Discovery of potent thiosemicarbazone inhibitors of rhodesain and cruzain

Herein we report the synthesis and evaluation of a series of thiosemicarbazones as potential inhibitors of cysteine proteases relevant to parasitic diseases. Derivatives of thiosemicarbazone 1 were discovered to be potent inhibitors of cruzain and rhodesain, crucial proteases in the life cycles of Trypanosoma cruzi and T. brucei rhodesiense, the organisms causing Chagas' disease and sleeping sickness. However, the entire series had only modest potency against falcipain-2, an essential protease for Plasmodium falciparum, the organism causing malaria. Among the active inhibitors, several potently inhibited proliferation of cultures of T. brucei. However, only modest activity was observed in inhibition of proliferation of T. cruzi or P. falciparum.     

Crystal structures of reversible ketone-Based inhibitors of the cysteine protease cruzain

The crystal structures of two hydroxymethyl ketone inhibitors complexed to the cysteine protease cruzain have been determined at 1.1 and 1.2 A resolution, respectively. These high resolution crystal structures provide the first structures of non-covalent inhibitors bound to cruzain. A series of compounds were prepared and tested based upon the structures providing further insight into the key binding interactions.     

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.     

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.     

The Trypanosoma cruzi protease cruzain mediates immune evasion

Trypanosoma cruzi is the causative agent of Chagas' disease. Novel chemotherapy with the drug K11777 targets the major cysteine protease cruzain and disrupts amastigote intracellular development. Nevertheless, the biological role of the protease in infection and pathogenesis remains unclear as cruzain gene knockout failed due to genetic redundancy. A role for the T. cruzi cysteine protease cruzain in immune evasion was elucidated in a comparative study of parental wild type- and cruzain-deficient parasites. Wild type T. cruzi did not activate host macrophages during early infection (<60 min) and no increase in ～P iκB was detected. The signaling factor NF-κB P65 colocalized with cruzain on the cell surface of intracellular wild type parasites, and was proteolytically cleaved. No significant IL-12 expression occurred in macrophages infected with wild type T. cruzi and treated with LPS and BFA, confirming impairment of macrophage activation pathways. In contrast, cruzain-deficient parasites induced macrophage activation, detectable iκB phosphorylation, and nuclear NF-κB P65 localization. These parasites were unable to develop intracellularly and survive within macrophages. IL 12 expression levels in macrophages infected with cruzain-deficient T. cruzi were comparable to LPS activated controls. Thus cruzain hinders macrophage activation during the early (<60 min) stages of infection, by interruption of the NF-κB P65 mediated signaling pathway. These early events allow T. cruzi survival and replication, and may lead to the spread of infection in acute 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.     

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.     

Crystal structure of the parasite protease inhibitor chagasin in complex with a host target cysteine protease

Chagasin is a protein produced by Trypanosoma cruzi, the parasite that causes Chagas' disease. This small protein belongs to a recently defined family of cysteine protease inhibitors. Although resembling well-known inhibitors like the cystatins in size (110 amino acid residues) and function (they all inhibit papain-like (C1 family) proteases), it has a unique amino acid sequence and structure. We have crystallized and solved the structure of chagasin in complex with the host cysteine protease, cathepsin L, at 1.75 A resolution. An inhibitory wedge composed of three loops (L2, L4, and L6) forms a number of contacts responsible for high-affinity binding (K(i), 39 pM) to the enzyme. All three loops interact with the catalytic groove, with the central loop L2 inserted directly into the catalytic center. Loops L4 and L6 embrace the enzyme molecule from both sides and exhibit distinctly different patterns of protein-protein recognition. Comparison with a 1.7 A structure of uncomplexed chagasin, also determined in this study, demonstrates that a conformational change of the first binding loop (L4) allows extended binding to the non-primed substrate pockets of the enzyme active site cleft, thereby providing a substantial part of the inhibitory surface. The mode of chagasin binding is generally similar, albeit distinctly different in detail, when compared to those displayed by cystatins and the cysteine protease inhibitory p41 fragment of the invariant chain. The chagasin-cathepsin L complex structure provides details of how the parasite protein inhibits a host enzyme of possible importance in host defense. The high level of structural and functional similarity between cathepsin L and the T. cruzi enzyme cruzipain gives clues to how the cysteine protease activity of the parasite can be targeted. This information will aid in the development of synthetic inhibitors for use as potential drugs for the treatment of Chagas disease.     

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.     

The substrate specificity of cruzipain 2, a cysteine protease isoform from Trypanosoma cruzi

Papain-like cysteine proteases are important for the survival of the flagellated protozoa Trypanosoma cruzi, the causative agent of Chagas' Disease. The lysosomal cysteine protease designated as cruzipain or cruzain, is the archetype of a multigene family of related isoforms. We investigated the substrate specificity of the cruzipain 2 isoform using internally quenched fluorogenic substrates. We found that cruzipain 2 and cruzain differ substantially regarding the specificity in the S2, S'1 and S'2 pockets. Our study indicates that cruzipain 2 has a more restricted specificity than cruzain, suggesting that these isoforms might act on distinct natural substrates.     

