1. Introduction
In the 1940’s, the development of penicillin as a potent broad-range antibiotic revolutionized the treatment of infectious disease and ushered in a prolific discovery period of natural small molecules produced by microorganisms that were antagonistic towards the growth of other bacteria. Antibiotics have generally been classified by their mechanism of action. For example, the β-lactam compounds, penicillin and cephalosporins, disrupt the synthesis of the peptidoglycan layer of the bacterial cell wall, whereas protein synthesis inhibitors, such as tetracycline and some aminoglycosides, bind to the 30S ribosomal subunit and block addition of amino acids to the growing peptide chain. By the 1960’s, the majority of all antibiotics in use today had been isolated and developed for public consumption, leading the U.S. Surgeon General to declare in 1968 that the war on infectious disease had been won.
Unfortunately, nature has found a way to thwart mankind’s effort to contain infectious disease. Under the selective pressure of antibiotics that target different cell processes, bacteria have evolved to become resistant to the lethal effects of many classes of antibiotics. One stark example that has emerged as a major public health threat is methicillin-resistant
There is no question that new strategies that target different aspects of pathogen function are urgently needed to combat multi-drug resistant bacteria. However, very few new scaffolds for drug discovery developed after the 1960s have been found to be effective [2]. To date, only four new classes of antibiotics, including mutilins and lipopeptides, have been introduced, but none of these have proven to be as effective as the panel of classic antibiotics. Instead, established scaffolds have been modified or re-purposed to develop successive generations of effective antibiotics. For example, the core structure of cephalosporins have been left intact to preserve activity, but the peripheral chemical groups have been modified to impart the molecule with the ability to penetrate the bacterial membrane more efficiently or be more resistant to β-lactamase [3]. Modifications of four classic antibiotics, cephalosporin, penicillin, quinolone, and macrolide, account for ~73% of the “new” antibiotics filed between 1981 and 2005 [4]. It is also important to note that small compounds need to exhibit not only anti-microbial activity, but also minimized cytotoxic properties to widen their therapeutic window.
Although advances in organic synthesis have extended the lifetime of classic antibiotics through synthetic modifications, new scaffolds are also needed. Recent efforts to search for new modalities amongst previously-overlooked natural sources, such as unmined bacterial taxa and ecological niches, have started to bear fruit. The increasingly rapid data acquisition and low cost of ultra high-throughput sequencing has provided rich coverage of bacterial genomes and transcriptomes. For example, genomic analyses of a vancomycin-resistant strain of
2. Methodology for high-throughput screens (HTS) using small molecule libraries
The workhorse platform for anti-bacterial drug discovery is a chemical genetics HTS approach using small molecule compound libraries to identify candidates that inhibit bacterial growth or the function of key bacterial enzymes. Small molecules, generally <500 molecular weight, have the potential to enter cells and selectively perturb specific protein activity, thus functioning as therapeutic agents against disease. In general, the precise mechanism of inhibitor activity remains unknown in the initial screen. Subsequent identification of the molecular targets of small molecules will have to be performed to implicate the specific bacterial functions that were inactivated in the screen. Thus, HTS can sample a large unbiased collection of structurally diverse molecules to select compounds that perturb the defined cell phenotype of interest. (Fig. 1)
Various chemical compound libraries are now available through commercial and public resources that include FDA-approved bioactive compounds, therapeutic agents, and natural products. To maximize the structural complexity and diversity of small molecule libraries, scientists have also employed diversity-oriented synthesis,
in which different scaffolds are modified with highly diverse functional groups. [7, 8]. To bolster academic research in chemical biology efforts for HTS-driven identification of bioactive compounds, the NIH launched the Molecular Libraries Program in 2005 to offer access to ten large-scale automated HTS centers in the Molecular Libraries Probe Production Centers Network, including diverse compound libraries through the Small Molecule Repository and information on biological activities of small molecules in the PubChem BioAssay public database.
