Crystal structure of chagasin, the endogenous cysteine-protease inhibitor from Trypanosoma cruzi

Trypanosoma cruzi chagasin belongs to a recently discovered family of cysteine protease inhibitors found in lower eukaryotes and prokaryotes but not in mammals. Chagasin binds tightly to cruzain, the major lysosomal T. cruzi cysteine protease, involved with infectivity and survival of the parasite in mammalian host cells. In the scope of a project to characterize proteins diferentially expressed during T. cruzi metacyclogenesis, we have determined the crystal structure of chagasin, which is now the first X-ray structure of a chagasin-like cysteine protease inhibitor to be reported. The structure was solved by the SIRAS method and refined at 1.7A resolution and a comparison with the two NMR structures available revealed some differences in the loops involved in binding to cysteine proteases. The highly flexible loop 4 could be entirely modeled and residues 29-33 from loop 2 form a 3(10)-helix structure that may be important to stabilize the loop conformation. Chagasin crystal structure was docked to the highest resolution structure available of cruzain and a model of chagasin-cruzain interaction was analyzed. The knowledge of the chagasin crystal structure may contribute to the elucidation of the molecular mechanism involved in the inhibition of cruzain and other T. cruzi cysteine proteases.     

Structural determinants of specificity in the cysteine protease cruzain.

The structure of cruzain, an essential protease from the parasite Trypanosoma cruzi, was determined by X-ray crystallography bound to two different covalent inhibitors. The cruzain S2 specificity pocket is able to productively bind both arginine and phenylalanine residues. The structures of cruzain bound to benzoyl-Arg-Ala-fluoromethyl ketone and benzoyl-Tyr-Ala-fluoromethyl ketone at 2.2 and 2.1 A, respectively, show a pH-dependent specificity switch. Glu 205 adjusts to restructure the S2 specificity pocket, conferring right binding to both hydrophobic and basic residues. Kinetic analysis of activated peptide substrates shows that substrates placing hydrophobic residues in the specificity pocket are cleaved at a broader pH range than hydrophilic substrates. These results demonstrate how cruzain binds both basic and hydrophobic residues and could be important for in vivo regulation of cruzain activity.     

A target within the target: probing cruzain's P1' site to define structural determinants for the Chagas' disease protease

BACKGROUND: Cysteine proteases of the papain superfamily are present in nearly all groups of eukaryotes and play vital roles in a wide range of biological processes and diseases, including antigen and hormone processing, bacterial infection, arthritis, osteoporosis, Alzheimer's disease and cancer-cell invasion. Because they are critical to the life-cycle progression of many pathogenic protozoa, they represent potential targets for selective inhibitors. Chagas' disease, the leading cause of death due to heart disease in Latin American countries, is transmitted by Trypanosoma cruzi. Cruzain is the major cysteine protease of T cruzi and has been the target of extensive structure-based drug design. RESULTS: High-resolution crystal structures of cruzain bound to a series of potent phenyl-containing vinyl-sulfone, sulfonate and sulfonamide inhibitors have been determined. The structures show a consistent mode of interaction for this family of inhibitors based on a covalent Michael addition formed at the enzyme's active-site cysteine, hydrophobic interactions in the S2 substrate-binding pocket and a strong constellation of hydrogen bonding in the S1' region. CONCLUSIONS: The series of vinyl-sulfone-based inhibitors examined in complex with cruzain was designed to probe recognition and binding potential of an aromatic-rich region of the enzyme. Analysis of the interactions formed shows that aromatic interactions play a less significant role, whereas the strength and importance of hydrogen bonding in the conformation adopted by the inhibitor upon binding to the enzyme was highlighted. A derivative of one inhibitor examined is currently under development as a therapeutic agent against Chagas' disease.     

Structure-based design, synthesis and evaluation of conformationally constrained cysteine protease inhibitors

