Synthetic compounds from an in house library as inhibitors of falcipain-2 from Plasmodium falciparum

Falcipain-2 (FP-2) is a key cysteine protease from the malaria parasite Plasmodium falciparum. Many previous studies have identified FP-2 inhibitors; however, none has yet met the criteria for an antimalarial drug candidate. In this work, we assayed an in-house library of non-peptidic organic compounds, including (E)-chalcones, (E)-N'-benzylidene-benzohydrazides and alkyl-esters of gallic acid, and assessed the activity toward FP-2 and their mechanisms of inhibition. The (E)-chalcones 48, 54 and 66 showed the lowest IC50 values (8.5±0.8μM, 9.5±0.2μM and 4.9±1.3μM, respectively). The best inhibitor (compound 66) demonstrated non-competitive inhibition, and using mass spectrometry and fluorescence spectroscopy assays, we suggest a potential allosteric site for the interaction of this compound, located between the catalytic site and the hemoglobin binding arm in FP-2. We combined structural biology tools and mass spectrometry to characterize the inhibition mechanisms of novel compounds targeting FP-2.Fil: Bertoldo, Jean Borges. Universidade Federal de Santa Catarina; BrasilFil: Chiaradia Delatorre, Louise Domeneghini. Universidade Federal de Santa Catarina; BrasilFil: Mascarello, Alessandra. Universidade Federal de Santa Catarina; BrasilFil: Leal, Paulo César. Universidade Federal de Santa Catarina; BrasilFil: Sechini Cordeiro, Marlon Norberto. Universidade Federal de Santa Catarina; BrasilFil: Nunes, Ricardo José. Universidade Federal de Santa Catarina; BrasilFil: Salas Sarduy, Emir. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de La Habana; CubaFil: Rosenthal, Philip Jon. University of California; Estados UnidosFil: Terenzi, Hernán. Universidade Federal de Santa Catarina; Brasi

Mycobacterium tuberculosis-secreted tyrosine phosphatases as targets against tuberculosis: exploring natural sources in searching for new drugs

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), which primarily affects the respiratory tract. Combinations of drugs are used for therapeutic synergism and to prevent the emergence of drug resistant strains, but even first-or second-choice drugs present some disadvantages, such as significant side effects and the need for long duration of treatments. Thus, new strategies for TB control and treatment are highly demanded. In this context, protein tyrosine phosphatases (PtpA and PtpB) are secreted by Mtb within the host macrophage and they have been shown to contribute to Mtb pathogenicity. The understanding of the role of these PTPs has led to interesting anti-TB drugs discovery. Here, we review the current knowledge on these two proteins as targets for novel anti-TB therapies, with particular emphasis on their mechanism of action and current advancements in developing small molecule inhibitors from natural sources

Discovery of <i>Mycobacterium tuberculosis</i> Protein Tyrosine Phosphatase B (PtpB) Inhibitors from Natural Products

<div><p>Protein tyrosine phosphatase B (PtpB) is one of the virulence factors secreted into the host cell by <i>Mycobacterium tuberculosis</i>. PtpB attenuates host immune defenses by interfering with signal transduction pathways in macrophages and, therefore, it is considered a promising target for the development of novel anti-tuberculosis drugs. Here we report the discovery of natural compound inhibitors of PtpB among an <i>in house</i> library of more than 800 natural substances by means of a multidisciplinary approach, mixing <i>in silico</i> screening with enzymatic and kinetics studies and MS assays. Six natural compounds proved to inhibit PtpB at low micromolar concentrations (< 30 µM) with Kuwanol E being the most potent with <i>K</i>_i = 1.6 ± 0.1 µM. To the best of our knowledge, Kuwanol E is the most potent natural compound PtpB inhibitor reported so far, as well as it is the first non-peptidic PtpB inhibitor discovered from natural sources. Compounds herein identified may inspire the design of novel specific PtpB inhibitors.</p> </div

Synthetic chalcones and sulfonamides as new classes of Yersinia enterocolitica YopH tyrosine phosphatase inhibitors

YopH plays a relevant role in three pathogenic species of Yersinia. Due to its importance in the prevention of the inflammatory response of the host, this enzyme has become a valid target for the identification and development of new inhibitors. In this work, an in-house library of 283 synthetic compounds was assayed against recombinant YopH from Yersinia enterocolitica. From these, four chalcone derivatives and one sulfonamide were identified for the first time as competitive inhibitors of YopH with binding affinity in the low micromolar range. Molecular modeling investigations indicated that the new inhibitors showed similar binding modes, establishing polar and hydrophobic contacts with key residues of the YopH binding site.CNPqCAPESFAPESCMCTIFINE