A variety of different molecular and cellular methods have been developed for HTS using small molecule libraries. Automated microscopy has been utilized for high-content, image-based screens of cells exposed to small molecules. Acquired cell images can be analyzed by automated image analysis software to quantitate physiological changes at the single-cell level, including phenotypes such as morphology and cell toxicity. Small molecule microarrays, in which ~10,000 small molecules are covalently bound to a glass slide, has been generated to detect high affinity binding to a protein of interest, as a potential inhibitor of function. Binding of the protein of interest to specific compounds on the microarray was then detected with fluorescent antibodies [9].
3. Disruption of host-pathogen interactions for novel drug discovery
Given the innovation gap in the discovery of novel antibiotics post-1960, strategies to inhibit novel targets are greatly needed to combat infectious disease. Multiple studies have identified small molecule inhibitors that target gene expression of pathogen TTSS components in
Research efforts have recently begun to focus on disruption of host-pathogen interactions as a new approach to identify potential targets for drug discovery, rather than solely on specific pathogen targets or processes. In particular, the screening of small molecule libraries to identify inhibitors that block pathogen infection of the host, using such phenotypes as pathogen invasion, host morphology, and pathogen replication in the host, is a powerful approach for therapeutic development that may uncover fundamental mechanisms of pathogenesis and potentially lead to discovery of new classes of anti-infective agents. Here, we describe case studies of the use of small molecules in host infection screens to identify novel inhibitors against infectious disease, including bacterial, viral, parasitic, and fungal infections. We will discuss these studies in the context of re-purposing known drugs, inhibitor specificity, and discovery of basic mechanisms of host-pathogen interactions. The screen results are summarized in Table 1.
Intracellular pathogens, including viruses, parasites, and some bacteria, manipulate specific host factors in order to downregulate the host immune response or modulate host actin cytoskeleton rearrangements to induce phagocytic uptake of the pathogen.
Parasites also employ a life cycle of host cell invasion, replication, and host cell lysis during onset of infection.
microscopy assay was developed to distinguish between extracellular and intracellular parasites in a BS-C-1 epithelial cell model, using differential labeling with fluorescent dyes [16]. Out of a 12,160 structurally-diverse small molecule library, 24 non-cytotoxic inhibitors were identified that reduced parasite invasion to <20% compared to control wells. These molecules inhibited different aspects of the infection process, including gliding motility and secretion of host cell adhesins. One of these inhibitors, tachypleginA, was found to post-translationally modify TgMLC1, a myosin light chain component of the
Many Gram-negative bacteria, including
Inhibitors of
Another pathogen family that employs the TTSS is
A screen to identify small molecule inhibitors of
The interaction between
Discovery of small molecule inhibitors has also been extended to plant pathogen systems as an approach to develop commercially-relevant chemicals to protect crops assets from disease. The Gram-negative pathogen
Given that viral pathogens are absolutely dependent on the host for propagation, even more so than bacterial pathogens, research in host-directed anti-virals has advanced at a faster pace than that for anti-bacterial agents. Human Immunodeficiency virus type 1 (HIV-1), a lentivirus of the retroviral family and the causative agent of AIDS, is the most-widely studied viral pathogen to date. HIV-1 infection causes a dramatic decline in host CD4+ T cell numbers and a progressive failure of the immune response, which makes the host susceptible to opportunistic infections and cancer. The highly glycosylated HIV-1 envelope, in combination with the extreme diversity of circulating viral strains, have presented daunting challenges for development of an effective vaccine. Furthermore, the virus establishes chronic infection that resists the highly active antiretroviral therapy (HAART). Conventional HAART for HIV-1 infection combines three main classes of anti-viral drugs:
nucleoside reverse transcriptase inhibitors (NRTIs),
non-nucleoside RT inhibitors (NNRTIs), which target the non-catalytic domain of RT, and
protease inhibitors (PIs).
HAART is usually patient-specific, and its formulation is determined by the viral load and drug resistance. A traditional HAART consists of two NRTIs and a NNRTI or a PI [25]. More advanced combination therapies include a fourth class of antiretroviral drugs, HIV entry inhibitors. HIV-1 entry into human cells is dependent on several sequential steps that include binding of viral envelope protein gp120 to the CD4 receptor, and conformational change in gp120 that increases its affinity to the chemokine co-receptors (CCR5 or CXCR4) and exposes gp41, an HIV envelope protein that executes the fusion of HIV and host cell membranes.
