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1.
Chemistry ; 28(62): e202201636, 2022 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-35852812

RESUMO

Rhodesain is the major cysteine protease of the protozoan parasite Trypanosoma brucei and a therapeutic target for sleeping sickness, a fatal neglected tropical disease. We designed, synthesized and characterized a bimodal activity-based probe that binds to and inactivates rhodesain. This probe exhibited an irreversible mode of action and extraordinary potency for the target protease with a kinac /Ki value of 37,000 M-1 s-1 . Two reporter tags, a fluorescent coumarin moiety and a biotin affinity label, were incorporated into the probe and enabled highly sensitive detection of rhodesain in a complex proteome by in-gel fluorescence and on-blot chemiluminescence. Furthermore, the probe was employed for microseparation and quantification of rhodesain and for inhibitor screening using a competition assay. The developed bimodal rhodesain probe represents a new proteomic tool for studying Trypanosoma pathobiochemistry and antitrypanosomal drug discovery.


Assuntos
Cisteína Proteases , Trypanosoma brucei brucei , Trypanosoma , Biotina , Fluorescência , Proteômica , Relação Estrutura-Atividade
2.
J Med Chem ; 64(16): 12322-12358, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34378914

RESUMO

Rhodesain is a major cysteine protease of Trypanosoma brucei rhodesiense, a pathogen causing Human African Trypanosomiasis, and a validated drug target. Recently, we reported the development of α-halovinylsulfones as a new class of covalent reversible cysteine protease inhibitors. Here, α-fluorovinylsulfones/-sulfonates were optimized for rhodesain based on molecular modeling approaches. 2d, the most potent and selective inhibitor in the series, shows a single-digit nanomolar affinity and high selectivity toward mammalian cathepsins B and L. Enzymatic dilution assays and MS experiments indicate that 2d is a slow-tight binder (Ki = 3 nM). Furthermore, the nonfluorinated 2d-(H) shows favorable metabolism and biodistribution by accumulation in mice brain tissue after intraperitoneal and oral administration. The highest antitrypanosomal activity was observed for inhibitors with an N-terminal 2,3-dihydrobenzo[b][1,4]dioxine group and a 4-Me-Phe residue in P2 (2e/4e) with nanomolar EC50 values (0.14/0.80 µM). The different mechanisms of reversible and irreversible inhibitors were explained using QM/MM calculations and MD simulations.


Assuntos
Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Sulfonas/farmacologia , Ácidos Sulfônicos/farmacologia , Tripanossomicidas/farmacologia , Compostos de Vinila/farmacologia , Animais , Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/toxicidade , Ensaios Enzimáticos , Feminino , Células HeLa , Humanos , Cinética , Masculino , Camundongos , Simulação de Acoplamento Molecular , Estrutura Molecular , Testes de Sensibilidade Parasitária , Ligação Proteica , Relação Estrutura-Atividade , Sulfonas/síntese química , Sulfonas/metabolismo , Sulfonas/toxicidade , Ácidos Sulfônicos/síntese química , Ácidos Sulfônicos/metabolismo , Ácidos Sulfônicos/toxicidade , Tripanossomicidas/síntese química , Tripanossomicidas/metabolismo , Tripanossomicidas/toxicidade , Trypanosoma brucei brucei/efeitos dos fármacos , Compostos de Vinila/síntese química , Compostos de Vinila/metabolismo , Compostos de Vinila/toxicidade
3.
J Biol Chem ; 296: 100565, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33745969

RESUMO

Rhodesain is the lysosomal cathepsin L-like cysteine protease of Trypanosoma brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood-brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating prodomain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression of T. brucei rhodesiense pro-rhodesain in Escherichia coli and determined its crystal structure. The trypanosomal prodomain differs from nonparasitic pro-cathepsins by a unique, extended α-helix that blocks the active site and whose side-chain interactions resemble those of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence correlation spectroscopy increase at low pH, where pro-rhodesain also undergoes autocleavage. Using the crystal structure, molecular dynamics simulations, and mutagenesis, we identify a conserved interdomain salt bridge that prevents premature intramolecular cleavage at higher pH values and may thus present a control switch for the observed pH sensitivity of proenzyme cleavage in (trypanosomal) CathL-like proteases.


Assuntos
Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Trypanosoma brucei rhodesiense/enzimologia , Ativação Enzimática , Concentração de Íons de Hidrogênio , Modelos Moleculares , Domínios Proteicos
4.
ACS Chem Biol ; 16(4): 661-670, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33719398

RESUMO

Viral and parasitic pathogens rely critically on cysteine proteases for host invasion, replication, and infectivity. Their inhibition by synthetic inhibitors, such as vinyl sulfone compounds, has emerged as a promising treatment strategy. However, the individual reaction steps of protease inhibition are not fully understood. Using the trypanosomal cysteine protease rhodesain as a medically relevant target, we design photoinduced electron transfer (PET) fluorescence probes to detect kinetics of binding of reversible and irreversible vinyl sulfones directly in solution. Intriguingly, the irreversible inhibitor, apart from its unlimited residence time in the enzyme, reacts 5 times faster than the reversible one. Results show that the reactivity of the warhead, and not binding of the peptidic recognition unit, limits the rate constant of protease inhibition. The use of a reversible inhibitor decreases the risk of off-target side effects not only by allowing its release from an off-target but also by reducing the rate constant of binding.


