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 optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Additional information

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

References

  1. 1.

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

  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).

  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).

  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).

  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).

  6. 6.

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

  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).

  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).

  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).

  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).

  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).

  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).

  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).

  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).

  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).

  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).

  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).

  20. 20.

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

  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).

  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).

  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).

  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).

  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).

  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).

  28. 28.

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

  29. 29.

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

  30. 30.

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

  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).

  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).

  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).

  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).

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

Author notes

    • Thomas D. Otto

    Present address: Centre of Immunobiology, Institute of Infection, Immunity & Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK

Affiliations

  1. Drug Discovery Unit, Wellcome Centre for Anti-Infectives Research, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, UK

    • Susan Wyllie
    • , Michael Thomas
    • , Stephen Patterson
    • , Manu De Rycker
    • , Michael D. Urbaniak
    • , Laste Stojanovski
    • , Frederick R. C. Simeons
    • , Sujatha Manthri
    • , Lorna M. MacLean
    • , Fabio Zuccotto
    • , Nadine Homeyer
    • , Lalitha Sastry
    • , Sebastian Albrecht
    • , David W. Gray
    • , Paul G. Wyatt
    • , Michael A. J. Ferguson
    • , Alan H. Fairlamb
    • , Kevin D. Read
    •  & Ian H. Gilbert
  2. Global Health R&D, GlaxoSmithKline, Tres Cantos, Spain

    • Sabrinia Crouch
    • , Jose M. Fiandor
    •  & Timothy J. Miles
  3. David Jack Centre for R&D, GlaxoSmithKline, Ware, UK

    • Rhiannon Lowe
    •  & Stephanie Gresham
  4. Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster, UK

    • Michael D. Urbaniak
  5. Wellcome Sanger Institute, Cambridge, UK

    • Thomas D. Otto
    •  & Matt Berriman
  6. Cellzome GmbH, A GlaxoSmithKline Company, Heidelberg, Germany

    • Hannah Pflaumer
    • , Markus Boesche
    • , Gerard Drewes
    •  & Sonja Ghidelli-Disse
  7. GlaxoSmithKline, New Frontiers Science Park, Harlow, UK

    • Paul Connolly
  8. Global Health R&D, GlaxoSmithKline, Stockley Park West, Uxbridge, UK

    • Susan Dixon

Authors

  1. Search for Susan Wyllie in:

  2. Search for Michael Thomas in:

  3. Search for Stephen Patterson in:

  4. Search for Sabrinia Crouch in:

  5. Search for Manu De Rycker in:

  6. Search for Rhiannon Lowe in:

  7. Search for Stephanie Gresham in:

  8. Search for Michael D. Urbaniak in:

  9. Search for Thomas D. Otto in:

  10. Search for Laste Stojanovski in:

  11. Search for Frederick R. C. Simeons in:

  12. Search for Sujatha Manthri in:

  13. Search for Lorna M. MacLean in:

  14. Search for Fabio Zuccotto in:

  15. Search for Nadine Homeyer in:

  16. Search for Hannah Pflaumer in:

  17. Search for Markus Boesche in:

  18. Search for Lalitha Sastry in:

  19. Search for Paul Connolly in:

  20. Search for Sebastian Albrecht in:

  21. Search for Matt Berriman in:

  22. Search for Gerard Drewes in:

  23. Search for David W. Gray in:

  24. Search for Sonja Ghidelli-Disse in:

  25. Search for Susan Dixon in:

  26. Search for Jose M. Fiandor in:

  27. Search for Paul G. Wyatt in:

  28. Search for Michael A. J. Ferguson in:

  29. Search for Alan H. Fairlamb in:

  30. Search for Timothy J. Miles in:

  31. Search for Kevin D. Read in:

  32. Search for Ian H. Gilbert in:

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.

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.

Corresponding authors

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

Extended data figures and tables

  1. 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.

  2. 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.

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

Supplementary information

  1. 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.

  2. Reporting Summary

  3. Supplementary Data

    This file contains Proteomic data from the work at Cellzome.

Source Data

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/s41586-018-0356-z

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.