Currently, there are two approved inhibitors of HIV-1 entry:
enfuvirtide, a peptide fusion inhibitor that binds to gp41 and
maraviroc, a small molecule entry inhibitor that prevents interaction between gp120 and CCR5.
The β-chemokine receptor CCR5 was found to act as a major co-receptor for the macrophage-tropic HIV-1 R5 strains, predominant in the early asymptomatic stages of virus infection, whereas the T-cell-tropic strains (using the CXCR4 co-receptor) become prevalent in the symptomatic stages concomitant with the decline of CD4+ T-cells [26]. CCR5 is an attractive target for development of HIV-1 entry inhibitors, given the discovery that HIV-1 non-progressors, individuals homozygous for a 32-bp deletion in the coding region of CCR5 gene (CCR5Δ32) were naturally resistant to infection with R5 HIV-1 [27]. Natural and synthetic CCR5 ligands such as RANTES, AOP-RANTES, Mip-1α, Mip-1β,and Met-RANTES were found to efficiently protect against R5 HIV-1 infection [28, 29]. Thus, the first published high throughput screen (HTS) for discovery of non- peptide inhibitors of HIV-1 entry was performed in a virus-free cell-based system using [125I]-labeled RANTES. A strong inhibitor of RANTES binding to CCR5 stably expressed on the surface of CHO cells was identified from the library of Takeda Chemical Industries. Further chemical modifications of the lead compound designated TAK-779 produced a potent (IC50 1.4 nM in CHO/CCR cells) and selective CCR5 antagonist capable of blocking R5 HIV-1 infection
The number of CCR5 inhibitors has significantly grown since the discovery of TAK-779, but very few compounds have entered clinical trials, and only maraviroc has been approved for clinical use [31]. A radiolabeled-chemokine binding assay similar to one applied for the identification of TAK-779 was used in a HTS of a small molecule library at Pfizer for the discovery of UK-107,543, which had become a scaffold for intensive medicinal chemistry, producing ~1,000 analog compounds, from which maraviroc (UK-427,857) was selected for its excellent preclinical pharmacokinetics (90% inhibitory concentration of 2 nM in pool of PBMCs from various donors) [32]. Despite its proven efficacy against HIV-1 R5 infection, maraviroc is vulnerable to gp120 escape mutations [33]. Site-directed mutagenesis and molecular modeling studies have identified a common binding pocket on CCR5 that is shared by various small-molecule CCR5 inhibitors [34, 35, 36]. Emerging details on gp120 and CCR5 points of interaction and binding thermodynamics provide valuable information that can be applied in developing tools for rational design of novel HIV-1 entry inhibitors [37, 38]. Efficient block of HIV entry into host cells is essential to curtail virus dissemination and is a key step towards eradication of HIV infection. The current HAART regiment can reduce HIV replication to very low levels (below 50 copies/ml plasma) and can lead to recovery of CD4+ T-cell counts but not cure the infection. Patients that have been successfully treated with HAART for years have experienced a rapid virus rebound upon termination of the therapeutic regiment [39, 40]. Such clinical cases present evidence that HIV establishes a chronic infection that resists current HAART designed to target actively replicating virus. A deliberate and controllable induction of HIV-1 replication from its latent reservoirs in combination with HAART is a novel and actively pursued approach that aims to eliminate both active and latent viral pools [41].
