Impact of the Recognition Part of Dipeptidyl Nitroalkene Compounds on the Inhibition Mechanism of Cysteine Proteases Cruzain and Cathepsin L

Cysteine proteases (CPs) are an important class of enzymes, many of which are responsible for several human diseases. For instance, cruzain of protozoan parasite Trypanosoma cruzi is responsible for the Chagas disease, while the role of human cathepsin L is associated with some cancers or is a potential target for the treatment of COVID-19. However, despite paramount work carried out during the past years, the compounds that have been proposed so far show limited inhibitory action against these enzymes. We present a study of proposed covalent inhibitors of these two CPs, cruzain and cathepsin L, based on the design, synthesis, kinetic measurements, and QM/MM computational simulations on dipeptidyl nitroalkene compounds. The experimentally determined inhibition data, together with the analysis and the predicted inhibition constants derived from the free energy landscape of the full inhibition process, allowed describing the impact of the recognition part of these compounds and, in particular, the modifications on the P2 site. The designed compounds and, in particular, the one with a bulky group (Trp) at the P2 site show promising in vitro inhibition activities against cruzain and cathepsin L for use as a starting lead compound in the development of drugs with medical applications for the treatment of human diseases and future designs.This work was supported by the Spanish Ministerio de Ciencia, Innovación y Universidades (Grant PGC2021-23332OB-C21), Generalitat Valenciana and the European Regional Funds (Grant PROMETEO CIPROM/2021/079, IDIFEDER/2021/027), and Universitat Jaume I (UJI-B2020-03 and UJI-2021-71). K.A. thanks Generalitat Valenciana (APOSTD/2020/015) for a postdoctoral contract. The authors thankfully acknowledge the local computational resources of the Servei d’Informàtica and Serveis Centrals d’Instrumentació Científica of Universitat Jaume I

Antioxidant and Anti-Protease Activities of Diazepinomicin from the Sponge-Associated Micromonospora Strain RV115

Diazepinomicin is a dibenzodiazepine alkaloid with an unusual structure among the known microbial metabolites discovered so far. Diazepinomicin was isolated from the marine sponge-associated strain Micromonospora sp. RV115 and was identified by spectroscopic analysis and by comparison to literature data. In addition to its interesting preclinical broad-spectrum antitumor potential, we report here new antioxidant and anti-protease activities for this compound. Using the ferric reducing antioxidant power (FRAP) assay, a strong antioxidant potential of diazepinomicin was demonstrated. Moreover, diazepinomicin showed a significant antioxidant and protective capacity from genomic damage induced by the reactive oxygen species hydrogen peroxide in human kidney (HK-2) and human promyelocytic (HL-60) cell lines. Additionally, diazepinomicin inhibited the proteases rhodesain and cathepsin L at an IC50 of 70–90 µM. It also showed antiparasitic activity against trypomastigote forms of Trypanosoma brucei with an IC50 of 13.5 µM. These results showed unprecedented antioxidant and anti-protease activities of diazepinomicin, thus further highlighting its potential as a future drug candidate

Identification of a potential allosteric site of Golgi α-mannosidase II using computer-aided drug design

Golgi α-mannosidase II (GMII) is a glycoside hydrolase playing a crucial role in the N-glycosylation pathway. In various tumour cell lines, the distribution of N-linked sugars on the cell surface is modified and correlates with the progression of tumour metastasis. GMII therefore is a possible molecular target for anticancer agents. Here, we describe the identification of a non-competitive GMII inhibitor using computer-aided drug design methods including identification of a possible allosteric binding site, pharmacophore search and virtual screening.publishedVersio

New cysteine protease inhibitors : electrophilic (het)arenes and unexpected prodrug identification for the trypanosoma protease rhodesain

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

Synthesis of SARS-CoV-2 Mpro inhibitors bearing a cinnamic ester warhead with in vitro activity against human coronaviruses

