The Impact of Activity-Based Protein Profiling in Malaria Drug Discovery

Activity-based protein profiling (ABPP) is an approach used at the interface of chemical biology and proteomics that uses small molecular probes to provide dynamic fingerprints of enzymatic activity in complex proteomes. Malaria is a disease caused by Plasmodium parasites with a significant death burden and for which new therapies are actively being sought. Here, we compile the main achievements from ABPP studies in malaria and highlight the probes used and the different downstream platforms for data analysis. ABPP has excelled at studying Plasmodium cysteine proteases and serine hydrolase families, the targeting of the proteasome and metabolic pathways, and in the deconvolution of targets and mechanisms of known antimalarials. Despite the major impact in the field, many antimalarials and enzymatic families in Plasmodium remain to be studied, which suggests ABPP will be an evergreen technique in the field.     

Activity-Based Protein Profiling (ABPP) of Oxidoreductases

Over the last two decades, activity-based protein profiling (ABPP) has been established as a tremendously useful proteomic tool for measuring the activity of proteins in their cellular context, annotating the function of uncharacterized proteins, and investigating the target profile of small-molecule inhibitors. Unlike hydrolases and other enzyme classes, which exhibit a characteristic nucleophilic residue, oxidoreductases have received much less attention in ABPP. In this minireview, the state of the art of ABPP of oxidoreductases is described and the scope and limitations of the existing approaches are discussed. It is noted that several ABPP probes have been described for various oxidases, but none so far for a reductase, which gives rise to opportunities for future research.     

Ligand-Directed Chemistry on Glycoside Hydrolases – A Proof of Concept Study

Selective covalent labelling of enzymes using small molecule probes has advanced the scopes of protein profiling. The covalent bond formation to a specific target is the key step of activity-based protein profiling (ABPP), a method which has become an indispensable tool for measuring enzyme activity in complex matrices. With respect to carbohydrate processing enzymes, strategies for ABPP so far involve labelling the active site of the enzyme, which results in permanent loss of activity. Here, we report in a proof of concept study the use of ligand-directed chemistry (LDC) for labelling glycoside hydrolases near – but not in – the active site. During the labelling process, the competitive inhibitor is cleaved from the probe, departs the active site and the enzyme maintains its catalytic activity. To this end, we designed a building block synthetic concept for small molecule probes containing iminosugar-based reversible inhibitors for labelling of two model β-glucosidases. The results indicate that the LDC approach can be adaptable for covalent proximity labelling of glycoside hydrolases.     

Activity-based profiling of retaining β-glucosidases: a comparative study

Activity-based protein profiling (ABPP) is a versatile strategy to report on enzyme activity in vitro, in situ, and in vivo. The development and use of ABPP tools and techniques has met with considerable success in monitoring physiological processes involving esterases and proteases. Activity-based profiling of glycosidases, on the other hand, has proven more difficult, and to date no broad-spectrum glycosidase activity-based probes (ABPs) have been reported. In a comparative study, we investigated both 2-deoxy-2-fluoroglycosides and cyclitol epoxides for their utility as a starting point towards retaining β-glucosidase ABP. We also investigated the merits of direct labeling and two-step bio-orthogonal labeling in reporting on glucosidase activity under various conditions. Our results demonstrate that 1) in general cyclitol epoxides are the superior glucosidase ABPs, 2) that direct labeling is the more efficient approach but it hinges on the ability of the glucosidase to be accommodated in the active site of the reporter (BODIPY) entity, and 3) that two-step bio-orthogonal labeling can be achieved on isolated enzymes but translating this protocol to cell extracts requires more investigation.     

STA-55, an Easily Accessible,Broad-Spectrum,Activity-Based Aldehyde Dehydrogenase Probe

Aldehyde dehydrogenases (ALDHs) convert aldehydes into carboxylic acids and are often upregulated in cancer. They have been linked to therapy resistance and are therefore potential therapeutic targets. However, only a few selective and potent inhibitors are currently available for this group of enzymes. Competitive activity-based protein profiling (ABPP) would aid the development and validation of new selective inhibitors. Herein, a broad-spectrum activity-based probe that reports on several ALDHs is presented. This probe was used in a competitive ABPP protocol against three ALDH inhibitors in lung cancer cells to determine their selectivity profiles and establish their target engagement.     