Researchers often seek new anti-infective agents amongst small molecules that have previously been approved for the treatment of cancer and neurological diseases, since they have well-established pharmacokinetics and in most cases, known molecular mechanisms of action. One example of this is the histone deacetylase (HDAC) inhibitor, valproic acid (VA), which had previously been approved for treatment of neurological and psychiatric disorders. HIV-1 has been shown to enter dormancy using epigenetic silencing via deaceylation of histones in the vicinity of the integrated viral genome [42]. Thus, VA was tested as a potential agent to disrupt HIV-1 latent infection. However, years of VA treatment in combination with HAART showed no clearance of the latent HIV reservoir [43]. A more potent HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), approved for treatment of cutaneous T-cell lymphoma, was subsequently tested as a potential agent that could ‘flush out’ HIV-1 from latently infected cells, based on its superior effect to VA in cell culture models [44, 45]. A substantial effort has also been invested in the design and synthesis of bryostatin chemical analogs, small molecules that activate protein kinase C (PKC) with single nanomolar concentration [46]. PKC activation leads to phosphorylation of nuclear factor κB (NFκB), a key transcription factor regulator of HIV-1 gene expression [47]. However, modulation of NFκB activity requires great caution, since abnormal NFκB signaling has been related to the pathophysiology of inflammatory diseases and neurodegenerative disorders [48].
A HTS of a small molecule library recently identified novel HIV latency activators [49]. The screen was performed using a lymphoma CD4+ T-cell line (SupT1) harboring latent recombinant HIV-1 and two reporters that reflect early and late virus gene expression incorporated in the HIV-1 genome [50]. A luminescent assay based on secreted alkaline phosphatase (SEAP) activity, incorporated in the late virus gene transcripts, was applied to screen a chemical library of ~200,000 compounds. Validation of 27 hits with diverse chemical structures demonstrated induction of latent virus from various cell models. Compounds with a selective index (CC50/EC50) above 25 were chosen for downstream medicinal chemistry modifications. Moreover, the lead compounds were shown to reactivate latent HIV from primary resting CD4+ T-cells with no induction of cell proliferation. Small molecule activators of latent HIV that act in concert using different mechanisms have a better chance of purging the virus out of infected cells [49]. Such pre-clinical data strongly suggests that successful treatment of HIV infection can be achieved only through combinational therapy consisting of diverse class of antiviral drugs.
4. Whole animal small molecule screens using C. elegans
In vitro high-throughput screens have several limitations for the discovery of therapeutic inhibitors with high efficacy. Synthetic compound libraries often contain toxic compounds with poor pharmacokinetic properties, and many
A small manual screen of 6000 synthetic compounds and 1136 natural extracts were analyzed in an immunocompromised mutant of
The development of automated sorting and handling of
A
The
An automated high-throughput screen using the COPAS Biosort was also applied to
5. Conclusion
Chemical library screens are a potent and valuable molecular tool for HTS identification of potential inhibitors of infectious disease. The long-standing paradigm to treat pathogen infection with small molecules that specifically target pathogen growth or metabolism has led to our current dilemma of microbial drug resistance and re-emergence of once-contained infectious diseases. Thus, new approaches to target pathogen virulence or host response factors rather than essential pathogen functions have become increasingly more attractive strategies that are less likely to induce microbial resistance. Some compounds, such as the FDA-approved anti-psychotic, pimozide, exhibited inhibitory properties against infection by several pathogens, suggesting that small molecules can potentially be developed as broad-spectrum anti-infectives. Although the molecular mechanism of inhibition by small molecules remains unknown in most cases, it may be possible to make an educated guess if targeted pathogens share a common virulence strategy, such as the Type III secretion system in Gram-negative bacteria. In other cases, identification of an inhibitor can lead to a molecular understanding of the infection mechanism. For example, the small molecule, tachypleginA, was found to post-translationally modify TgMLC1, a myosin light chain component, to drive host cell penetration by the parasite
From the various studies detailed in this review, it is apparent that the library screens represent a first step on the road of drug discovery. There has been a growing realization that fundamental discovery of biological mechanisms oftentimes reaches a ‘valley of death’, in which potential translation avenues into clinical therapies and diagnostics for disease treatment comes to a standstill and is lost. NIH is addressing this widening gap between basic and clinical research with the establishment of Clinical and Translational Science Centers across the country. The research community will have to remain pro-active to move promising leads from the initial screen stage into downstream validation and development modes in a timely manner. As with any drug development strategy, there still remain multiple technical challenges that need to be overcome before small molecule inhibitors can successfully transition into the clinic. Researchers will need to assess such parameters as compound toxicity, pharmacokinetics and pharmacodynamics, and validation in animal models. However, FDA-approved small molecule libraries can be applied to HTS as a cost-effective method to identify existing licensed drugs for repurposing from diseases unrelated to microbial infection. Furthermore, the development of the
Acknowledgments
The writing of this review was supported by a Los Alamos National Laboratory LDRD-DR grant to study development of novel inhibitors that block host-pathogen interactions.