COVID-19 now ranks among the most devastating global pandemics in history. The causative virus, SARS-CoV-2, is a new human coronavirus (hCoV) that spreads among humans and animals. Great efforts have been made to develop therapeutic agents to treat COVID-19, and among the available viral molecular targets, the cysteine protease SARS-CoV-2 Mpro is considered the most appealing one due to its essential role in viral replication. However, the inhibition of Mpro activity is an interesting challenge and several small molecules and peptidomimetics have been synthesized for this purpose. In this work, the Michael acceptor cinnamic ester was employed as an electrophilic warhead for the covalent inhibition of Mpro by endowing some peptidomimetic derivatives with such a functionality. Among the synthesized compounds, the indole-based inhibitors 17 and 18 efficiently impaired the in vitro replication of beta hCoV-OC-43 in the low micromolar range (EC50 = 9.14 μM and 10.1 μM, respectively). Moreover, the carbamate derivative 12 showed an antiviral activity of note (EC50 = 5.27 μM) against another hCoV, namely hCoV-229E, thus suggesting the potential applicability of such cinnamic pseudopeptides also against human alpha CoVs. Taken together, these results support the feasibility of considering the cinnamic framework for the development of new Mpro inhibitors endowed with antiviral activity against human coronaviruses

Identification of a potential allosteric site of Golgi α-mannosidase II using computer-aided drug design

Golgi α-mannosidase II (GMII) is a glycoside hydrolase playing a crucial role in the N-glycosylation pathway. In various tumour cell lines, the distribution of N-linked sugars on the cell surface is modified and correlates with the progression of tumour metastasis. GMII therefore is a possible molecular target for anticancer agents. Here, we describe the identification of a non-competitive GMII inhibitor using computer-aided drug design methods including identification of a possible allosteric binding site, pharmacophore search and virtual screening

Naphthoquinones as covalent reversible inhibitors of cysteine proteases : studies on inhibition mechanism and kinetics

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

New Tetromycin Derivatives with Anti-Trypanosomal and Protease Inhibitory Activities †

Four new tetromycin derivatives, tetromycins 1–4 and a previously known one, tetromycin B (5) were isolated from Streptomyces axinellae Pol001T cultivated from the Mediterranean sponge Axinella polypoides. Structures were assigned using extensive 1D and 2D NMR spectroscopy as well as HRESIMS analysis. The compounds were tested for antiparasitic activities against Leishmania major and Trypanosoma brucei, and for protease inhibition against several cysteine proteases such as falcipain, rhodesain, cathepsin L, cathepsin B, and viral proteases SARS-CoV Mpro, and PLpro. The compounds showed antiparasitic activities against T. brucei and time-dependent inhibition of cathepsin L-like proteases with Ki values in the low micromolar range

Peptidyl Nitroalkene Inhibitors of Main Protease (Mpro) rationalized by Computational/1 Crystallographic 2 Investigations as Antivirals against SARS-CoV-2

