and H30N/F98Y) S. aureus DHFR with several novel propargyl-linked antifolates show that the structural effects of the resistance mutations are subtle [51,52]. Development of compounds that have more interactions within the DHFR active site has been the predominant strategy to overcome TMP resistance. Iclaprim, which underwent Phase III trials but did not continue thereafter in development, achieved nanomolar affinity for the wild-type and mutant enzymes and showed a lower propensity for resistance [53]. Crystal structures of iclaprim bound to the wild-type and mutant enzymes show additional hydrophobic interactions in a highly conserved area of the substrate-binding site [54]. Reported propargylbased inhibitors also interact more substantially with the active site [51,52] than TMP and are less affected by the mutations that cause TMP resistance; further experiments to assess their propensity for resistance are ongoing.

topoisomerases catalyze either a transient single-strand break (Type I) or a double-strand break (Type II) in supercoiled DNA. In bacteria, gyrase introduces negative supercoils for the entire chromosome, creating condensed packages of genetic material that can be divided correctly during cell division [56,57]. Fluoroquinolones are an established class of drugs (Fig. 1d) that stabilize the covalent DNAgyrase complex and the homologous DNA-topoIV complex. The prototypical quinolone antibiotic nalidixic acid was discovered in the early 1960s and has since been replaced by compounds with higher potency and improved dosing modalities such as moxifloxicin and ciprofloxacin. Resistance has emerged to the quinolone antibiotics in Gram-positive and Gram-negative strains. A summary of hospital surveys from 1997 [(Figure_3)TD$IG]

to 2001 showed an expanding rate of resistance across infectious species [58]. Resistance mechanisms include increasing drug efflux or mutating the genes that code for DNA gyrase and topoisomerase IV. Structural studies of a DNA–topoIV cleavage complex with bound moxifloxicin (Fig. 3) reveal the effects of amino acid mutations in the quinolone-resistancedetermining region (QRDR), located in the GyrA subunit [59]. Other drug-binding models attempt to explain the effects of other resistance-conferring mutations [60,61]. Compounds that inhibit DNA cleavage via a new binding mechanism that evades the fluoroquinolone resistance mutations and compounds that simultaneously inhibit GyrB/ParE ATPase function represent promising new approaches to overcome resistance.

DNA gyrase and topoisomerase IV inhibitors In Gram-negative and Gram-positive bacteria gyrase and topoisomerase IV, respectively, are important drug targets [55] that serve parallel functions and are often targeted with the same compounds. To perform the processes of transcription, replication, repair and recombination, 758

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FIGURE 3

Crystal structure of moxifloxicin bound to GyrA [59].

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FIGURE 4

Crystal structure of GSK299423 bound to ParB [62].

The topoIV/GyrA subunit inhibitor GSK299423 (Fig. 4) inhibits the turnover of the DNA cleaved complex by stabilizing the enzyme–DNA complex before cleavage and inhibiting separation of the DNA strands. In this way, GSK299423 employs a mechanism that is significantly different from that of the fluoroquinolones [62]. The binding site for GSK299423 is highly conserved in bacteria and does not overlap with the known binding sites for fluoroquinolones, creating a potent inhibitor that shows antibacterial activity against a range of resistant strains with mutations in DNA gyrase and topoisomerase IV. Using the topology of the binding sites of DNA gyrase (GyrB) and topoisomerase IV (ParE), (reviewed in Ref. [63]), novel classes of inhibitors of the ATPase activity have been designed to circumvent the resistance observed with quinolones. Interestingly, these compounds also overcome the resistance associated with the coumarins and cyclothialidines [64,65] that bind in a region that overlaps the ATP-binding site [66,67]. These ATPase inhibitors inhibit gyrase and topoisomerase IV simultaneously, thus yielding an advantage in that the emergence of resistance would require the unlikely occurrence of simultaneous mutations. Combinations of virtual screening and HTS led to compound A (Fig. 1e), which was of modest potency. Compound A was optimized using structure-based design to arrive at B, a compound that displayed nanomolar Ki values against S. aureus and Escherichia coli gyrase and topoisomerase IV, and impressive MIC90 values of 0.03–0.12 mg/ml against a range of bacteria. This compound has since spawned a wide variety of related chemotypes from several other companies [65,68–

70], such as compound C, recently reported by Pfizer [69].