Achyranthes bidentata Polypeptides Reduces Oxidative Stress and Exerts Protective Effects against Myocardial Ischemic/Reperfusion Injury in Rats

Achyranthes bidentata, a Chinese medicinal herb, is reported to be neuroprotective. However, its role in cardioprotection remains largely unknown. Our present study aimed to investigate the effects of Achyranthes bidentata polypeptides (ABPP) preconditioning on myocardial ischemia/reperfusion (MI/R) injury and to test the possible mechanisms. Rats were treated with ABPP (10 mg/kg/d, i.p.) or saline once daily for one week. Afterward, all the animals were subjected to 30 min of myocardial ischemia followed by 4 h of reperfusion. ABPP preconditioning for one week significantly improved cardiac function following MI/R. Meanwhile, ABPP reduced infarct size, plasma creatine kinase (CK)/lactate dehydrogenase (LDH) activities and myocardial apoptosis at the end of reperfusion in rat hearts. Moreover, ABPP preconditioning significantly inhibited superoxide generation, gp91^phox expression, malonaldialdehyde formation and enhanced superoxide dismutase activity in I/R hearts. Furthermore, ABPP treatment inhibited PTEN expression and increased Akt phosphorylation in I/R rat heart. PI3K inhibitor wortmannin blocked Akt activation, and abolished ABPP-stimulated anti-oxidant effect and cardioprotection. Our study demonstrated for the first time that ABPP reduces oxidative stress and exerts cardioprotection against MI/R injury in rats. Inhibition of PTEN and activation of Akt may contribute to the anti-oxidant capacity and cardioprotection of ABPP.     

Two-Step Bioorthogonal Activity-Based Protein Profiling of Individual Human Proteasome Catalytic Sites

Bioorthogonal chemistry allows the selective modification of biomolecules in complex biological samples. One application of this methodology is in two-step activity-based protein profiling (ABPP), a methodology that is particularly attractive where direct ABPP using fluorescent or biotinylated probes is ineffective. Herein we describe a set of norbornene-modified, mechanism-based proteasome inhibitors aimed to be selective for each of the six catalytic sites of human constitutive proteasomes and immunoproteasomes. The probes developed for β1i, β2i, β5c, and β5i proved to be useful two-step ABPs that effectively label their developed proteasome subunits in both Raji cell extracts and living Raji cells through inverse-electron-demand Diels–Alder (iEDDA) ligation. The compound developed for β1c proved incapable of penetrating the cell membrane, but effectively labels β1c in vitro. The compound developed for β2c proved not selective, but its azide-containing analogue LU-002c proved effective in labeling of β2c via azide–alkyne click ligation chemistry both in vitro and in situ. In total, our results contribute to the growing list of proteasome activity tools to include five subunit-selective activity-based proteasome probes, four of which report on proteasome activities in living cells.     

Zinc(II) coordination complexes as membrane-active fluorescent probes and antibiotics

Molecular probes with zinc(II)-(2,2'-dipicolylamine) coordination complexes associate with oxyanions in aqueous solution and target biomembranes that contain anionic phospholipids. This study examines a new series of coordination complexes with 2,6-bis(zinc(II)-dipicolylamine)phenoxide as the molecular recognition unit. Two lipophilic analogues are observed to partition into the membranes of zwitterionic and anionic vesicles and induce the transport of phospholipids and hydrophilic anions (carboxyfluorescein). These lipophilic zinc complexes are moderately toxic to mammalian cells. A more hydrophilic analogue does not exhibit mammalian cell toxicity (LD(50) >50 microg mL(-1)), but it is highly active against the Gram-positive bacteria Staphylococcus aureus (MIC of 1 microg mL(-1)). Furthermore, it is active against clinically important S. aureus strains that are resistant to various antibiotics, including vancomycin and oxacillin. The antibiotic action is attributed to its ability to depolarize the bacterial cell membrane. The intense bacterial staining that was exhibited by a fluorescent conjugate suggests that this family of zinc coordination complexes can be used as molecular probes for the detection and imaging of bacteria.     

