Microarray Compound Screening (μARCS) to Identify Inhibitors of HIV Integrase

David, Caroline A.; Middleton, Tim; Montgomery, Debra; Lim, Hock B.; Kati, Warren M.; Molla, Akhter; Xuei, Xiaoling; Warrior, Usha; Kofron, James L.; Burns, David J.

doi:10.1177/108705710200700309

Cited by 28 publications

(26 citation statements)

References 10 publications

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“…Traditionally, integrase activities have been measured by low-throughput gel-based assays involving radioactively labeled oligonucleotides. 14,15 More recently, however, highthroughput screening (HTS)-compatible assays for HIV-IN have been described, among them 1 gel-based assay using microarrayed compound libraries 16 and several microtiter-based solid-phase assays. 8,[17][18][19][20][21] These solid-phase assays typically monitor the integrase-mediated fusion of an immobilized oligonucleotide mimicking an HIV-1 LTR terminus with a 2nd tagged target oligonucleotide.…”

Section: +mentioning

confidence: 99%

Homogeneous High-Throughput Screening Assays for HIV-1 Integrase 3'-Processing and Strand Transfer Activities

et al. 2005

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HIV-1 integrase (HIV-IN) is a well-validated antiviral drug target catalyzing a multistep reaction to incorporate the HIV-1 provirus into the genome of the host cell. Small molecule inhibitors of HIV-1 integrase that specifically target the strand transfer step have demonstrated efficacy in the suppression of virus propagation. However, only few specific strand transfer inhibitors have been identified to date, and the need to screen for novel compound scaffolds persists. Here, the authors describe 2 homogeneous time-resolved fluorescent resonance energy transfer-based assays for the measurement of HIV-1 integrase 3′-processing and strand transfer activities. Both assays were optimized for high-throughput screening formats, and a diverse library containing more than 1 million compounds was screened in 1536-well plates for HIV-IN strand transfer inhibitors. As a result, compounds were found that selectively affect the enzymatic strand transfer reaction over 3′ processing. Moreover, several bioactive molecules were identified that inhibited HIV-1 reporter virus infection in cellular model systems. In conclusion, the assays presented herein have proven their utility for the identification of mechanistically interesting and biologically active inhibitors of HIV-1 integrase that hold potential for further development into potent antiviral drugs. (Journal of Biomolecular Screening 2005:456-462)

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Section: +mentioning

confidence: 99%

Homogeneous High-Throughput Screening Assays for HIV-1 Integrase 3'-Processing and Strand Transfer Activities

et al. 2005

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“…With 1536-well plates experiments have been able to reduce screening costs by reducing reactant volumes and processing step, but this plate-based format is still reliant on liquid and plate handling robotics. Here, we build on existing microscale technologies including toxicology assays [13, 14] and microarrays [15, 16] to design a generalized microarray platform for benchtop HTS. Furthermore, we aim to design a platform in which arrayed chemical libraries can be fabricated and stored separately from the assay experiments, so that a pre-fabricated arrayed chemical library can be printed at a central facility and used at a remote location.…”

Section: Introductionmentioning

confidence: 99%

A sandwiched microarray platform for benchtop cell-based high throughput screening

Wu¹,

Wheeldon²,

Guo³

et al. 2011

Biomaterials

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The emergence of combinatorial chemistries and the increased discovery of natural compounds have led to the production of expansive libraries of drug candidates and vast numbers of compounds with potentially interesting biological activities. Despite broad interest in high throughput screening (HTS) across varied fields of biological research, there has not been an increase in accessible HTS technologies. Here, we present a simple microarray sandwich system suitable for screening chemical libraries in cell-based assays at the benchtop. The microarray platform delivers chemical compounds to isolated cell cultures by ‘sandwiching’ chemical-laden arrayed posts with cell-seeded microwells. In this way, an array of sealed cell-based assays was generated without cross-contamination between neighboring assays. After chemical exposure, cell viability was analyzed by fluorescence detection of cell viability indicator assays on a per microwell basis in a standard microarray scanner. We demonstrate the efficacy of the system by generating four hits from toxicology screens towards MCF-7 human breast cancer cells. Three of the hits were identified in a combinatorial screen of a library of natural compounds in combination with verapamil, a P-glycoprotein inhibitor. A fourth hit, 9-methoxy-camptothecin, was identified by screening the natural compound library in the absence of verapamil. The method developed here miniaturizes existing HTS systems and enables the screening of a wide array of individual or combinatorial libraries in a reproducible and scalable manner. We anticipate broad application of such a system as it is amenable to combinatorial drug screening in a simple, robust and portable platform.

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“…22 The strand transfer IC 50 value (μM) and cell-based (TZM-bl) cytotoxicity CC 50 value (μM) at 48 h were determined for all derivatives (Table 1). It was observed that dihydroisoquinolines having 7-fluoro function 4a− d were found to be less potent while the IC 50 and CC 50 values of 7-nitro derivatives of 1,2-dihydroisoquinoline 4e−h showed a values were in the range of 8.5 to 50.9 μM for both earlier mentioned derivatives.…”

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confidence: 99%

Design, Synthesis, and Biological Evaluation of 1,2-Dihydroisoquinolines as HIV-1 Integrase Inhibitors

Tandon

Urvashi

Yadav

et al. 2015

ACS Med. Chem. Lett.

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6-Endo-dig-cyclization is an efficient method for the synthesis of 1,2-dihydroisoquinolines. We have synthesized few 1,2-dihydroisoquinolines having different functionality at the C-1, C-3, C-7, and N-2 positions for evaluation against HIV-1 integrase (HIV1-IN) inhibitory activity. A direct nitro-Mannich condensation of o-alkynylaldimines and dual activation of o-alkynyl aldehydes by inexpensive cobalt chloride yielded desired compounds. Out of 24 compounds, 4m and 6c came out as potent integrase inhibitors in in vitro strand transfer (ST) assay, with IC 50 value of 0.7 and 0.8 μM, respectively. Molecular docking of these compounds in integrase revealed strong interaction between metal and ligands, which stabilizes the enzyme−inhibitor complex. The ten most active compounds were subjected to antiviral assay. Out of those, 6c reduced the level of p24 viral antigen by 91%, which is comparable to RAL in antiviral assay. Interestingly, these compounds showed similar ST inhibitory activity in G140S mutant, suggesting they can act against resistant strains.

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Microarray Compound Screening (μARCS) to Identify Inhibitors of HIV Integrase

Cited by 28 publications

References 10 publications

Homogeneous High-Throughput Screening Assays for HIV-1 Integrase 3'-Processing and Strand Transfer Activities

Homogeneous High-Throughput Screening Assays for HIV-1 Integrase 3'-Processing and Strand Transfer Activities

A sandwiched microarray platform for benchtop cell-based high throughput screening

Design, Synthesis, and Biological Evaluation of 1,2-Dihydroisoquinolines as HIV-1 Integrase Inhibitors

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