Drug resistance in enzymes targeted by antiviral agents Most antiviral therapy is rightly focused on vaccine development. However, for a growing number of viruses vaccines have remained elusive, or the virus evolves so quickly that their scope is limited. Although some successful antiviral agents target viral entry, several target the viral enzymes that include: proteases, transcriptases, integrases and neuraminidases. As with all quickly evolving diseases there are challenges in developing inhibitors that are robust; one strategy is to target enzymes that are evolutionarily constrained. The viral proteases, which cleave several diverse substrates, represent an example of such a class and, to retain activity, the protease must cleave all the substrates.

HIV protease inhibitors HIV protease, which cleaves Gag and Gag-Pro-Pol polyproteins at ten varied sites necessary for the maturation of virus [71], is a major therapeutic target for antiviral drugs. During the past 20 years, structure-based drug discovery efforts have led to the development of nine approved competitive active site protease inhibitors (PIs). These inhibitors are the most potent anti-HIV drugs and essential components of highly active antiretroviral therapy (HAART) [72,73]. The development of drug resistance is a major reason for the failure of PI therapy. The virus accumulates many mutations within the protease that prevent PIs from binding to the protease. More than half the residues within the

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protease mutate in different combinations and lead to drug resistance [74,75]. Drug resistance is a subtle change in the balance of recognition events: the protease is still able to recognize and process the natural substrate sites in the Gag and Gag-Pro-Pol polyprotein, at the same time no longer being effectively inhibited by the competitive inhibitor. When resistance emerges the interactions of the protease with an inhibitor are significantly altered, whereas the interactions with a natural substrate are maintained. Crystallographic studies of the wild-type protease bound to inhibitor molecules have shown that most of the PIs occupy a similar volume and contact similar residues within the active site of the protease. Drug resistance occurs where inhibitor atoms protrude beyond the substrate envelope and contact protease residues [76]. Thus, mutations at these sites would specifically impact inhibitor binding while maintaining substrate cleavage. The observation that many of the drug-resistant mutations in the active site do not contact the substrates has led to the development of the substrate envelope hypothesis: inhibitors that fit well within the substrate envelope would be less susceptible to drug resistance because a mutation that affects inhibitor binding would simultaneously impact the recognition and processing of the majority of the substrates [76]. Of the currently prescribed inhibitors the most potent is darunivar (DRV) and, although not designed with the substrate envelope constraint, DRV fits well within this volume [2,77]. DRV in combination with ritinovir has a high genetic barrier to resistance [78]. Developing robust HIV-1 PIs that avoid drug resistance has proven to be a challenging task, and the substrate envelope hypothesis provides an approach to solving this problem. A survey of five approved drugs using quantitative measures of the bound inhibitor outside the substrate envelope indicated that the exterior volume of the inhibitors correlated with the loss of affinity to mutant proteases [79]. The ability of the substrate envelope to correlate with resistance mutations prompted the prospective design of new inhibitors with a lower likelihood of developing resistance [3,80–83]. These studies assist in validating the use of the substrate envelope hypothesis [84] in structure-based drug design of novel HIV PIs and have yielded several leads for potential new drugs.

Hepatitis C NS/3A protease inhibitors Drug resistance is also a major obstacle in the treatment of hepatitis C virus (HCV). An estimated 180 million people worldwide are infected with HCV [85]. The essential HCV NS3/ www.drugdiscoverytoday.com

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4A protease is an attractive therapeutic target responsible for cleaving at least four sites along the viral polyprotein. Many PIs are currently in clinical trials; however, multidrug resistance is widespread and arises very quickly. A recent study [86] compares the cocrystal structures of substrate structures with cocrystal structures of inhibitor complexes and shows that, as in the case of HIV-1 protease [77,79,81–83,87,88], primary drug resistance occurs in HCV NS3/4A where the inhibitors protrude away from the substrate envelope. These findings suggest a general model for using the substrate envelope approach to predict patterns of drug resistance in other quickly evolving diseases. For drug resistance to occur, mutations must selectively weaken enzyme affinity for an inhibitor without significantly altering its activity. Mutations occur outside the substrate envelope to achieve this effect, because these molecular changes can selectively alter inhibitor binding without compromising enzyme activity. Whenever the interaction of a drug target with its biological substrates can be structurally characterized, we predict that drugs designed to fit within the substrate envelope will be less susceptible to resistance. Structure-based design strategies can incorporate this as an added constraint to develop inhibitors that fit within the substrate envelope. As a general paradigm, design efforts incorporating the substrate envelope approach can lead to the development of more-robust inhibitors that are less susceptible to resistance.