Activity‐Based Protein Profiling of Rhomboid Proteases in Liposomes

Although activity‐based protein profiling (ABPP) has been used to study a variety of enzyme classes, its application to intramembrane proteases is still in its infancy. Intramembrane proteolysis is an important biochemical mechanism for activating proteins residing within the membrane in a dormant state. Rhomboid proteases (intramembrane serine proteases) are embedded in the lipid bilayers of membranes and occur in all phylogenetic domains. The study of purified rhomboid proteases has mainly been performed in detergent micelle environments. Here we report on the reconstitution of rhomboids in liposomes. Using ABPP, we have been able to detect active rhomboids in large and giant unilamellar vesicles. We have found that the inhibitor profiles of rhomboids in micelles and liposomes are similar, thus validating previous inhibitor screenings. Moreover, fluorescence microscopy experiments on the liposomes constitute the first steps towards activity‐based imaging of rhomboid proteases in membrane environments.     

Synthetic Approaches of Epoxysuccinate Chemical Probes

Synthetic chemical probes are powerful tools for investigating biological processes. They are particularly useful for proteomic studies such as activity-based protein profiling (ABPP). These chemical methods initially used mimics of natural substrates. As the techniques gained prominence, more and more elaborate chemical probes with increased specificity towards given enzyme/protein families and amenability to various reaction conditions were used. Among the chemical probes, peptidyl-epoxysuccinates represent one of the first types of compounds used to investigate the activity of the cysteine protease papain-like family of enzymes. Structurally derived from the natural substrate, a wide body of inhibitors and activity- or affinity-based probes bearing the electrophilic oxirane unit for covalent labeling of active enzymes now exists. Herein, we review the literature regarding the synthetic approaches to epoxysuccinate-based chemical probes together with their reported applications, from biological chemistry and inhibition studies to supramolecular chemistry and the formation of protein arrays.     

Chemical Proteomics for Expanding the Druggability of Human Disease

Abstract . Over the past decade, chemical proteomics has emerged as a powerful technique to understand small molecule and protein function in the physiological system and plays a key role in unravelling the cellular targets of pharmacological modulators. Chemical proteomics that integrates activity-based protein profiling (ABPP) with mass spectrometry has been introduced to evaluate small-molecule and protein interaction and expand the druggable proteome. A much larger fraction of the human proteome can now be targeted by small molecules than estimated by past predictions of protein druggability.     

Copper‐Assisted Click Reactions for Activity‐Based Proteomics: Fine‐Tuned Ligands and Refined Conditions Extend the Scope of Application

Copper‐catalysed alkyne–azide 1,3‐dipolar cycloaddition (CuAAC) is the predominantly used bioconjugation method in the field of activity‐based protein profiling (ABPP). Several limitations, however, including conversion efficiency, protein denaturation and buffer compatibility, restrict the scope of established procedures. We introduce an ABPP customised click methodology based on refined CuAAC conditions together with new accelerating copper ligands. A screen of several triazole compounds revealed the cationic quaternary {3‐[4‐({bis[(1‐tert‐butyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]amino}methyl)‐1H‐1,2,3‐triazol‐1‐yl]propyl}trimethylammonium trifluoroacetate (TABTA) to be a superior ligand. TABTA exhibited excellent in vitro conjugation kinetics and optimal ABPP labelling activity while almost exclusively preserving the native protein fold. The application of this CuAAC‐promoting system is amenable to existing protocols with minimal perturbations and is even compatible with previously unusable buffer systems such as Tris ? HCl.     

