Development of α-keto-based inhibitors of cruzain, a cysteine protease implicated in Chagas disease

doi:10.1016/j.bmc.2004.12.053

Bioorganic & Medicinal Chemistry

Volume 13, Issue 6, 15 March 2005, Pages 2141-2156

https://doi.org/10.1016/j.bmc.2004.12.053 Get rights and content

Abstract

Trypanosoma cruzi, a protozoan parasite, is the causative agent of Chagas disease, a major cause of cardiovascular disease in many Latin American countries. There is an urgent need to develop an improved therapy due to the toxicity of existing drugs and emerging drug resistance. Cruzain, the primary cysteine protease of T. cruzi, is essential for the survival of the parasite in host cells and therefore is an important target for the development of inhibitors as potential therapeutics. A novel series of α-ketoamide-, α-ketoacid-, α-ketoester-, and aldehyde-based inhibitors of cruzain has been developed. The inhibitors were identified by screening protease targeted small molecule libraries and systematically optimizing the P1, P2, P3, and P1′ residues using specific structure-guided methods. A total of 20 compounds displayed picomolar potency in in vitro assays and three inhibitors representing different α-keto-based inhibitor scaffolds demonstrated anti-trypanosomal activity in cell culture. A 2.3 Å crystallographic structure of cruzain bound with one of the α-ketoester analogs is also reported. The structure and kinetic assay data illustrate the covalent binding, reversible inhibition mechanism of the inhibitor. Information on the compounds reported here will be useful in the development of new lead compounds as potential therapeutic agents for the treatment of Chagas disease and as biological probes to study the role that cruzain plays in the pathology. This study also demonstrates the validity of structure-guided approaches to focused library design and lead compound optimization.

Graphical abstract

A series of novel α-keto based inhibitors has been developed that covalently binds to the active site of cruzain, a cysteine protease crucial for the life cycle of Trypanosoma cruzi, the causative agent for Chagas disease.

Introduction

Chagas disease or American trypanosomiasis, is one of the most threatening endemics in Central and South America. Approximately 16–18 million people are infected, resulting in adverse health events such as heart failure and more than 50,000 deaths each year.¹ It is thought that another 100 million people are at risk of infection.¹ Due to the toxicity of existing drugs combined with the advent of drug resistance, treatment for this debilitating, often chronic illness is woefully inadequate.¹ Trypanosoma cruzi, a parasitic protozoan, is the causative agent of the disease. Its infectious trypomastigote form is transmitted to human hosts through the triatomine ‘kissing bug’ vectors. Trypomastigotes transform into the intracellular amastigotes after invading cardiac muscle cells through the bloodstream. Amastigotes complete the infectious life cycle by multiplying in the cell, transforming back to infectious trypomastigotes and rupturing the cell to release an amplified quantity of the infectious form back into the bloodstream.²

Cruzain, the primary cysteine protease of T. cruzi, is expressed throughout the life cycle and is known to be essential for the survival of the parasite within host cells.³ In addition to its obvious role in parasite nutrition, cruzain has been proposed to be involved in the penetration of the parasite into the host cell and in digestion of immunoglobulins as a defense mechanism. Some inhibitors of cruzain have been shown to successfully treat animal models of Chagas disease by blocking the parasitic life cycle.⁴ Thus, cruzain presents itself as an interesting target for the development of potential therapeutics for the treatment of the disease. Cruzain was initially discovered from cell-free extracts,⁵ and subsequently heterologously expressed in Escherichia coli.⁶ The X-ray crystallographic structures of a recombinant truncated form of the enzyme in complex with several small molecule inhibitors have been determined.⁷ The overall folding pattern and the arrangement of the active site residues are similar to those in papain. As in most papain family proteases, the S2 subsite of the enzyme is known to be the predominating specificity determinant. Cruzain contains a unique C-terminal extension of 130 amino acids, which is retained in native forms. The enzyme is inhibited by organomercurial reagents, E-64, Tos-Lys-CH₂Cl, leupeptin, a number of peptidyl chloromethanes, and peptidyl fluoromethane derivatives, vinyl sulfones, thio semicarbazones, cystatins, stefins, and kininogens.2, 4 Covalent inhibitors such as peptidyl epoxy ketones,⁸ aldehydes,⁹ and vinyl sulfones,⁹ have also been reported to inhibit cruzain. However, published information on cruzain inhibitory activity from α-keto based compounds has been very limited.10, 11

