Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Jan 8;22(2):575.
doi: 10.3390/ijms22020575.

Combinatorial Drug Treatments Reveal Promising Anticytomegaloviral Profiles for Clinically Relevant Pharmaceutical Kinase Inhibitors (PKIs)

Markus Wild  1 , Jintawee Kicuntod  1 , Lisa Seyler  2 , Christina Wangen  1 , Luca D Bertzbach  3 , Andelé M Conradie  3 , Benedikt B Kaufer  3 , Sabrina Wagner  1 , Detlef Michel  4 , Jan Eickhoff  5 , Svetlana B Tsogoeva  6 , Tobias Bäuerle  2 , Friedrich Hahn  1 , Manfred Marschall  1
Affiliations

Combinatorial Drug Treatments Reveal Promising Anticytomegaloviral Profiles for Clinically Relevant Pharmaceutical Kinase Inhibitors (PKIs)

Markus Wild et al. Int J Mol Sci. .

Abstract

Human cytomegalovirus (HCMV) is a human pathogenic herpesvirus associated with a variety of clinical symptoms. Current antiviral therapy is not always effective, so that improved drug classes and drug-targeting strategies are needed. Particularly host-directed antivirals, including pharmaceutical kinase inhibitors (PKIs), may help to overcome problems of drug resistance. Here, we focused on utilizing a selection of clinically relevant PKIs and determined their anticytomegaloviral efficacies. Particularly, PKIs directed to host or viral cyclin-dependent kinases, i.e., abemaciclib, LDC4297 and maribavir, exerted promising profiles against human and murine cytomegaloviruses. The anti-HCMV in vitro activity of the approved anti-cancer drug abemaciclib was confirmed in vivo using our luciferase-based murine cytomegalovirus (MCMV) animal model in immunocompetent mice. To assess drug combinations, we applied the Bliss independence checkerboard and Loewe additivity fixed-dose assays in parallel. Results revealed that (i) both affirmative approaches provided valuable information on anti-CMV drug efficacies and interactions, (ii) the analyzed combinations comprised additive, synergistic or antagonistic drug interactions consistent with the drugs' antiviral mode-of-action, (iii) the selected PKIs, especially LDC4297, showed promising inhibitory profiles, not only against HCMV but also other α-, β- and γ-herpesviruses, and specifically, (iv) the combination treatment with LDC4297 and maribavir revealed a strong synergism against HCMV, which might open doors towards novel clinical options in the near future. Taken together, this study highlights the potential of therapeutic drug combinations of current developmental/preclinical PKIs.

Keywords: activity in vitro and in vivo; antiviral drugs; combinatorial drug analyses; human cytomegalovirus; new synergistic combinations; pharmaceutical kinase inhibitors (PKIs).