References
- 1.
Klevens R. M. Morrison M. A. Nadle J. Petit S. Gershman K. Ray S. Harrison L. H. Lynfield R. Dumyati G. Townes J. M. Craig A. S. Zell E. R. Fosheim G. E. Mc Dougal L. K. Carey R. B. Fridkin S. K. 2007 Invasive methicillin-resistant Staphylococcus aureus infections in the United States. 298 1763 71 - 2.
Fischbach M. A. Walsh C. T. 2009 Antibiotics for emerging pathogens. 325 1089 93 - 3.
Neu H. C. Fu K. P. 1978 Cefuroxime, a beta-lactamase-resistant cephalosporin with a broad spectrum of gram-positive and-negative activity. 13 657 64 - 4.
Newman D. J. Cragg G. M. 2007 Natural products as sources of new drugs over the last 25 years. 70 461 77 - 5.
Banskota A. H. Mc Alpine J. B. Sorensen D. Ibrahim A. Aouidate M. Piraee M. Alarco A. M. Farnet C. M. Zazopoulos E. 2006 Genomic analyses lead to novel secondary metabolites 59 533 42 - 6.
Bister B. Bischoff D. Strobele M. Riedlinger J. Reicke A. Wolter F. Bull A. T. Zahner H. Fiedler H. P. Sussmuth R. D. 2004 Abyssomicin C-A polycyclic antibiotic from a marine Verrucosispora strain as an inhibitor of the p-aminobenzoic acid/tetrahydrofolate biosynthesis pathway 43 2574 6 - 7.
Schreiber S. L. 2000 Target-oriented and diversity-oriented organic synthesis in drug discovery. 287 1964 9 - 8.
Tan D. S. 2005 Diversity-oriented synthesis: exploring the intersections between chemistry and biology. 1 74 84 - 9.
Kuruvilla F. G. Shamji A. F. Sternson S. M. Hergenrother P. J. Schreiber S. L. 2002 Dissecting glucose signalling with diversity-oriented synthesis and small-molecule microarrays. 416 653 7 - 10.
Aiello D. Williams J. D. Majgier-Baranowska H. Patel I. Peet N. P. Huang J. Lory S. Bowlin T. L. Moir D. T. 2010 Discovery and characterization of inhibitors of Pseudomonas aeruginosa type III secretion. 54 1988 99 - 11.
Gauthier A. Robertson M. L. Lowden M. Ibarra J. A. Puente J. L. Finlay B. B. 2005 Transcriptional inhibitor of virulence factors in enteropathogenic Escherichia coli Antimicrob Agents Chemother49 4101 9 - 12.
Pan N. J. Brady M. J. Leong J. M. Goguen J. D. 2009 Targeting type III secretion in Yersinia pestis. 53 385 92 - 13.
Hung D. T. Shakhnovich E. A. Pierson E. Mekalanos J. J. 2005 Small-molecule inhibitor of Vibrio cholerae virulence and intestinal colonization. 310 670 4 - 14.
Felise H. B. Nguyen H. V. Pfuetzner R. A. Barry K. C. Jackson S. R. Blanc M. P. Bronstein P. A. Kline T. Miller S. I. 2008 An inhibitor of gram-negative bacterial virulence protein secretion. 4 325 36 - 15.
Lieberman L. A. Higgins D. E. 2009 A small-molecule screen identifies the antipsychotic drug pimozide as an inhibitor of Listeria monocytogenes infection. 53 756 64 - 16.
Carey K. L. Westwood N. J. Mitchison T. J. Ward G. E. 2004 A small-molecule approach to studying invasive mechanisms of Toxoplasma gondii. 101 7433 8 - 17.