Naturally occurring Diels-Alder-type adducts from Morus nigra as potent inhibitors of Mycobacterium tuberculosis protein tyrosine phosphatase B

Mycobacterium tuberculosis (Mtb) protein tyrosine phosphatases A and B (PtpA and PtpB) have been recognized as potential molecular targets for the development of new therapeutic strategies against tuberculosis (TB). In this context, we have recently reported that the naturally occurring Diels-Alder-type adduct Kuwanol E is an inhibitor of PtpB (Ki= 1.6 ± 0.1 μM). Here, we describe additional Diels-Alder-type adducts isolated from Morus nigra roots bark that inhibit PtpB at sub-micromolar concentrations. The two most potent compounds, namely Kuwanon G and Kuwanon H, showed Kivalues of 0.39 ± 0.27 and 0.20 ± 0.01 μM, respectively, and interacted with the active site of the enzyme as suggested by kinetics and mass spectrometry studies. Molecular docking coupled with intrinsic fluorescence analysis and isothermal titration calorimetry (ITC) further characterized the interaction of these promising PtpB inhibitors. Notably, in an Mtb survival assay inside macrophages, Kuwanon G showed inhibition of Mtb growth by 61.3%. All these results point to the common Diels-Alder-type adduct scaffold, and highlight its relevance for the development of PtpB inhibitors as candidate therapeutics for TB

Peptide mass fingerprints.

<p>Peptide mass fingerprinting of PtpB recorded by MS in absence (A) and presence of 300 µM KuwE (B). The tryptic peptide <i>m/z</i> 2224 corresponds to the complete sequence of the catalytic site ((R145)VVTLLAAGRPVLT<u>HCFAGKDR</u>(T167)) and the tryptic peptide <i>m/z</i> 1953 corresponds to a part of the catalytic site ((R145)VVTLLAAGRPVLT<u>HCFAGK</u>(D165)), which include the His159 and the catalytic Cys160 residues.</p

Protection of PtpB proteolytic cleavage by KuwE.

<p>Schematic representation of the region protected by KuwE in PtpB structure. The amino acid sequence VVTLLAAGRPVLTHCFAGKDR (<i>m/z</i> 2224) identified by mass spectrometry is highlighted in blue sticks and cartoon. KuwE is showed as orange sticks while PtpB is represented as green (alpha-helix) and magenta (beta-chain) cartoon.</p

Kinetics measurements of PtpB inhibitors.

<p>Lineweaver-Burk double-reciprocal plots representing inhibitory profiles of compounds KuwE, PirIII, Ega1, 6016, Ac3 and ∆3 against PtpB. Kinetic experiments were conducted in the presence of increasing concentrations of inhibitors: 0 µM (), 1 µM (), 2 µM (), 3 µM (), 6 µM (), 10 µM (), 20 µM (), 25 µM (), 30 µM (), 35 µM (), 40 µM (), 45 µM (); <i>p</i>NPP was used as substrate in all experiments. For KuwE, Ac3 and 6016, all lines converged at the <i>y</i>-axis (1/<i>V</i>_max), whereas the slope (K_Mapp/<i>V</i>_max) and <i>x</i>-axis interception (1/<i>K</i>_Mapp) varies according to the inhibitor concentration; the constant value of <i>V</i>_max and the increased values of <i>K</i>_Mapp are consistent with a competitive inhibition mechanism. For PirIII, Ega1 and Δ3, all lines converge at the <i>x</i>-axis (1/<i>K</i>_Mapp) and the <i>y</i>-axis interception (1/<i>V</i>_max) varies as a function of the inhibitor concentration; the constant value of <i>K</i>_Mapp and the increased values of <i>V</i>_max indicate that these compounds are noncompetitive inhibitors. </p

Differences between PtpB and PTP1B.

<p>Sequence alignment of Mtb PtpB (UniProtKB code: P96830, 276 aa complete sequence) and human PTP1B (UniProtKB code: P18031, 435 aa complete sequence). Not conserved amino acids of the PtpB active site motif, which may be exploited to design selective Mtb PtpB inhibitors, are highlighted by a red box. Sequence alignment was performed with ClustalX. Sequence numbering corresponds to human PTP1B. Bars below the sequence alignment correspond to the degree of amino acid conservation between the two sequence (full bar: residues identity; empty bar: completely different residues). </p

Structures of PtpB inhibitors.

<p>Chemical structure of PtpB inhibitors showing an IC₅₀ < 100 µM. Below the line are the two common chemical scaffolds: Scaffold A present in KuwE, Ega1, M2 and M2H; Scaffold B present in PirIII, Δ3, 6016 and Ac3.</p