Cyclin-dependent kinase 12 is a drug target for visceral leishmaniasis

Abstract

Visceral leishmaniasis causes considerable mortality and morbidity in many parts of the world. There is an urgent need for the development of new, effective treatments for this disease. Here we describe the development of an anti-leishmanial drug-like chemical series based on a pyrazolopyrimidine scaffold. The leading compound from this series (7, DDD853651/GSK3186899) is efficacious in a mouse model of visceral leishmaniasis, has suitable physicochemical, pharmacokinetic and toxicological properties for further development, and has been declared a preclinical candidate. Detailed mode-of-action studies indicate that compounds from this series act principally by inhibiting the parasite cdc-2-related kinase 12 (CRK12), thus defining a druggable target for visceral leishmaniasis.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: The evolution of the pyrazolopyrimidine series to give the development compound 7.
Fig. 2: Efficacy of compound 7 in a mouse model of visceral leishmaniasis.
Fig. 3: Studies to validate the molecular target of the pyrazolopyrimidine series.
Fig. 4: Identification of cyclin-dependent related kinases as targets of the pyrazolopyrimidine series using a chemoproteomic approach.
Fig. 5: Docking studies for compounds 4 and 7.

References

  1. 1.

    Alvar, J. et al. Leishmaniasis worldwide and global estimates of its incidence. PLoS ONE 7, e35671 (2012).

    ADS  Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. 2.

    Ritmeijer, K. & Davidson, R. N. Royal Society of Tropical Medicine and Hygiene joint meeting with Médecins Sans Frontières at Manson House, London, 20 March 2003: field research in humanitarian medical programmes. Médecins Sans Frontières interventions against kala-azar in the Sudan, 1989–2003. Trans. R. Soc. Trop. Med. Hyg. 97, 609–613 (2003).

    Article  PubMed  CAS  Google Scholar 

  3. 3.

    Sundar, S. et al. Efficacy of miltefosine in the treatment of visceral leishmaniasis in India after a decade of use. Clin. Infect. Dis. 55, 543–550 (2012).

    Article  PubMed  CAS  Google Scholar 

  4. 4.

    den Boer, M. L., Alvar, J., Davidson, R. N., Ritmeijer, K. & Balasegaram, M. Developments in the treatment of visceral leishmaniasis. Expert Opin. Emerg. Drugs 14, 395–410 (2009).

    Article  CAS  Google Scholar 

  5. 5.

    Mueller, M. et al. Unresponsiveness to AmBisome in some Sudanese patients with kala-azar. Trans. R. Soc. Trop. Med. Hyg. 101, 19–24 (2007).

    Article  PubMed  CAS  Google Scholar 

  6. 6.

    Khare, S. et al. Proteasome inhibition for treatment of leishmaniasis, Chagas disease and sleeping sickness. Nature 537, 229–233 (2016).

    ADS  Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. 7.

    Don, R. & Ioset, J.-R. Screening strategies to identify new chemical diversity for drug development to treat kinetoplastid infections. Parasitology 141, 140–146 (2014).

    Article  PubMed  Google Scholar 

  8. 8.

    Woodland, A. et al. From on-target to off-target activity: identification and optimisation of Trypanosoma brucei GSK3 inhibitors and their characterisation as anti-Trypanosoma brucei drug discovery lead molecules. ChemMedChem 8, 1127–1137 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. 9.

    De Rycker, M. et al. Comparison of a high-throughput high-content intracellular Leishmania donovani assay with an axenic amastigote assay. Antimicrob. Agents Chemother. 57, 2913–2922 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. 10.

    Nühs, A. et al. Development and validation of a novel Leishmania donovani screening cascade for high-throughput screening using a novel axenic assay with high predictivity of leishmanicidal intracellular activity. PLoS Negl. Trop. Dis. 9, e0004094 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. 11.

    Henderson, C. J., Pass, G. J. & Wolf, C. R. The hepatic cytochrome P450 reductase null mouse as a tool to identify a successful candidate entity. Toxicol. Lett. 162, 111–117 (2006).

    Article  PubMed  CAS  Google Scholar 

  12. 12.

    Miles, T. J. & Thomas, M. G. Pyrazolo[3,4-d]pyrimidin derivative and its use for the treatment of leishmaniasis. WIPO patent WO/2016/116563 (2016).

  13. 13.

    Ding, Q., Jiang, N. & Roberts, J. L. Pyrazolo pyrimidines. WIPO patent WO/2005/121107 (2005).

