Disrupting the Constitutive, Homodimeric Protein–Protein Interface in CK2β Using a Biophysical Fragment-Based Approach

Seetoh, Wei-Guang; Abell, Chris

doi:10.1021/jacs.6b07440

Cited by 21 publications

(18 citation statements)

References 62 publications

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“…Indeed, the L86A/L87A substitution reduced binding to GST-UL44 in pulldown assays (Figure 1A), and greatly reduced protein stability in TSA (Figure 1B). The latter observation is consistent with the fact that shown that substitutions affecting protein multimerization decrease the Tm (41). Similarly, the L86A/L87A substitution reduced FRET efficiency and the B 50 value for UL44 self-interaction, but had little or no effect on the ability of the protein to bind to its catalytic subunit UL54 (Figures 2 and 3), therefore suggesting that the protein was still properly folded.…”

Section: Discussionsupporting

confidence: 90%

“…On the other hand, the fraction of His 6 -UL44-L86A/L87A pulled down was clearly reduced, indicating impaired ability to associate with GST-UL44, and thus further supporting the validity of the previously published crystal structure (Figure 2A, right panel ). Since single amino acids substitutions impairing self-association of multimeric proteins are known to decrease protein stability (41), we decided to further corroborate our findings by comparing the stability of His 6 -UL44 and its L86A/L87A derivative by thermal shift assays (TSAs). To this end, we initially performed a small-scale screening to identify the most suitable conditions to assess UL44 stability by TSA (Supplementary Figure S1).…”

Section: Resultsmentioning

confidence: 70%

“…Most of such inhibitors target PPIs in which partner proteins are characterized by short primary sequences at the interface (37, 38) and the hot-spots residues are concentrated in small binding pockets (39, 40). Recent studies have also reported the inhibition of protein dimerization, with a focus on proteins overexpressed in cancer or involved in viral replication (30, 41–43).…”

Section: Introductionmentioning

confidence: 99%

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Divide et Impera: Identification of Small-Molecule Inhibitors of HCMV Replication Interfering with Dimerization of DNA Polymerase Processivity Factor UL44

Ghassabian

Falchi

Antonio

et al. 2020

Preprint

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16Human cytomegalovirus (HCMV) is a leading cause of severe diseases in immunocompromised 17 individuals, including AIDS and transplanted patients, and in congenitally infected newborns. Despite 18 the availability of several antiviral drugs, their utility is limited by poor bioavailability, toxicity, and 19 resistant strains emergence. Therefore, it is crucial to identify new targets of therapeutic intervention. 20 The dimerization of HCMV DNA polymerase processivity factor UL44 plays an essential role in the 21 viral life cycle being required for oriLyt-dependent DNA replication. We validated the existence of 22 UL44 homodimers both in vitro and in living cells by a variety of approaches, including GST 23 pulldown, thermal shift, FRET and BRET assays. Dimerization occurred with an affinity comparable 24 to that of the UL54/UL44 interaction, and was impaired by amino acid substitutions at the 25 dimerization interface. Subsequently, we performed an in-silico screening to select 18 small 26 molecules (SMs) potentially interfering with UL44 homodimerization. Antiviral assays using 27 recombinant HCMV TB4-UL83-YFP in the presence of the 18 selected SMs led to the identification 28 of four active SMs. The most active one also inhibited AD169 in plaque reduction assays, and 29 impaired replication of an AD169-GFP reporter virus and its ganciclovir-resistant counterpart to a 30 similar extent. As assessed by Western blotting experiments, treatment of infected cells specifically 31 reduced viral gene expression starting from 48 h post infection, consistent with activity on viral DNA 32 synthesis. Therefore, SMs inhibitors of UL44 dimerization could represent a new class of HCMV 33 inhibitors, alternative to those targeting the DNA polymerase catalytic subunit or the viral terminase 34 complex. 35 IMPORTANCE 36 HCMV is a ubiquitous infectious agent causing life-lasting infections in humans. HCMV primary 37 infections and reactivation in non-immunocompetent individuals often result in life-threatening 38 conditions. Antiviral therapy mainly targets the DNA polymerase catalytic subunit UL54 and is often 39 limited by toxicity and selection of drug-resistant viral strains, making the identification of new 40 targets of therapeutic intervention crucial for a successful management of HCMV infections. The 41 49The b-Herpesvirinae member human cytomegalovirus (HCMV) is a major human pathogen, causing 50 severe and life-threatening infections in immunocompromised patients (1) and in congenitally 51 infected newborns (2). Herpesviruses are opportunistic double-stranded DNA viruses, whose genome 52 transcription, replication, and packaging occur in the host cell nucleus (3). The molecular mechanisms 53 involved in herpesvirus DNA replication and its regulation have been widely studied as they provide 54 important models for the study of eukaryotic DNA replication and because viral enzymes involved 55 in the process represent targets for antiviral therapy. HCMV DNA polymerase holoenzyme is a multi-56 functional enzyme that play...