34 p.-10 fig.-2 tab.-2 schem.The coronavirus disease 2019 (COVID-19) pandemic continues to represent a global public health issue. The viral main protease (Mpro) represents one of the most attractive targets for the development of antiviral drugs. Herein we report peptidyl nitroalkenes exhibited enzyme inhibitory activity against Mpro (Ki: 1-10 μM) and three of them good anti-SARS-CoV-2 infection activity in the low micromolar range (EC50: 1-12 μM) without significant toxicity. Additional kinetic studies of compounds FGA145, FGA146 and FGA147 show that all three compounds inhibit Cathepsin L, denoting a possible multitarget effect of these compounds in the antiviral activity. QM/MM computer simulations assisted in the design and in elucidating the way of action. Finally, structural analysis shows, in agreement with the computer predictions, the binding mode of FGA146 and FGA147 to the active site of the protein. Our results illustrate that peptidyl nitroalkenes are potent covalent reversible inhibitors of the Mpro and cathepsin L, and that inhibitors FGA145, FGA146 and FGA147 prevent infection becoming promising drugs against SARS-CoV-2.This research was funded by the Consejo Superior de Investigaciones Científicas, grant number PIE- 202020E224, the Spanish Ministerio de Ciencia e Innovación (ref. PID2021-123332OB-C21 and PID2019- 107098RJ-I00), the Generalitat Valenciana (PROMETEO with ref. CIPROM/2021/079, and SEJI/2020/007), Universitat Jaume I (UJI-B2020-03, UJI-B2021-71 and SomUJIcontracovid crowdfunding campaign).K.Ś.thanks to Ministerio de Ciencia e Innovación and Fondo Social Europeo for a Ramon y Cajal contract (Ref. RYC2020-030596-I). The authors wish to thank the staff of beamlines ID30B (ESRF Synchrotron) and BL13- XALOC (ALBA Synchrotron) for their generous and much appreciated support, and the Serveis Centrals d’Instrumentació Científica of Universitat Jaume I for technical support. Finally, the authors acknowledge the computer resources at Mare Nostrum of the Barcelona Supercomputing Center (QH-2022-2-0004 and QH- 2022-3-0008), as well as the local computational resources founded by Generalitat Valenciana - European Regional Development Fund (REF: IDIFEDER/2021/02).Peer reviewe

Peptidyl nitroalkene inhibitors of main protease rationalized by computational and crystallographic investigations as antivirals against SARS-CoV-2

16 p.-10 fig.-2 tab.The coronavirus disease 2019 (COVID-19) pandemic continues to represent a global public health issue. The viral main protease (Mpro) represents one of the most attractive targets for the development of antiviral drugs. Herein we report peptidyl nitroalkenes exhibiting enzyme inhibitory activity against Mpro (Ki: 1–10 μM) good anti-SARS-CoV-2 infection activity in the low micromolar range (EC50: 1–12 μM) without significant toxicity. Additional kinetic studies of compounds FGA145, FGA146 and FGA147 show that all three compounds inhibit cathepsin L, denoting a possible multitarget effect of these compounds in the antiviral activity. Structural analysis shows the binding mode of FGA146 and FGA147 to the active site of the protein. Furthermore, our results illustrate that peptidyl nitroalkenes are effective covalent reversible inhibitors of the Mpro and cathepsin L, and that inhibitors FGA145, FGA146 and FGA147 prevent infection against SARS-CoV-2.This research was funded by the Consejo Superior de Investigaciones Científicas, grant number PIE-202020E224, the Spanish Ministerio de Ciencia e Innovación (ref. PID2021-123332OB-C21 and PID2019-107098RJ-I00), the Generalitat Valenciana (PROMETEO with ref. CIPROM/2021/079, and SEJI/2020/007), Universitat Jaume I (UJI-B2020-03, UJI-B2021-71 and SomUJIcontracovid crowdfunding campaign). K.Ś. thanks to Ministerio de Ciencia e Innovación and Fondo Social Europeo for a Ramon y Cajal contract (Ref. RYC2020-030596-I). The authors wish to thank the staff of beamlines ID30B (ESRF Synchrotron) and BL13-XALOC (ALBA Synchrotron) for their generous and much appreciated support, and the Serveis Centrals d’Instrumentació Científica of Universitat Jaume I for technical support. The work was also supported by a Research Grant of the University Medical Center Giessen and Marburg (UKGM, to C.M.), the von Behring-Röntgen-Stiftung (project 71_0016, to C.M.) the Deutsche Forschungsgemeinschaſt (DFG, project 530813989, to C.M.). Finally, the authors acknowledge the computer resources at Mare Nostrum of the Barcelona Supercomputing Center (QH-2022-2-0004 and QH-2022-3-0008), as well as the local computational resources founded by Generalitat Valenciana - European Regional Development Fund (REF: IDIFEDER/2021/02).Peer reviewe