The inhibition of cysteine proteases is being studied as a strategy to combat parasitic diseases such as Chagas' disease, leishmaniasis, and malaria. Cruzain is the major cysteine protease of Trypanosoma cruzi, the etiologic agent of Chagas' disease. A crystal structure of cruzain, covalently inactivated by fluoromethyl ketone inhibitor 1 (Cbz-Phe-Ala-FMK), was used as a template to design potential inhibitors. Conformationally constrained γ-lactams containing electrophilic aldehyde (12, 17, 18, 25, 26, and 29) or vinyl sulfone (43, 44, and 46) units were synthesized. Constrained lactam 26 had IC₅₀ values of ca. 20 nM against the Leishmania major protease and ca. 50 nM versus falcipain, an important cysteine protease isolated from Plasmodium falciparum. However, all of the conformationally constrained inhibitors were weak inhibitors of cruzain, compared to unconstrained peptide aldehyde (e.g. 5) and vinyl sulfone inhibitors (e.g. 48, which proved to be an excellent inhibitor of cruzain with an apparent second order inhibition rate constant (k_inact/K_i) of 634,000 s⁻¹M⁻¹). A significant reduction in activity was also observed with acyclic inhibitors 30 and 51 containing -methyl phenylalanine residues at the P₂ position. These data indicate that the pyrrolidinone ring, especially the quarternary center at P₂, interferes with the normal substrate binding mode with cruzain, but not with falcipain or the leishmania protease.     

Molecular Design,Synthesis and Trypanocidal Activity of Dipeptidyl Nitriles as Cruzain Inhibitors

A series of compounds based on the dipeptidyl nitrile scaffold were synthesized and assayed for their inhibitory activity against the T. cruzi cysteine protease cruzain. Structure activity relationships (SARs) were established using three, eleven and twelve variations respectively at the P1, P2 and P3 positions. A K _i value of 16 nM was observed for the most potent of these inhibitors which reflects a degree of non-additivity in the SAR. An X-ray crystal structure was determined for the ligand-protein complex for the structural prototype for the series. Twenty three inhibitors were also evaluated for their anti-trypanosomal effects and an EC₅₀ value of 28 μM was observed for the most potent of these. Although there remains scope for further optimization, the knowledge gained from this study is also transferable to the design of cruzain inhibitors based on warheads other than nitrile as well as alternative scaffolds.     

Potent second generation vinyl sulfonamide inhibitors of the trypanosomal cysteine protease cruzain

A new family of potent N-alkoxyvinylsulfonamide inhibitors of cruzain have been developed. Inhibitor 13 has a second order inactivation rate constant of 6,480,000s(-1)M(-1) versus cruzain, and is also highly effective against Trypanosoma cruzi trypomastigotes in a tissue culture assay.     

2D QSAR and similarity studies on cruzain inhibitors aimed at improving selectivity over cathepsin L

Hologram quantitative structure-activity relationships (HQSAR) were applied to a data set of 41 cruzain inhibitors. The best HQSAR model (Q(2)=0.77; R(2)=0.90) employing Surflex-Sim, as training and test sets generator, was obtained using atoms, bonds, and connections as fragment distinctions and 4-7 as fragment size. This model was then used to predict the potencies of 12 test set compounds, giving satisfactory predictive R(2) value of 0.88. The contribution maps obtained from the best HQSAR model are in agreement with the biological activities of the study compounds. The Trypanosoma cruzi cruzain shares high similarity with the mammalian homolog cathepsin L. The selectivity toward cruzain was checked by a database of 123 compounds, which corresponds to the 41 cruzain inhibitors used in the HQSAR model development plus 82 cathepsin L inhibitors. We screened these compounds by ROCS (Rapid Overlay of Chemical Structures), a Gaussian-shape volume overlap filter that can rapidly identify shapes that match the query molecule. Remarkably, ROCS was able to rank the first 37 hits as being only cruzain inhibitors. In addition, the area under the curve (AUC) obtained with ROCS was 0.96, indicating that the method was very efficient to distinguishing between cruzain and cathepsin L inhibitors.     

Uncovering false positives on a virtual screening search for cruzain inhibitors

Some unexpected promiscuous inhibitors were observed in a virtual screening protocol applied to select cruzain inhibitors from the ZINC database. Physical-chemical and pharmacophore model filters were used to reduce the database size. The selected compounds were docked into the cruzain active site. Six hit compounds were tested as inhibitors. Although the compounds were designed to be nucleophilically attacked by the catalytic cysteine of cruzain, three of them showed typical promiscuous behavior, revealing that false positives are a prevalent concern in VS programs.     

Crystal structures of the complex of porcine pancreatic elastase with two valine-derived benzoxazinone inhibitors

The crystal structures of porcine pancreatic elastase complexed to two similar benzoxazinone inhibitors are reported to 2.09 A and 1.76 A resolution, and refined to conventional R factors of 0.153 and 0.172.     

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

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 alpha-ketoamide-, alpha-ketoacid-, alpha-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 alpha-keto-based inhibitor scaffolds demonstrated anti-trypanosomal activity in cell culture. A 2.3A crystallographic structure of cruzain bound with one of the alpha-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.     