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Section: Discussionsupporting

confidence: 90%

Section: Resultsmentioning

confidence: 70%

Section: Introductionmentioning

confidence: 99%

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Divide et Impera: Identification of Small-Molecule Inhibitors of HCMV Replication Interfering with Dimerization of DNA Polymerase Processivity Factor UL44

Ghassabian

Falchi

Antonio

et al. 2020

Preprint

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“…However, small-molecule inhibitors have been shown to cause negative shifts in the Tm values of target proteins by disrupting their oligomeric interfaces, leading to thermal destabilization and subsequent loss of interaction between the protein subunits (23,24). Some examples include 6hydroxy-DL-dopa binding to RAD52 (25) and SPD304 binding to TNF-α (26).…”

Section: Mechanism Of Action Of Tfq Against Sars-cov-2 M Promentioning

confidence: 99%

Inhibition of Severe Acute Respiratory Syndrome Coronavirus 2 main protease by tafenoquinein vitro

Chen

Yang

Huang

et al. 2020

Preprint

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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing the current pandemic, coronavirus disease 2019 (COVID-19), has taken a huge toll on human lives and the global economy. Therefore, effective treatments against this disease are urgently needed. Here, we established a fluorescence resonance energy transfer (FRET)-based high-throughput screening platform to screen compound libraries to identify drugs targeting the SARS-CoV-2 main protease (Mpro), in particular those which are FDA-approved, to be used immediately to treat patients with COVID-19. Mpro has been shown to be one of the most important drug targets among SARS-related coronaviruses as impairment of Mpro blocks processing of viral polyproteins which halts viral replication in host cells. Our findings indicate that the anti-malarial drug tafenoquine (TFQ) induces significant conformational change in SARS-CoV-2 Mpro and diminishes its protease activity. Specifically, TFQ reduces the alpha-helical content of Mpro, which converts it into an inactive form. Moreover, TFQ greatly inhibits SARS-CoV-2 infection in cell culture system. Hence, the current study provides a mechanistic insight into the mode of action of TFQ against SARS-CoV-2 Mpro. Moreover, the low clinical toxicity of TFQ and its strong antiviral activity against SARS-CoV-2 should warrant further testing in clinical trials.

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“…3, a-c). Because compounds were initially selected for their capacity to occupy preferential sites on the dimerization interface of p8, the decrease in T m values suggests that these four fragments would destabilize the homodimeric state (19). To complete the validation stage, we next employed the saturation transfer difference NMR (STD-NMR) approach (20).…”

Section: Two Fragments Turned Out To Destabilize the Dimerization Intmentioning

confidence: 99%

Small molecule–based targeting of TTD-A dimerization to control TFIIH transcriptional activity represents a potential strategy for anticancer therapy

Gervais

Muller

Mari

et al. 2018

Journal of Biological Chemistry

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The human transcription factor TFIIH is a large complex composed of 10 subunits that form an intricate network of protein-protein interactions critical for regulating its transcriptional and DNA repair activities. The trichothiodystrophy group A protein (TTD-A or p8) is the smallest TFIIH subunit, shuttling between a free and a TFIIH-bound state. Its dimerization properties allow it to shift from a homodimeric state, in the absence of a functional partner, to a heterodimeric structure, enabling dynamic binding to TFIIH. Recruitment of p8 at TFIIH stabilizes the overall architecture of the complex, whereas p8's absence reduces its cellular steady-state concentration and consequently decreases basal transcription, highlighting that p8 dimerization may be an attractive target for down-regulating transcription in cancer cells. Here, using a combination of molecular dynamics simulations to study p8 conformational stability and a >3000-member library of chemical fragments, we identified small-molecule compounds that bind to the dimerization interface of p8 and provoke its destabilization, as assessed by biophysical studies. Using quantitative imaging of TFIIH in living mouse cells, we found that these molecules reduce the intracellular concentration of TFIIH and its transcriptional activity to levels similar to that observed in individuals with trichothiodystrophy owing to mutated Our results provide a proof of concept of fragment-based drug discovery, demonstrating the utility of small molecules for targeting p8 dimerization to modulate the transcriptional machinery, an approach that may help inform further development in anticancer therapies.

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Disrupting the Constitutive, Homodimeric Protein–Protein Interface in CK2β Using a Biophysical Fragment-Based Approach

Cited by 21 publications

References 62 publications

Divide et Impera: Identification of Small-Molecule Inhibitors of HCMV Replication Interfering with Dimerization of DNA Polymerase Processivity Factor UL44

Divide et Impera: Identification of Small-Molecule Inhibitors of HCMV Replication Interfering with Dimerization of DNA Polymerase Processivity Factor UL44

Inhibition of Severe Acute Respiratory Syndrome Coronavirus 2 main protease by tafenoquinein vitro

Small molecule–based targeting of TTD-A dimerization to control TFIIH transcriptional activity represents a potential strategy for anticancer therapy

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