  14. 14.

    Bantscheff, M. et al. Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors. Nat. Biotechnol. 25, 1035–1044 (2007).

    Article  PubMed  CAS  Google Scholar 

  15. 15.

    Terstappen, G. C., Schlüpen, C., Raggiaschi, R. & Gaviraghi, G. Target deconvolution strategies in drug discovery. Nat. Rev. Drug Discov. 6, 891–903 (2007).

    Article  PubMed  CAS  Google Scholar 

  16. 16.

    Park, J., Koh, M. & Park, S. B. From noncovalent to covalent bonds: a paradigm shift in target protein identification. Mol. Biosyst. 9, 544–550 (2013).

    Article  PubMed  CAS  Google Scholar 

  17. 17.

    Lee, H. & Lee, J. W. Target identification for biologically active small molecules using chemical biology approaches. Arch. Pharm. Res. 39, 1193–1201 (2016).

    Article  PubMed  CAS  Google Scholar 

  18. 18.

    Ursu, A. & Waldmann, H. Hide and seek: identification and confirmation of small molecule protein targets. Bioorg. Med. Chem. Lett. 25, 3079–3086 (2015).

    Article  PubMed  CAS  Google Scholar 

  19. 19.

    Urbaniak, M. D., Guther, M. L. S. & Ferguson, M. A. J. Comparative SILAC proteomic analysis of Trypanosoma brucei bloodstream and procyclic lifecycle stages. PLoS ONE 7, e36619 (2012).

    ADS  Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. 20.

    Liu, Y. & Gray, N. S. Rational design of inhibitors that bind to inactive kinase conformations. Nat. Chem. Biol. 2, 358–364 (2006).

    Article  PubMed  CAS  Google Scholar 

  21. 21.

    Zhang, L. et al. Design, synthesis, and biological evaluation of pyrazolopyrimidine-sulfonamides as potent multiple-mitotic kinase (MMK) inhibitors (part I). Bioorg. Med. Chem. Lett. 21, 5633–5637 (2011).

    Article  PubMed  CAS  Google Scholar 

  22. 22.

    Freyne, E. J. E. et al. Pyrazolopyrimidines as cell cycle kinase inhibitors. WIPO patent WO/2006/074984 (2006).

  23. 23.

    Rogers, M. B. et al. Chromosome and gene copy number variation allow major structural change between species and strains of Leishmania. Genome Res. 21, 2129–2142 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. 24.

    Downing, T. et al. Whole genome sequencing of multiple Leishmania donovani clinical isolates provides insights into population structure and mechanisms of drug resistance. Genome Res. 21, 2143–2156 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. 25.

    Monnerat, S. et al. Identification and functional characterisation of CRK12:CYC9, a novel cyclin-dependent kinase (CDK)–cyclin complex in Trypanosoma brucei. PLoS ONE 8, e67327 (2013).

    ADS  Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. 26.

    Hassan, P., Fergusson, D., Grant, K. M. & Mottram, J. C. The CRK3 protein kinase is essential for cell cycle progression of Leishmania mexicana. Mol. Biochem. Parasitol. 113, 189–198 (2001).

    Article  PubMed  CAS  Google Scholar 

  27. 27.

    Tu, X. & Wang, C. C. Pairwise knockdowns of cdc2-related kinases (CRKs) in Trypanosoma brucei identified the CRKs for G1/S and G2/M transitions and demonstrated distinctive cytokinetic regulations between two developmental stages of the organism. Eukaryot. Cell 4, 755–764 (2005).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. 28.

    Médard, G. et al. Optimized chemical proteomics assay for kinase inhibitor profiling. J. Proteome Res. 14, 1574–1586 (2015).

    Article  PubMed  CAS  Google Scholar 

  29. 29.

    Bergamini, G. et al. A selective inhibitor reveals PI3Kγ dependence of TH17 cell differentiation. Nat. Chem. Biol. 8, 576–582 (2012).

    Article  PubMed  CAS  Google Scholar 

  30. 30.

    Bantscheff, M. et al. Chemoproteomics profiling of HDAC inhibitors reveals selective targeting of HDAC complexes. Nat. Biotechnol. 29, 255–265 (2011).

    Article  PubMed  CAS  Google Scholar 

  31. 31.

    Bradley, D. J. & Kirkley, J. Regulation of Leishmania populations within the host. I. The variable course of Leishmania donovani infections in mice. Clin. Exp. Immunol. 30, 119–129 (1977).