Assuntos
Cisteína Endopeptidases/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Fluorescência , Cinética , Ligantes
5.
Molecules ; 25(9)2020 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-32354191

RESUMO

The facile synthesis and detailed investigation of a class of highly potent protease inhibitors based on 1,4-naphthoquinones with a dipeptidic recognition motif (HN-l-Phe-l-Leu-OR) in the 2-position and an electron-withdrawing group (EWG) in the 3-position is presented. One of the compound representatives, namely the acid with EWG = CN and with R = H proved to be a highly potent rhodesain inhibitor with nanomolar affinity. The respective benzyl ester (R = Bn) was found to be hydrolyzed by the target enzyme itself yielding the free acid. Detailed kinetic and mass spectrometry studies revealed a reversible covalent binding mode. Theoretical calculations with different density functionals (DFT) as well as wavefunction-based approaches were performed to elucidate the mode of action.


Assuntos
Cisteína Proteases/química , Inibidores de Cisteína Proteinase/síntese química , Naftoquinonas/química , Tripanossomicidas/farmacologia , Catepsina L/química , Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/química , Dipeptídeos , Elétrons , Ésteres , Hidrólise , Concentração Inibidora 50 , Cinética , Espectrometria de Massas , Pró-Fármacos/química , Teoria Quântica , Relação Estrutura-Atividade , Trypanosoma brucei brucei/efeitos dos fármacos
6.
Molecules ; 25(6)2020 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-32210166

RESUMO

Electrophilic (het)arenes can undergo reactions with nucleophiles yielding π- or Meisenheimer (σ-) complexes or the products of the SNAr addition/elimination reactions. Such building blocks have only rarely been employed for the design of enzyme inhibitors. Herein, we demonstrate the combination of a peptidic recognition sequence with such electrophilic (het)arenes to generate highly active inhibitors of disease-relevant proteases. We further elucidate an unexpected mode of action for the trypanosomal protease rhodesain using NMR spectroscopy and mass spectrometry, enzyme kinetics and various types of simulations. After hydrolysis of an ester function in the recognition sequence of a weakly active prodrug inhibitor, the liberated carboxylic acid represents a highly potent inhibitor of rhodesain (Ki = 4.0 nM). The simulations indicate that, after the cleavage of the ester, the carboxylic acid leaves the active site and re-binds to the enzyme in an orientation that allows the formation of a very stable π-complex between the catalytic dyad (Cys-25/His-162) of rhodesain and the electrophilic aromatic moiety. The reversible inhibition mode results because the SNAr reaction, which is found in an alkaline solvent containing a low molecular weight thiol, is hindered within the enzyme due to the presence of the positively charged imidazolium ring of His-162. Comparisons between measured and calculated NMR shifts support this interpretation.


Assuntos
Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase , Proteínas de Protozoários , Trypanosoma/química , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/química , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Relação Estrutura-Atividade
7.
Front Genet ; 10: 876, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31608115

RESUMO

Modification mapping from cDNA data has become a tremendously important approach in epitranscriptomics. So-called reverse transcription signatures in cDNA contain information on the position and nature of their causative RNA modifications. Data mining of, e.g. Illumina-based high-throughput sequencing data, is therefore fast growing in importance, and the field is still lacking effective tools. Here we present a versatile user-friendly graphical workflow system for modification calling based on machine learning. The workflow commences with a principal module for trimming, mapping, and postprocessing. The latter includes a quantification of mismatch and arrest rates with single-nucleotide resolution across the mapped transcriptome. Further downstream modules include tools for visualization, machine learning, and modification calling. From the machine-learning module, quality assessment parameters are provided to gauge the suitability of the initial dataset for effective machine learning and modification calling. This output is useful to improve the experimental parameters for library preparation and sequencing. In summary, the automation of the bioinformatics workflow allows a faster turnaround of the optimization cycles in modification calling.

8.
ACS Med Chem Lett ; 7(12): 1073-1076, 2016 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-27994740

RESUMO

Dipeptidyl nitroalkenes are potent reversible inhibitors of cysteine proteases. Inhibitor 11 resulted to be the most potent one with Ki values of 0.49 and 0.44 nM against rhodesain and cruzain, respectively. According to enzymatic dilution and dialysis experiments, as well as computational and NMR studies, dipeptidyl nitroalkenes are tightly binding covalent reversible inhibitors.

9.
J Am Chem Soc ; 138(27): 8332-5, 2016 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-27347738

RESUMO

We propose a structure-based protocol for the development of customized covalent inhibitors. Starting from a known inhibitor, in the first and second steps appropriate substituents of the warhead are selected on the basis of quantum mechanical (QM) computations and hybrid approaches combining QM with molecular mechanics (QM/MM). In the third step the recognition unit is optimized using docking approaches for the noncovalent complex. These predictions are finally verified by QM/MM or molecular dynamic simulations. The applicability of our approach is successfully demonstrated by the design of reversible covalent vinylsulfone-based inhibitors for rhodesain. The examples show that our approach is sufficiently accurate to identify compounds with the desired properties but also to exclude nonpromising ones.

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