In Vitro and In Vivo Studies of the Trypanocidal Properties of WRR-483 against Trypanosoma cruzi

Background

Cruzain, the major cysteine protease of Trypanosoma cruzi, is an essential enzyme for the parasite life cycle and has been validated as a viable target to treat Chagas'' disease. As a proof-of-concept, K11777, a potent inhibitor of cruzain, was found to effectively eliminate T. cruzi infection and is currently a clinical candidate for treatment of Chagas'' disease.

Methodology/Principal Findings

WRR-483, an analog of K11777, was synthesized and evaluated as an inhibitor of cruzain and against T. cruzi proliferation in cell culture. This compound demonstrates good potency against cruzain with sensitivity to pH conditions and high efficacy in the cell culture assay. Furthermore, WRR-483 also eradicates parasite infection in a mouse model of acute Chagas'' disease. To determine the atomic-level details of the inhibitor interacting with cruzain, a 1.5 Å crystal structure of the protease in complex with WRR-483 was solved. The structure illustrates that WRR-483 binds covalently to the active site cysteine of the protease in a similar manner as other vinyl sulfone-based inhibitors. Details of the critical interactions within the specificity binding pocket are also reported.

Conclusions

We demonstrate that WRR-483 is an effective cysteine protease inhibitor with trypanocidal activity in cell culture and animal model with comparable efficacy to K11777. Crystallographic evidence confirms that the mode of action is by targeting the active site of cruzain. Taken together, these results suggest that WRR-483 has potential to be developed as a treatment for Chagas'' disease.     

Identification of Structure-Stabilizing Interactions in Enzymes: A Novel Mechanism to Impact Enzyme Activity

Cruzain, a cysteine protease in the cathepsin family, is pivotal to the life-cycle of Trypanosoma cruzi, the etiological agent in Chagas disease. Current inhibitors of cruzain suffer from drawbacks involving gastrointestinal and neurological side effects and as a result have spurred the search for alternative anti-trypanocidals. Through sequence alignment studies and intra-residue interaction analysis of the pro-protein of cruzain (pro-cruzain), we have identified a host of non-active site residues that are conserved among the cathepsins. We hypothesize that these conserved amino acids play a critical role in structure-stabilizing interactions among the cathepsins and are therefore crucial for eventually gaining protease activity. As predicted, mutation of selected conserved non-active site amino-acid candidates in cruzain resulted in a compromised structural stability and a corresponding loss in enzymatic activity relative to wild-type enzyme. By advancing the discovery of novel, non-active-site-based targets to arrest enzymatic activity our results potentially open the field of alternative inhibitor design. The advantages of defining such a non-active-site inhibitor design space is discussed.     

Probing the cruzain S2 recognition subsite: a kinetic and binding energy calculation study

Cysteine proteases are relevant to several aspects of the parasite life cycle and the parasite-host relationship. Moreover, they appear as promising targets for antiparasite chemotherapy. Here, a quantitative investigation on the catalytic properties of cruzain, the papain-like cysteine protease from epimastigotes of Trypanosoma cruzi, is reported. The results indicate that kinetics for the cruzain catalyzed hydrolysis of N-alpha-benzyloxycarbonyl-l-arginyl-l-alanine-(7-amino-4-methylcoumarin), N-alpha-benzyloxycarbonyl-l-phenylalanyl-l-alanine-(7-amino-4-methylcoumarin), and N-alpha-benzyloxycarbonyl-l-tyrosyl-l-alanine-(7-amino-4-methylcoumarin) can be consistently fitted to the minimum three-step mechanism of cysteine proteases involving the acyl.enzyme intermediate E.P; the deacylation step is rate-limiting in enzyme catalysis. Remarkably, these substrates show identical catalytic parameters. This reflects the ability of the cruzain Glu205 residue, located at the bottom of the S(2) subsite, to neutralize the substrate/inhibitor polar P(2) residues (e.g., Arg or Tyr) and to be solvent-exposed when substrate/inhibitor nonpolar P(2) residues (e.g., Phe) fit the S(2) subsite. More complex catalytic mechanisms are also discussed. Binding free-energy calculation provides a quantitative framework for the interpretation of these results; in particular, direct evidence for the compensatory effect between Coulomb interaction(s) and solvation effect(s) is reported. These results appear of general significance for a deeper understanding of (macro)molecular recognition and for the rational design of novel inhibitors of parasitic cysteine proteases.     