Due to its essential function in the parasite’s life cycle and therefore in the propagation of Chagas disease, cruzain was elected as a target to develop inhibitors through in vitro screening of a series of protease specific small molecule libraries and structure-guided optimization. The libraries were designed to initially focus screening on known pharmacologically active and target class specific small molecules.¹⁰ Additional efficiencies could also be realized from this approach due to the comprehensive understanding of the automated process chemistry.12, 13 The in vitro screening of these libraries for cruzain inhibitory activity led to the identification of novel α-ketoamide-based cruzain inhibitors. The screening also facilitated the exploration of closely related α-ketoamide-, α-ketoacid-, α-ketoester-, and aldehyde-based analogs. Identification of an inhibitor in the arrayed analog libraries provides rapid access to a database of structure–activity relationships (SAR). This information is then used to guide the systematic optimization of each inhibitor scaffold. Since the papain family cysteine proteases including cruzain (clan CA) share a common three-dimensional structure and very similar biochemical characteristics, this approach and the inhibitor scaffolds developed for cruzain can be readily applied to other physiologically important clan CA proteases.

Section snippets

Identification of α-ketoamide-based inhibitors of cruzain

The in vitro screening of protease targeted libraries for inhibitory activity against cruzain identified novel peptidyl α-ketoamide-based compounds as covalent inhibitors of the enzyme (Fig. 1). Further exploration of this structural motif led to the synthesis of a small set of novel α-ketoester-, α-ketoacid, and α-ketoamide-based compounds (Table 1). A modified Dakin–West reaction was used for the preparation of the compounds.¹⁴ It should be noted that this synthetic approach produces a

Discussion

Herein, we have described the synthesis, identification, optimization, and crystallographically determined binding mode of a new series of α-keto-based cruzain inhibitors. As various essential roles are revealed that proteases play in both normal and pathological states, specific protease inhibitors are being developed and are proving to be promising lead compounds for drug development. The approach has been greatly augmented by the advance of structural biology, high-speed parallel synthesis,

Conclusion

A novel series of peptide based α-ketoamide, α-ketoester, α-ketoacid, and aldehyde analogs has been developed and shown to be potent inhibitors of cruzain, a major cysteine protease involved in Chagas disease. To aid the development and the optimization of the inhibitors at the P1, P2, P3, and P1′ positions, a specific SAR has been established using high speed chemistry, three-dimensional enzyme structure guided modeling, and docking approaches. A total of 20 compounds displayed picomolar

Materials

Cruzain was expressed, purified as previously described and stored in 20 mM bis–tris buffer pH 5.8 at 4 °C.6, 16, 29, 30 All chemicals for buffer preparation were purchased from Sigma unless described otherwise. The substrate, Cbz-Phe-Arg-AMC, was purchased from molecular probes and 10 mM stock solution in neat DMSO was stored at −20 °C.

In vitro cruzain activity assay

A 96-well formatted fluorescent assay was used to monitor the time course of the activity assay. For K_m determination, initial velocities at a total of 11 substrate

Acknowledgements

The authors would like to thank the Analytical and Production Groups at ArQule, Inc. for their support. This work was supported by a NIH grant P01-AI35707 to C.S.C. and J.C.E.

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^†: These authors contributed equally to this work.

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Development of α-keto-based inhibitors of cruzain, a cysteine protease implicated in Chagas disease

Abstract

Graphical abstract

Introduction

Section snippets

Identification of α-ketoamide-based inhibitors of cruzain

Discussion

Conclusion

Materials

In vitro cruzain activity assay

Acknowledgements

J. Biol. Chem.

J. Mol. Biol.

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem.

Struct. Fold. Des.

Bioorg. Med. Chem.

Arch. Biochem. Biophys.

J. Mol. Biol.

Chem. Biol.

J. Biol. Chem.

J. Med. Chem.

J. Cell Sci.

J. Exp. Med.

J. Protozool.

Med. Chem. Res.

J. Comb. Chem.

Curr. Drug Discov.

J. Med. Chem.