Looking at the proteases from a simple perspective

Proteases have received enormous interest from the research and medical communities because of their significant roles in several human diseases. Some examples include the involvement of thrombin in thrombosis, HIV-1 protease in Acquired Immune Deficiency Syndrome, cruzain in Trypanosoma cruzi infection, and membrane-type 1 matrix metalloproteinase in tumor invasion and metastasis. Many efforts has been undertaken to design effective inhibitors featuring potent inhibitory activity, specificity, and metabolic stability to those proteases involved in such pathologies. Protease inhibitors usually target the active site, but some of them act by other inhibitory mechanisms. The understanding of the structure-function relationships of proteases and inhibitors has an impact on new inhibitor drugs designing. In this paper, the structures of four proteases (thrombin, HIV-protease, cruzain, and a matrix metalloproteinase) are briefly reviewed, and used as examples of the importance of proteases for the development of new treatment strategies, leading to a longer and healthier life.     

Crystal structures of human dipeptidyl peptidase IV in its apo and diprotin B-complexed forms

Dipeptidyl peptidase IV （DPPIV）, which belongs to the prolyl oligopeptidase family of serine proteases, is known to have a variety of regulatory biological functions and has been shown to be implicated in type 2 diabetes. It is therefore important to develop selective human DPPIV （hDPPIV） inhibitors. In this study, we determined the crystal structure of apo hDPPIV at 1.9A resolution. Our high-resolution crystal structure of apo hDPPIV revealed the presence of sodium ion and glycerol molecules at the active site. In order to elucidate the hDPPIV binding mode and substrate specificity, we determined the crystal structure of hDPPIV-diprotin B （Val-Pro-Leu） complex at 2.1A resolution, and clarified the difference in binding mode between diprotin B and diprotin A （Ile-Pro-Ile） into the active site of hDPPIV. Comparison between our crystal structures and the reported apo hDPPIV structures revealed that positively charged functional groups and conserved water molecules contributed to the interaction of ligands with hDPPIV. These results are useful for the design of potent hDPPIV inhibitors.     

Novel non-peptidic vinylsulfones targeting the S2 and S3 subsites of parasite cysteine proteases

We describe here the identification of non-peptidic vinylsulfones that inhibit parasite cysteine proteases in vitro and inhibit the growth of Trypanosoma brucei brucei parasites in culture. A high resolution (1.75 Å) co-crystal structure of 8a bound to cruzain reveals how the non-peptidic P2/P3 moiety in such analogs bind the S2 and S3 subsites of the protease, effectively recapitulating important binding interactions present in more traditional peptide-based protease inhibitors and natural substrates.     

CoMFA and HQSAR of acylhydrazide cruzain inhibitors

An approach combining CoMFA and HQSAR methods was used to describe QSAR models for a series of cruzain inhibitors having the acylhydrazide framework. A CoMFA study using two alignment orientations (I and II), three different probe atoms and changes of the lattice spacing (1 and 2 A) was performed. Alignment II and an sp3 probe carbon atom yielded good cross-validation (q2=0.688) employing lattice spacing of 1 A. The best HQSAR model was generated using atoms, bond, and connectivity as fragment distinction and fragment size default (4-5) showing similar cross-validated value of CoMFA (q2=0.689). Based upon the information derived from CoMFA and HQSAR, we have identified some key features that may be used to design new acylhydrazide derivatives that may be more potent cruzain inhibitors.     

General solid-phase method to prepare novel cyclic ketone inhibitors of the cysteine protease cruzain

A series of constrained ketone-based inhibitors has been developed that show low nanomolar Ki values. These ketone inhibitors showed promising activity towards cruzain, the cysteine protease implicated in Chagas' disease. This series of constrained inhibitors, which can be accessed quickly and efficiently using a solid-phase combinatorial strategy, should be applicable to other members of the cysteine protease class.     

Crystal structure of phosphoglucose isomerase from Trypanosoma brucei complexed with glucose-6-phosphate at 1.6 A resolution

Enzymes of glycolysis in Trypanosoma brucei have been identified as potential drug targets for African sleeping sickness because glycolysis is the only source of ATP for the bloodstream form of this parasite. Several inhibitors were previously reported to bind preferentially to trypanosomal phosphoglucose isomerase (PGI, the second enzyme in glycolysis) than to mammalian PGIs, which suggests that PGI might make a good target for species-specific drug design. Herein, we report recombinant expression, purification, crystallization and X-ray crystal structure determination of T. brucei PGI. One structure solved at 1.6 A resolution contains a substrate, D-glucose-6-phosphate, in an extended conformation in the active site. A second structure solved at 1.9 A resolution contains a citrate molecule in the active site. The structures are compared with the crystal structures of PGI from humans and from Leishmania mexicana. The availability of recombinant tPGI and its first high-resolution crystal structures are initial steps in considering this enzyme as a potential drug target.     