    PubMed  PubMed Central  CAS  Google Scholar 

  32. 32.

    Croft, S. L., Snowdon, D. & Yardley, V. The activities of four anticancer alkyllysophospholipids against Leishmania donovani, Trypanosoma cruzi and Trypanosoma brucei. J. Antimicrob. Chemother. 38, 1041–1047 (1996).

    Article  PubMed  CAS  Google Scholar 

  33. 33.

    Seifert, K. & Croft, S. L. In vitro and in vivo interactions between miltefosine and other antileishmanial drugs. Antimicrob. Agents Chemother. 50, 73–79 (2006).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. 34.

    Escobar, P., Yardley, V. & Croft, S. L. Activities of hexadecylphosphocholine (miltefosine), AmBisome, and sodium stibogluconate (Pentostam) against Leishmania donovani in immunodeficient scid mice. Antimicrob. Agents Chemother. 45, 1872–1875 (2001).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgements

The authors acknowledge the Wellcome Trust for funding (grants 092340, 105021, 100476, 101842, 079838 and 098051).

Reviewer information

Nature thanks R. Guy, J. Mottram and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Affiliations

Authors

Contributions

In brief, S.W., M.D.U., T.D.O, H.P., M.Bo. and S.M. carried out the mode of action, genomic and proteomic studies. M.T., S.P. and S.A. carried out the chemistry studies. M.D.R., S.M., L.M.M. and L.Sa. carried out the parasite screening. S.C., L.S., F.R.C.S. and P.C. carried out the drug metabolism and pharmacokinetic studies. F.Z. and N.H. carried out the molecular modelling. R.L. and S.G. carried out the safety studies. S.W., M.T., S.P., M.D.R., R.L., S.G., M.D.U., L.M.M., F.Z., M.Be., G.D., D.W.G., S.G.-D., S.D., J.M.F., P.W.G., M.A.J.F., A.H.F., T.J.M., K.D.R. and I.H.G. designed experiments, managed parts of the project and contributed to the writing. See Supplementary Information for further details.

Corresponding authors

Correspondence to Timothy J. Miles or Kevin D. Read or Ian H. Gilbert.

Ethics declarations

Competing interests

These authors have shares in GlaxoSmithKline: P.G.W., S.D., T.J.M., K.D.R., S.C., R.L., S.G., M.Bo., H.P., P.C., G.D., D.G., S.G.-D. and J.M.F. The other authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Rate-of-kill of L. donovani axenic amastigotes by compound 7.

Chart shows relative luminescence units (RLU) versus time from axenic amastigote rate-of-kill experiment with compound 7 (representative results for one of two independent experiments are shown; data are mean and s.d. of three technical replicates). Concentrations are as follows (µM): 50, open circles; 16.7, closed circles; 5.6, open squares; 1.85, closed squares; 0.62, open triangles; 0.21, closed triangles; 0.069, open inverted triangles; 0.023, closed inverted triangles, 0.0076, open diamonds and 0.0025, closed diamonds.

Extended Data Fig. 2 Linker-containing target molecules synthesized for chemical proteomic experiments and their corresponding EC50 values.

Potencies of the compounds in the cidal axenic and intra-macrophage assays are shown; data are from at least three independent replicates.

Extended Data Table 1 Activity of compound 7 and miltefosine against a panel of Leishmania clinical isolates
Extended Data Table 2 Solubility of compound 7 in simulated physiological media
Extended Data Table 3 In vitro metabolic stability data for compound 7
Extended Data Table 4 Drug metabolism and pharmacokinetics data for compound 7
Extended Data Table 5 Sensitivity of wild-type and drug-resistant promastigotes to compounds within the series
Extended Data Table 6 Sensitivity of wild-type and compound 5-resistant intra-macrophage amastigotes to the compound series

Supplementary information

Supplementary Information

This file contains author contributions, methods, characterisation of compounds and ethical statements. It also contains supplementary figures S1-S74 and supplementary tables S1-S9.

Reporting Summary

Supplementary Data

This file contains Proteomic data from the work at Cellzome.

Source Data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wyllie, S., Thomas, M., Patterson, S. et al. Cyclin-dependent kinase 12 is a drug target for visceral leishmaniasis. Nature 560, 192–197 (2018). https://doi.org/10.1038/s41586-018-0356-z

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.