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Dose-response curve of ABE against HCMV in vitro. Data are presented as mean relative HCMV replication compared to control ±SD over three independent experiments each consisting of biological duplicates.
Figure 2
Figure 2
In vivo antiviral drug dosage assessment: ABE-mediated concentration-dependent reduction of spleen-specific MCMV viral load. Oral application of ABE in dosages of (A) 20 mg/kg/d over 4 days and (B) 50 mg/kg/d or 100 mg/kg/d over 5 days, resulting in a reduction in viral load of 44%, 74% or 87%, respectively, compared to vehicle-treated animals. Data are presented as mean + SD of viral replication across five animals per group as measured using in vitro luciferase assay performed on spleen homogenates.
Figure 3
Figure 3
Reduction of viral load by PKI treatment. (A) Viral load in spleen, liver and lung was measured using in vitro luciferase assay, in vivo luciferase imaging and viral genome-specific qPCR and was compared to the mean value of viral load of six vehicle-treated mice. No, moderate or strong reduction is indicated by black, grey or white colored boxes, respectively, as indicated in panel B. Grey bars on the right indicate mean values + SD of viral load for each animal in all eight analyzed organs/methods (see also percentages given above of each bar). (B) Exemplary pictures of in vivo luciferase imaging. Mice were fixed, sedated and injected with luciferin solution. Organ-specific luciferase activity was measured according to regions of interest marked in image a. Pictures show exemplary animals for the categories used in panel A, i.e., no reduction (image b), moderate reduction (image c), strong reduction (image d). (C) Weight of treated animals as determined on days 0, 2, 4 and 6. Data are given as mean weight in each treatment group. VGCV, valganciclovir.
Figure 4
Figure 4
Characteristics of the Bliss independence checkerboard (A,B) and the Loewe additivity fixed-dose assays (C,D) adapted to the assessment of anticytomegaloviral drug combinations in vitro. (A) Schematic depiction of exemplary concentration combinations of drugs A (blue) and B (grey) employed in the Bliss independence checkerboard assay. (B) Exemplary synergistic result of a checkerboard assay. Positive synergy volume (volume above the 0 plane, green) as well as negative synergy volume (volume below 0 plane, red) are given in the table below. (C) Schematic depiction of exemplary concentration combinations of drugs A (blue) and B (grey) employed in the Loewe additivity fixed-dose assay. (D) Exemplary synergistic result of a fixed-dose assay. CI values at 50%, 75%, 90% and 95% virus inhibition as well as the calculated weighted CI value (CIwt) are given in the table below. CIwt was calculated as (1 × CI50 + 2 × CI75 + 3 × CI90 + 4 × CI95)/10. Note that the data shown in panels B and C are exemplary data from Figure 5 and Figure 6. CI, combination index.
Figure 5
Figure 5
Bliss independence checkerboard assay results (HCMV-infected HFFs in all cases). (A) MBV + GCV (replicate I); (B) MBV + GCV (replicate II); (C) ABE + GCV (replicate I); (D) ABE + GCV (replicate II); (E) ABE + LDC4297 (replicate I); (F) ABE +LDC4297 (replicate II); (G) ABE + MBV (replicate I); (H) ABE + MBV (replicate II); (I) TF27 + GCV (replicate I); (K) TF27 + GCV (replicate II); (L) TF27 + LDC4297; (M) TF27 + LMV; (N) MBV + LDC4297 (replicate I); (O) MBV + LDC4297 (replicate II).
Figure 5
Figure 5
Bliss independence checkerboard assay results (HCMV-infected HFFs in all cases). (A) MBV + GCV (replicate I); (B) MBV + GCV (replicate II); (C) ABE + GCV (replicate I); (D) ABE + GCV (replicate II); (E) ABE + LDC4297 (replicate I); (F) ABE +LDC4297 (replicate II); (G) ABE + MBV (replicate I); (H) ABE + MBV (replicate II); (I) TF27 + GCV (replicate I); (K) TF27 + GCV (replicate II); (L) TF27 + LDC4297; (M) TF27 + LMV; (N) MBV + LDC4297 (replicate I); (O) MBV + LDC4297 (replicate II).
Figure 6
Figure 6
Loewe additivity fixed-dose assay results. Numbers in brackets indicate fixed ratio of drugs in the respective combination. (A) MBV + GCV (1:1) against HCMV in HFFs; (B) ABE + GCV (1:1) against HCMV in HFFs; (C) ABE + GCV (1:1) against MCMV in MEFs; (D) ABE + LDC4297 (100:1) against HCMV in HFFs; (E) GCV + LDC4297 (100:1) against HCMV in HFFs; (F) GCV + LDC4297 (100:1) against MCMV in MEFs; (G) TF27 + GCV (1:100) against HCMV in HFFs; (H) MBV + LDC4297 (50:1) against HCMV in HFFs; (I) MBV + LDC4297 (100:1) against HCMV in HFFs; (K) MBV + LDC4297 (100:1) against MCMV-UL97 in MEFs. (L) MBV + LDC4297 (100:1) against HCMV TB40 in ARPE-19 cells. Data are presented as mean CI values ± SD, extrapolated to 50%, 75%, 90% and 95% virus inhibition across the number of individual experiments (n = 1 to n = 3).
Figure 7
Figure 7
Comparative alignment of HCMV-specific Loewe fixed-dose and Bliss checkerboard results. X-axis value of each drug combination indicates mean synergy volume across checkerboard assays (positive and negative synergy volumes were added together to acquire one plottable value). Y-axis value indicates mean CIwt across fixed-dose assays. Colored bars represent ranges of antagonistic (blue), additive (white) and synergistic (red) interactions for each approach (specified in Table 2 and Table 3). Solid blue/red fields within the chart indicate overlapping antagonistic/synergistic range of both approaches, respectively; dashed blue/red areas designate antagonistic or synergistic ranges in one method with additive values in the other method.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Cannon M.J., Schmid D.S., Hyde T.B. Review of cytomegalovirus seroprevalence and demographic characteristics associated with infection. Rev. Med. Virol. 2010;20:202–213. doi: 10.1002/rmv.655. - DOI - PubMed
    1. Mocarski E.S., Shenk T., Griffiths P.D., Pass R.F. Fields Virology. 6th ed. Wolters Kluwer Health/Lippincott Williams & Wilkins; Philadelphia, PA, USA: 2013. Cytomegaloviruses; pp. 1960–2014.
    1. Griffiths P., Baraniak I., Reeves M. The pathogenesis of human cytomegalovirus. J. Pathol. 2015;235:288–297. doi: 10.1002/path.4437. - DOI - PubMed
    1. Sever J.L., Rakusan T.A., Ellaurie M., Frenkel N., Wyatt L.S., Campos J.M., O’Donnell R.M., Price M.V. Coinfection with herpesviruses in young children of HIV-infected women. Pediatr. AIDS HIV Infect. 1995;6:75–82. - PubMed
    1. Meesing A., Razonable R.R. Pharmacologic and immunologic management of cytomegalovirus infection after solid organ and hematopoietic stem cell transplantation. Expert Rev. Clin. Pharmacol. 2018;11:773–788. doi: 10.1080/17512433.2018.1501557. - DOI - PubMed

MeSH terms