Heaslip A. T. Leung J. M. Carey K. L. Catti F. Warshaw D. M. Westwood N. J. Ballif BA Ward G. E. 2010 A small-molecule inhibitor of T. gondii motility induces the posttranslational modification of myosin light chain-1 and inhibits myosin motor activity 6 e1000720 - 18.
Lee V. T. Pukatzki S. Sato H. Kikawada E. Kazimirova AA Huang J. Li X. Arm J. P. Frank D. W. Lory S. 2007 Pseudolipasin A is a specific inhibitor for phospholipase A2 activity of Pseudomonas aeruginosa cytotoxin ExoU. 75 1089 98 - 19.
Arnoldo A. Curak J. Kittanakom S. Chevelev I. Lee V. T. Sahebol-Amri M. Koscik B. Ljuma L. Roy P. J. Bedalov A. Giaever G. Nislow C. Merrill A. R. Lory S. Stagljar I. 2008 Identification of small molecule inhibitors of Pseudomonas aeruginosa exoenzyme S using a yeast phenotypic screen. 4 e1000005 - 20.
Harmon D. E. Davis A. J. Castillo C. Mecsas J. 2010 Identification and characterization of small-molecule inhibitors of Yop translocation in Yersinia pseudotuberculosis Antimicrob Agents Chemother54 3241 54 - 21.
Zhu P. J. Hobson J. P. Southall N. Qiu C. Thomas C. J. Lu J. Inglese J. Zheng W. Leppla S. H. Bugge T. H. Austin CP Liu S. 2009 Quantitative high-throughput screening identifies inhibitors of anthrax-induced cell death. 17 5139 45 - 22.
Lee Y. S. Bergson P. He W. S. Mrksich M. Tang W. J. 2004 Discovery of a small molecule that inhibits the interaction of anthrax edema factor with its cellular activator, calmodulin. 11 1139 46 - 23.
Schreiber K. Ckurshumova W. Peek J. Desveaux D. 2008 A high-throughput chemical screen for resistance to Pseudomonas syringae in Arabidopsis Plant J54 522 31 - 24.
Schreiber K. J. Nasmith C. G. Allard G. Singh J. Subramaniam R. Desveaux D. 2011 Found in translation: high-throughput chemical screening in Arabidopsis thaliana identifies small molecules that reduce Fusarium head blight disease in wheat 24 640 8 - 25.
Hammer S. M. Eron J. J. Jr Reiss P. Schooley R. T. Thompson M. A. Walmsley S. Cahn P. Fischl M. A. Gatell J. M. Hirsch M. S. Jacobsen D. M. Montaner J. S. Richman D. D. Yeni P. G. Volberding P. A. 2008 Antiretroviral treatment of adult HIV infection: 2008 recommendations of the International AIDS Society-USA panel. 300 555 70 - 26.
Berger E. A. Murphy P. M. Farber J. M. 1999 Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. 17 657 700 - 27.
Liu R. Paxton W. A. Choe S. Ceradini D. Martin S. R. Horuk R. Mac Donald. ME Stuhlmann H. Koup R. A. Landau N. R. 1996 Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. 86 367 77 - 28.
Alkhatib G. Combadiere C. Broder C. C. Feng Y. Kennedy P. E. Murphy P. M. Berger E. A. 1996 CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1. 272 1955 8 - 29.
Simmons G. Clapham P. R. Picard L. Offord R. E. Rosenkilde MM Schwartz T. W. Buser R. Wells T. N. Proudfoot A. E. 1997 Potent inhibition of HIV-1 infectivity in macrophages and lymphocytes by a novel CCR5 antagonist. 276 276 9 - 30.
Baba M. Nishimura O. Kanzaki N. Okamoto M. Sawada H. Iizawa Y. Shiraishi M. Aramaki Y. Okonogi K. Ogawa Y. Meguro K. Fujino M. 1999 A small-molecule, nonpeptide CCR5 antagonist with highly potent and selective anti-HIV-1 activity 96 5698 703 - 31.
Kuhmann S. E. Hartley O. 2008 Targeting chemokine receptors in HIV: a status report 48 425 61 - 32.