Concluding remarks Drug resistance negatively impacts the lives of millions of patients and costs our society billions of dollars by limiting the longevity of many of our most potent drugs. Drug resistance develops so rapidly that new drugs quickly become less effective, as witnessed by the evolution of resistance to existing anticancer and antimicrobial agents (antibiotics, antivirals, antifungals and antiprotozoals). A recent survey of European intensive care doctors indicated that 50% have treated at least one patient with a Gram-negative pathogen that was totally or almost totally resistant to all antibiotics during the previous six months’ [89]. Drug resistance research has been very much a disease-specific endeavor, which has limited intellectual progress and breakthroughs. However, as shown here, we have highlighted many parallels among quickly evolving diseases, such as cancer and infectious disease. No single approach to develop drugs that are less prone to resistance will work for every drug target. Rather, 760

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several approaches will be needed to successfully develop new drugs that will work on targets where resistance is an issue. One such approach that we have highlighted in this Feature is to define targets that are resilient and to then develop robust inhibitors for those targets.

Acknowledgements The authors thank John D. Williams, PhD, and Bing Li, PhD, of Microbiotix, for technical assistance with this manuscript. National Institute of Health supports the investigators as follows: ACA – R01 AI073375, R01 GM067542; MPP – R01 AI082577; CAS – R01 AI085051, P01 GM66524, R01 GM65347. References 1 Peet, N.P. (2010) Drug resistance: a growing problem. Drug Discov. Today 15, 583–586 2 Lefebvre, E. and Schiffer, C.A. (2008) Resilience to resistance of HIV-1 protease inhibitors: profile of darunavir. AIDS Rev. 10, 131–142 3 Nalam, M.N. and Schiffer, C.A. (2008) New approaches to HIV protease inhibitor drug design II: testing the substrate envelope hypothesis to avoid drug resistance and discover robust inhibitors. Curr. Opin. HIV AIDS 3, 642–646 4 Oerlemans, R. et al. (2008) Molecular basis of bortezomib resistance: proteasome subunit beta5 (PSMB5) gene mutation and overexpression of PSMB5 protein. Blood 112, 2489–2499 5 Lu, S. et al. (2008) Overexpression of the PSMB5 gene contributes to bortezomib resistance in Tlymphoblastic lymphoma/leukemia cells derived from Jurkat line. Exp. Hematol. 36, 1278–1284 6 Kute, T. et al. (2004) Development of Herceptin resistance in breast cancer cells. Cytometry A 57, 86–93 7 Shah, N.P. et al. (2008) Transient potent BCR-ABL inhibition is sufficient to commit chronic myeloid leukemia cells irreversibly to apoptosis. Cancer Cell 14, 485–493 8 Huse, M. and Kuriyan, J. (2002) The conformational plasticity of protein kinases. Cell 109, 275–282 9 Liu, Y. and Gray, N.S. (2006) Rational design of inhibitors that bind to inactive kinase conformations. Nat. Chem. Biol. 2, 358–364 10 Hubbard, S.R. et al. (1994) Crystal structure of the tyrosine kinase domain of the human insulin receptor. Nature 372, 746–754 11 Nigg, E.A. (2001) Mitotic kinases as regulators of cell division and its checkpoints. Nat. Rev. Mol. Cell Biol. 2, 21–32 12 Druker, B.J. et al. (2001) Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N. Engl. J. Med. 344, 1038–1042 13 Gorre, M.E. et al. (2001) Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 293, 876–880 14 Sawyers, C.L. (2010) Even better kinase inhibitors for chronic myeloid leukemia. N. Engl. J. Med. 362, 2314– 2315 15 Quintas-Cardama, A. et al. (2007) Flying under the radar: the new wave of BCR-ABL inhibitors. Nat. Rev. Drug Discov. 6, 834–848

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Amy C. Anderson1,2 Michael P. Pollastri1,3 Celia A. Schiffer1,4,* Norton P. Peet1,5 1 Institute for Drug Resistance, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605-2324, USA 2 Department of Pharmaceutical Sciences, University of Connecticut, 69 N. Eagleville Road, Storrs, CT 06269, USA 3 Department of Chemistry & Chemical Biology Northeastern University, 417 Egan Research Center, 360 Huntingdon Avenue, Boston, MA 02115, USA 4 Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605-2324, USA 5 Microbiotix, Inc., One Innovation Drive, Worcester, MA 01605-4306, USA www.drugdiscoverytoday.com

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Drug Discovery Today  Volume 16, Numbers 17/18  September 2011