Structural basis for the activity of drugs that inhibit phosphodiesterases

Phosphodiesterases (PDEs) comprise a large family of enzymes that catalyze the hydrolysis of cAMP or cGMP and are implicated in various diseases. We describe the high-resolution crystal structures of the catalytic domains of PDE4B, PDE4D, and PDE5A with ten different inhibitors, including the drug candidates cilomilast and roflumilast, for respiratory diseases. These cocrystal structures reveal a common scheme of inhibitor binding to the PDEs: (i) a hydrophobic clamp formed by highly conserved hydrophobic residues that sandwich the inhibitor in the active site; (ii) hydrogen bonding to an invariant glutamine that controls the orientation of inhibitor binding. A scaffold can be readily identified for any given inhibitor based on the formation of these two types of conserved interactions. These structural insights will enable the design of isoform-selective inhibitors with improved binding affinity and should facilitate the discovery of more potent and selective PDE inhibitors for the treatment of a variety of diseases.     

Inhibition of cysteine proteases by a natural biflavone: behavioral evaluation of fukugetin as papain and cruzain inhibitor

Cruzain is the major cysteine protease of Trypanosoma cruzi, the infectious agent responsible for Chagas disease, and cruzain inhibitors display considerable antitrypanosomal activity. In the present work we elucidated crystallographic data of fukugetin, a biflavone isolated from Garcinia brasiliensis, and investigated the role of this molecule as cysteine protease inhibitor. The kinetic analyses demonstrated that fukugetin inhibited cruzain and papain by a slow reversible type inhibition with K_I of 1.1 and 13.4 µM, respectively. However, cruzain inhibition was about 12 times faster than papain inhibition. Lineweaver–Burk plots demonstrated partial competitive inhibition for cruzain and hyperbolic mixed-type inhibition for papain. Furthermore, the docking results showed that the biflavone binds to ring C′ in the S2 pocket and to ring C in the S3 pocket through hydrophobic interactions and hydrogen bonds. Finally, fukugetin also presented inhibitory activity on proteases of the T. cruzi extract, with IC₅₀ of 7 µM.     

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.     

Design, synthesis, and biochemical evaluation of novel cruzain inhibitors with potential application in the treatment of Chagas' disease

A series of compounds bearing tetrahydronaphthalene, benzophenone, propiophenone, and related rigid molecular skeletons functionalized with thiosemicarbazone or unsaturated carbonyl moieties were prepared by chemical synthesis and evaluated for their ability to inhibit the enzyme cruzain. As potential treatment agents for Chagas' disease, three compounds from the group demonstrate potent inhibition of cruzain with IC(50) values of 17, 24, and 80 nM, respectively.     

Mapping the active site polarity in structures of endothelial nitric oxide synthase heme domain complexed with isothioureas

Analyzing the active site topology and plasticity of nitric oxide synthase (NOS) and understanding enzyme-drug interactions are crucial for the development of potent, isoform-selective NOS inhibitors. A small hydrophobic pocket in the active site is identified in the bovine eNOS heme domain structures complexed with potent isothiourea inhibitors: seleno analogue of S-ethyl-isothiourea, S-isopropyl-isothiourea, and 2-aminothiazoline, respectively. These structures reveal the importance of nonpolar van der Waals contacts in addition to the well-known hydrogen bonding interactions between inhibitor and enzyme. The scaffold of a potent NOS inhibitor should be capable of donating hydrogen bonds to as well as making nonpolar contacts with amino acids in the NOS active site.     

Design of inhibitors of scytalone dehydratase: probing interactions with an asparagine carboxamide

Among the active-site residues of scytalone dehydratase, the side-chain carboxamide of asparagine 131 has the greatest potential for strong electrostatic interactions. Structure-based inhibitor design aimed at enhancing interactions with this residue led to the synthesis of a series of highly potent inhibitors that have a five- or six-membered ring containing a carbonyl functionality for hydrogen bonding. To achieve a good orientation for hydrogen bonding, the inhibitors incorporate a phenyl substituent that displaces a phenylalanine residue away from the five- or six-membered rings. Without the phenyl substituent, inhibitor binding potency is diminished by three orders of magnitude. Larger K_i values of a site-directed mutant (Asn131Ala) of scytalone dehydratase in comparison to those of wild-type enzyme validate the design concept. The most potent inhibitor (K_i=15 pM) contains a tetrahydrothiophenone that can form a single hydrogen bond with the asparagine carboxamide. Inhibitors with a butyrolactam that can form two hydrogen bonds with the asparagine carboxamide demonstrate excellent in vivo fungicidal activity.     

Intramolecular hydrogen bonding: a potential strategy for more bioavailable inhibitors of neuronal nitric oxide synthase

Selective neuronal nitric oxide synthase (nNOS) inhibitors have therapeutic applications in the treatment of numerous neurodegenerative diseases. Here we report the synthesis and evaluation of a series of inhibitors designed to have increased cell membrane permeability via intramolecular hydrogen bonding. Their potencies were examined in both purified enzyme and cell-based assays; a comparison of these results demonstrates that two of the new inhibitors display significantly increased membrane permeability over previous analogs. NMR spectroscopy provides evidence of intramolecular hydrogen bonding under physiological conditions in two of the inhibitors. Crystal structures of the inhibitors in the nNOS active site confirm the predicted non-intramolecular hydrogen bonded binding mode. Intramolecular hydrogen bonding may be an effective approach for increasing cell membrane permeability without affecting target protein binding.