Dorr P. Westby M. Dobbs S. Griffin P. Irvine B. Macartney M. Mori J. Rickett G. Smith-Burchnell C. Napier C. Webster R. Armour D. Price D. Stammen B. Wood A. Perros M. 2005 Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum anti-human immunodeficiency virus type 1 activity 49 4721 32 - 33.
Roche M. Jakobsen M. R. Sterjovski J. Ellett A. Posta F. Lee B. Jubb B. Westby M. Lewin S. R. Ramsland P. A. Churchill MJ Gorry P. R. 2011 HIV-1 escape from the CCR5 antagonist maraviroc associated with an altered and less-efficient mechanism of gp120-CCR5 engagement that attenuates macrophage tropism 85 4330 42 - 34.
Dragic T. Trkola A. Thompson D. A. Cormier E. G. Kajumo F. A. Maxwell E. Lin S. W. Ying W. Smith S. O. Sakmar T. P. Moore J. P. 2000 A binding pocket for a small molecule inhibitor of HIV-1 entry within the transmembrane helices of CCR5 97 5639 44 - 35.
Nishikawa M. Takashima K. Nishi T. Furuta R. A. Kanzaki N. Yamamoto Y. Fujisawa J. 2005 Analysis of binding sites for the new small-molecule CCR5 antagonist TAK-220 on human CCR5. 49 4708 15 - 36.
Maeda K. Das D. Ogata-Aoki H. Nakata H. Miyakawa T. Tojo Y. Norman R. Takaoka Y. Ding J. Arnold G. F. Arnold E. Mitsuya H. 2006 Structural and molecular interactions of CCR5 inhibitors with CCR5. 281 12688 98 - 37.
Huang C. C. Lam S. N. Acharya P. Tang M. Xiang S. H. Hussan S. S. Stanfield R. L. Robinson J. Sodroski J. Wilson I. A. Wyatt R. Bewley C. A. Kwong P. D. 2007 Structures of the CCR5 N terminus and of a tyrosine-sulfated antibody with HIV-1 gp120 and CD4. 317 1930 4 - 38.
Brower E. T. Schon A. Klein J. C. Freire E. 2009 Binding thermodynamics of the N-terminal peptide of the CCR5 coreceptor to HIV-1 envelope glycoprotein gp120 48 779 85 - 39.
Chun T. W. Davey R. T. Jr Ostrowski M. Shawn Justement. J. Engel D. Mullins J. I. Fauci AS 2000 Relationship between pre-existing viral reservoirs and the re-emergence of plasma viremia after discontinuation of highly active anti-retroviral therapy. 6 757 61 - 40.
Brooks D. G. Hamer D. H. Arlen P. A. Gao L. Bristol G. Kitchen C. M. Berger E. A. Zack J. A. 2003 Molecular characterization, reactivation, and depletion of latent HIV. 19 413 23 - 41.
Richman D. D. Margolis D. M. Delaney M. Greene W. C. Hazuda D. Pomerantz R. J. 2009 The challenge of finding a cure for HIV infection 323 1304 7 - 42.
Lehrman G. Hogue I. B. Palmer S. Jennings C. Spina CA Wiegand A. Landay A. L. Coombs R. W. Richman D. D. Mellors J. W. Coffin J. M. Bosch R. J. Margolis D. M. 2005 Depletion of latent HIV-1 infection in vivo: a proof-of-concept study. 366 549 55 - 43.
Siliciano J. D. Lai J. Callender M. Pitt E. Zhang H. Margolick J. B. Gallant J. E. Cofrancesco J. Jr Moore R. D. Gange S. J. Siliciano R. F. 2007 Stability of the latent reservoir for HIV-1 in patients receiving valproic acid. 195 833 6 - 44.
Edelstein L. C. Micheva-Viteva S. Phelan B. D. Dougherty J. P. 2009 Short communication: activation of latent HIV type 1 gene expression by suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor approved for use to treat cutaneous T cell lymphoma. 25 883 7 - 45.
Archin N. M. Espeseth A. Parker D. Cheema M. Hazuda D. Margolis D. M. 2009 Expression of latent HIV induced by the potent HDAC inhibitor suberoylanilide hydroxamic acid 25 207 12 - 46.