Bacteriological evaluation of goat milk and cheese distributed in the Metropolitan Area of San José, Costa Rica

In the last years, there has been an increase in the production, industrialization and consumption of goat's milk and derivate products, including cheese, worldwide. Nevertheless, in Costa Rica there is no study of these products, reason why the objective of this work was to determine the microbiological characteristics of goat's milk and fresh cheese distributed in the Metropolitan Area of San José, Costa Rica, in order to evaluate its impact in the economical field and as a potential risk for Public Health. A total of 25 raw goat's milk samples, obtained by manual milking from 5 different producers tested in five different dates and 15 cheese samples, elaborated with pasteurized milk, commercially available and coming from three different producers were analyzed. The study included the analysis of spoilage bacteria (total aeobic count and lactic bacteria count), indicators of hygiene (total coliforms), fecal contamination (fecal coliforms), manipulation (Staphylococcus aureus) and pathogens (Listeria monocytogenes and Salmonella spp). High results were obtained for the total aerobic count and lactic bacteria count of the milk and cheese samples, showing a reduced shelf life. Total coliforms, in limits beyond the established ones by the Costa Rican legislation for human consumption raw milk, were found in 100% of milk samples, as well as for fecal coliforms in 76% of them. All cheese samples, except one, were negative for these indicators, suggesting good manufacturing practices. S. aureus counts were low and both Salmonella spp. and L. monocytogenes were not isolated from samples analyzed.     

两种含二茂铁基schiff 碱的合成及其抑菌活性

通过乙酰基二茂铁与两种杂环胺(4-氨基安替比林,2-氨基-5-巯基-1,3,4-噻二唑)缩合合成了两种含二茂铁基的Schiff碱(a、b),产率分别为85.8%和72.6%,并通过IR、1HNMR及元素分析对其结构进行了确证。最后,将合成出的两种Schiff碱分别对3种细菌(金黄色葡萄球菌、大肠杆菌、枯草芽孢杆菌)进行了初步抑菌实验,将其结果与非杂环类Schiff碱Fc-C(CH3)NC6H5=(c)的抑菌性能做了比较。结果表明,Schiff碱对上述3种细菌都有抑制作用,且抑菌效果随着Schiff碱浓度的增大而增强。此外,杂环类Schiff碱(a、b)对上述3种细菌的抑菌活性明显优于非杂环类Schiff碱(c)。     

Synthesis and Biological Evaluation of 3-Arylindazoles as Selective MEK4 Inhibitors

Herein we report the discovery of a novel series of highly potent and selective mitogen-activated protein kinase kinase 4 (MEK4) inhibitors. MEK4 is an upstream kinase in MAPK signaling pathways that phosphorylates p38 MAPK and JNK in response to mitogenic and cellular stress queues. MEK4 is overexpressed and induces metastasis in advanced prostate cancer lesions. However, the value of MEK4 as an oncology target has not been pharmacologically validated because selective chemical probes targeting MEK4 have not been developed. Optimization of this series via structure–activity relationships and molecular modeling led to the identification of compound 6 ff (4-(6-fluoro-2H-indazol-3-yl)benzoic acid), a highly potent and selective MEK4 inhibitor. This series of inhibitors is the first of its kind in both activity and selectivity and will be useful in further defining the role of MEK4 in prostate and other cancers.     

Multifaceted Target Specificity Analysis as a Tool in Antimicrobial Drug Development: Type III Pantothenate Kinases as a Case Study

The research and development of a new antimicrobial drug using a target-based approach raises the question of whether any resulting hits will also show activity against the homologous target in other closely related organisms. While an assessment of the similarities of the predicted interactions between the identified inhibitor and the various targets is an obvious first step in answering this question, no clear and consistent framework has been proposed for how this should be done. Here we developed Multifaceted Target Specificity Analysis (MTSA) and applied it to type III pantothenate kinase (PanK_III) – an essential enzyme required for coenzyme A biosynthesis in a wide range of pathogenic bacteria – as a case study to establish if targeting a specific organism's PanK_III would lead to a narrow- or broad-spectrum agent. We propose that MTSA is a useful tool and aid for directing new target-based antimicrobial drug development initiatives.     

