Assay Development and High-Throughput Screening for Inhibitors of Kaposi’s Sarcoma–Associated Herpesvirus N-Terminal Latency-Associated Nuclear Antigen Binding to Nucleosomes

Abstract

Kaposi's sarcoma associated herpesvirus (KSHV) has a causative role in several human malignancies, especially in immunocompromised hosts. KSHV latently infects tumor cells and persists as an extrachromosomal episome (plasmid). KSHV latency-associated nuclear antigen (LANA) mediates KSHV episome persistence. LANA binds specific KSHV sequence to replicate viral DNA. In addition, LANA tethers KSHV genomes to mitotic chromosomes to efficiently segregate episomes to daughter nuclei after mitosis. N-terminal LANA binds histones H2A/H2B to attach to chromosomes. Currently, there are no specific inhibitors of KSHV latent infection. To enable high throughput screening of inhibitors of N-LANA binding to nucleosomes, here we develop, miniaturize, and validate a fluorescence polarization (FP) assay that detects fluorophore labeled N-LANA peptide binding to nucleosomes. We also miniaturize a counterscreen to identify DNA intercalators that nonspecifically inhibit N-LANA binding to nucleosomes, and also develop an ELISA to assess N-LANA binding to nucleosomes in the absence of fluorescence. High throughput screening of libraries containing more than 350,000 compounds identified multiple compounds that inhibited N-LANA binding to nucleosomes. However, no compounds survived all counterscreens. More complex small molecule libraries will likely be necessary to identify specific inhibitors of N-LANA binding to histones H2A/H2B; these assays should prove useful for future screens.

Non-stoichiometric inhibition in integrated lead finding – a literature review

“…While non-stoichiometric inhibitors constitute a major component of raw HTS hit lists [54] and confirmed stoichiometric inhibitors are relatively few, [55,73] this is not surprising if truly novel inhibition modes are sought. It is simply not realistic to assume that there are thousands of truly stoichiometric and specific inhibitors against unprecedented targets in standard screening libraries, which are frequently fed with compounds from earlier medicinal chemistry efforts against well-established target classes.…”

“…The efficacy of this approach is demonstrated by an extreme case study in which the hit list was completely annihilated by such filtering. [73] This was without doubt disappointing for the investigators but much preferable to further, wasted efforts as in the notorious case of the presumed sirtuin activator resveratrol. [94] However, as most of the assays focus on just one type of non-stoichiometric inhibition, it is hardly possible to identify each and every of the many different mechanisms.…”

“…For example, screening with assay protocols involving nucleosomes or other nucleic-acid components might benefit from a counterscreen to remove DNA intercalators [73] that would make little sense for other enzymatic targets such as proteases or kinases. In case of doubt, pilot studies, [91] if possible in dose-response mode, [92] or a retrospective analysis of appropriately annotated historical data [93] can provide first impressions of assay sensitivity and liabilities and help to explore meaningful counterscreens.…”

Featuring the nucleosome surface as a therapeutic target

“…An attempt to identify specific inhibitors of KSHV was recently conducted focused on small molecules with the ability to displace the LANA peptide from the acidic patch [19]. High-throughput screening of large chemical libraries with more than 350 000 compounds identified many small molecules that prevented LANA binding to the nucleosome.…”

Site‐Specific Studies of Nucleosome Interactions by Solid‐State NMR Spectroscopy

“…[7] LANAb inds to the acidic patch, an egatively charged region on the nucleosome surface formed by acidic residues from H2A and H2B. [1a, 2c] A2 0residue peptide derived from the LANA N-terminus binds tightly (K D = 0.16 mm)t on ucleosomes,i n agreement with previous studies using longer fragments [21] ( Figure S6 A). TheL ANAp eptide (2 kDa) was added in fivefold molar excess to ad ilute solution of H2A-labeled nucleosomes from the same batch as the sample used for assignment, followed by sedimentation into the rotor.C omparison of the remaining supernatant and pure LANA peptide solution by NMR spectroscopy suggested that the peptide had been successfully co-sedimented with the nucleosome (Figure S6 B).…”