De Christopher B. A. Loy B. Marsden M. Schrier A. J. Zack J. A. Wender P. A. 2012 Designed, synthetically accessible bryostatin analogues potently induce activation of latent HIV reservoirs in vitro. - 47.
Williams S. A. Chen L. F. Kwon H. Fenard D. Bisgrove D. Verdin E. Greene W. C. 2004 Prostratin antagonizes HIV latency by activating NF-kappaB. 279 42008 17 - 48.
Pikarsky E. Porat R. M. Stein I. Abramovitch R. Amit S. Kasem S. Gutkovich-Pyest E. Urieli-Shoval S. Galun E. Ben-Neriah Y. 2004 NF-kappaB functions as a tumour promoter in inflammation-associated cancer. 431 461 6 - 49.
Micheva-Viteva S. Kobayashi Y. Edelstein L. C. Pacchia A. L. Lee H. L. Graci J. D. Breslin J. Phelan B. D. Miller L. K. Colacino J. M. Gu Z. Ron Y. Peltz S. W. Dougherty J. P. 2011 High-throughput screening uncovers a compound that activates latent HIV-1 and acts cooperatively with a histone deacetylase (HDAC) inhibitor. 286 21083 91 - 50.
Micheva-Viteva S. Pacchia A. L. Ron Y. Peltz S. W. Dougherty J. P. 2005 Human immunodeficiency virus type 1 latency model for high-throughput screening. 49 5185 8 - 51.
Moy T. I. Ball A. R. Anklesaria Z. Casadei G. Lewis K. Ausubel F. M. 2006 Identification of novel antimicrobials using a live-animal infection model 103 10414 9 - 52.
Moy T. I. Conery A. L. Larkins-Ford J. Wu G. Mazitschek R. Casadei G. Lewis K. Carpenter A. E. Ausubel F. M. 2009 High-throughput screen for novel antimicrobials using a whole animal infection model. 4 527 33 - 53.
Junker L. M. Clardy J. 2007 High-throughput screens for small-molecule inhibitors of Pseudomonas aeruginosa biofilm development. 51 3582 90 - 54.
Zhou Y. M. Shao L. Li J. A. Han L. Z. Cai W. J. Zhu C. B. Chen D. J. 2011 An efficient and novel screening model for assessing the bioactivity of extracts against multidrug-resistant Pseudomonas aeruginosa using Caenorhabditis elegans Biosci Biotechnol Biochem75 1746 51 - 55.
Breger J. Fuchs B. B. Aperis G. Moy T. I. Ausubel F. M. Mylonakis E. 2007 Antifungal chemical compounds identified using a C. elegans pathogenicity assay 3 e18 - 56.
Watabe M. Hishikawa K. Takayanagi A. Shimizu N. Nakaki T. 2004 Caffeic acid phenethyl ester induces apoptosis by inhibition of NFkappaB and activation of Fas in human breast cancer MCF-7 cells. 279 6017 26 - 57.
Park J. H. Lee J. K. Kim H. S. Chung S. T. Eom J. H. Kim K. A. Chung S. J. Paik S. Y. Oh H. Y. 2004 Immunomodulatory effect of caffeic acid phenethyl ester in Balb/c mice. 4 429 36 - 58.
Okoli I. Coleman J. J. Tampakakis E. An W. F. Holson E. Wagner F. Conery A. L. Larkins-Ford J. Wu G. Stern A. Ausubel F. M. Mylonakis E. 2009 Identification of antifungal compounds active against Candida albicans using an improved high-throughput Caenorhabditis elegans assay. 4 e7025 - 59.
Fox D. S. Cruz M. C. Sia R. A. Ke H. Cox G. M. Cardenas M. E. Heitman J. 2001 Calcineurin regulatory subunit is essential for virulence and mediates interactions with FKBP12-FK506 in Cryptococcus neoformans. 39 835 49 - 60.
Steinbach W. J. Schell W. A. Blankenship J. R. Onyewu C. Heitman J. Perfect J. R. 2004 In vitro interactions between antifungals and immunosuppressants against Aspergillus fumigatus Antimicrob Agents Chemother48 1664 9