Non-peptidic Cruzain Inhibitors with Trypanocidal Activity Discovered by Virtual Screening and In Vitro Assay

A multi-step cascade strategy using integrated ligand- and target-based virtual screening methods was developed to select a small number of compounds from the ZINC database to be evaluated for trypanocidal activity. Winnowing the database to 23 selected compounds, 12 non-covalent binding cruzain inhibitors with affinity values (K _i) in the low micromolar range (3–60 µM) acting through a competitive inhibition mechanism were identified. This mechanism has been confirmed by determining the binding mode of the cruzain inhibitor Nequimed176 through X-ray crystallographic studies. Cruzain, a validated therapeutic target for new chemotherapy for Chagas disease, also shares high similarity with the mammalian homolog cathepsin L. Because increased activity of cathepsin L is related to invasive properties and has been linked to metastatic cancer cells, cruzain inhibitors from the same library were assayed against it. Affinity values were in a similar range (4–80 µM), yielding poor selectivity towards cruzain but raising the possibility of investigating such inhibitors for their effect on cell proliferation. In order to select the most promising enzyme inhibitors retaining trypanocidal activity for structure-activity relationship (SAR) studies, the most potent cruzain inhibitors were assayed against T. cruzi-infected cells. Two compounds were found to have trypanocidal activity. Using compound Nequimed42 as precursor, an SAR was established in which the 2-acetamidothiophene-3-carboxamide group was identified as essential for enzyme and parasite inhibition activities. The IC₅₀ value for compound Nequimed42 acting against the trypomastigote form of the Tulahuen lacZ strain was found to be 10.6±0.1 µM, tenfold lower than that obtained for benznidazole, which was taken as positive control. In addition, by employing the strategy of molecular simplification, a smaller compound derived from Nequimed42 with a ligand efficiency (LE) of 0.33 kcal mol⁻¹ atom⁻¹ (compound Nequimed176) is highlighted as a novel non-peptidic, non-covalent cruzain inhibitor as a trypanocidal agent candidate for optimization.     

Re-engineering of human urokinase provides a system for structure-based drug design at high resolution and reveals a novel structural subsite

Inhibition of urokinase has been shown to slow tumor growth and metastasis. To utilize structure-based drug design, human urokinase was re-engineered to provide a more optimal crystal form. The redesigned protein consists of residues Ile(16)-Lys(243) (in the chymotrypsin numbering system; for the urokinase numbering system it is Ile(159)-Lys(404)) and two point mutations, C122A and N145Q (C279A and N302Q). The protein yields crystals that diffract to ultra-high resolution at a synchrotron source. The native structure has been refined to 1.5 A resolution. This new crystal form contains an accessible active site that facilitates compound soaking, which was used to determine the co-crystal structures of urokinase in complex with the small molecule inhibitors amiloride, 4-iodo-benzo(b)thiophene-2-carboxamidine and phenylguanidine at 2. 0-2.2 A resolution. All three inhibitors bind at the primary binding pocket of urokinase. The structures of amiloride and 4-iodo-benzo(b)thiophene-2-carboxamidine also reveal that each of their halogen atoms are bound at a novel structural subsite adjacent to the primary binding pocket. This site consists of residues Gly(218), Ser(146), and Cys(191)-Cys(220) and the side chain of Lys(143). This pocket could be utilized in future drug design efforts. Crystal structures of these three inhibitors in complex with urokinase reveal strategies for the design of more potent nonpeptidic urokinase inhibitors.     

Synthesis of macrocyclic trypanosomal cysteine protease inhibitors

The importance of cysteine proteases in parasites, compounded with the lack of redundancy compared to their mammalian hosts makes proteases attractive targets for the development of new therapeutic agents. The binding mode of K11002 to cruzain, the major cysteine protease of Trypanosoma cruzi was used in the design of conformationally constrained inhibitors. Vinyl sulfone-containing macrocycles were synthesized via olefin ring-closing metathesis and evaluated against cruzain and the closely related cysteine protease, rhodesain.