Activity‐Based Protein Profiling: Recent Advances in Probe Development and Applications

The completion of the human genome sequencing project has provided a wealth of new information regarding the genomic blueprint of the cell. Although, to date, there are roughly 20 000 genes in the human genome, the functions of only a handful of proteins are clear. The major challenge lies in translating genomic information into an understanding of their cellular functions. The recently developed activity‐based protein profiling (ABPP) is an unconventional approach that is complementary for gene expression analysis and an ideal utensil in decoding this overflow of genomic information. This approach makes use of synthetic small molecules that covalently modify a set of related proteins and subsequently facilitates identification of the target protein, enabling rapid biochemical analysis and inhibitor discovery. This tutorial review introduces recent advances in the field of ABPP and its applications.     

Evaluation of the antimicrobial action of honey against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, Salmonella enteritidis, Listeria monocytogenes and Aspergillus niger. Evaluation of its microbiological charge

The evaluation of the microbiological charge present in Costa Rican samples as the evaluation of its antimicrobial activity over different microorganisms, including those associated to wound infections, will allow to emit criteria referred to its use in therapeutic treatments, specially as alternative therapy for cases involving antibiotic resistant bacteria. The microbiological charge of 25 honey samples, acquired in Costa Rican markets was evaluated through several indicators including total plate aerobic count, total plate anaerobic count, total aerobic spore count, total anaerobic spore count and molds and yeast count. Also, samples were inoculated in tubes with chopped meat media and plated in egg yolk agar in order to determine the presence of Clostridium botulinum. For the antimicrobial activity evaluation, the diffusion method in Muller Hinton agar was performed, testing different honey concentrations (100, 75, 50, 25, 12,5 and 6,25 % v/v) against Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (UCR 2902), Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC25922), Salmonella enteritidis (ATCC 13076), Listeria monocytogenes (ATCC 19116) and Aspergillus niger. The results obtained for the microbiological characterization of honey show that 91% of samples had counts equal or lower than 1,0 x 10(1) CFU/g. No positive result was obtained for the isolation of C. botulinum. 24 of the samples analyzed inhibited the growth of S. aureus even in a 25% v/v concentration, nevertheless, A. niger was no inhibited by any of the samples tested.     

O-羧甲基壳聚糖抗菌性的研究

以金黄色葡萄球菌为实验菌种,研究了羧甲基化度对Ｏ－羧甲基壳聚糖抗菌性的影响,并与相应的壳聚糖的抗菌性对比,结果表明：Ｏ－羧甲基壳聚糖的抗菌性随着羧甲基化度的升高呈现出先升后降的规律,并且在比较宽的羧甲基化度范围内表现出较壳聚糖更好的抗菌性。     

Evaluation of the effect of Lactobacillus rhamnosus probiotic culture added to yogurt over Staphylococcus aureus, Escherichia coli O157:H7, Listeria monocytogenes and Salmonella enteritidis populations

The effect of different types of probiotics present in yogurt over known populations of Staphylococcus aureus, Escherichia coli O157:H7, Listeria monocytogenes and Salmonella enteritidis was evaluated. The three types of yogurt used were: without added probiotics, with added probiotics (Lactobacillus casei CRL_431 and L. acidophilus CRL_730 CHR HANSEN) and another one with the same probiotics mentioned above and Lactobacillus rhamnosus (LR-35) culture. About 10(9) CFU/ mL of each potentially pathogenic bacteria was added to each type of yogurt tested, and kept in refrigeration at 4 degrees C during its shelf life, about 30 days. Bacterial count was done the initial day and every four days. Results obtained show that there is a difference in the inhibition between yogurts without added probiotics and the commercial yogurt with added probiotics; there is a clear inhibitory effect of the last one over S. aureus, E. coli O157:H7 and Listeria monocytogenes. The yogurt with added probiotics and L. rhamnosus did not show any additional inhibitory effect over the bacteria tested when compared with the yogurt with added probiotics. S. enteritidis could not be evaluated because it was not detectable in any yogurt samples evaluated four days after its inoculation. This study confirms the antagonic effect of probiotic cultures over potentially pathogenic bacteria for human beings and animals that may be present in food. Nevertheless, the use of L. rhamnosus did not produce any additional inhibitory effect.