Structure-guided approach to identify a novel class of anti-leishmaniasis diaryl sulfide compounds targeting the trypanothione metabolism

Leishmania protozoans are the causative agent of leishmaniasis, a neglected tropical disease consisting of three major clinical forms: visceral leishmaniasis (VL), cutaneous leishmaniasis, and mucocutaneous leishmaniasis. VL is caused by Leishmania donovani in East Africa and the Indian subcontinent and by Leishmania infantum in Europe, North Africa, and Latin America, and causes an estimated 60,000 deaths per year. Trypanothione reductase (TR) is considered to be one of the best targets to find new drugs against leishmaniasis. This enzyme is fundamental for parasite survival in the human host since it reduces trypanothione, a molecule used by the tryparedoxin/tryparedoxin peroxidase system of Leishmania to neutralize the hydrogen peroxide produced by host macrophages during infection. Recently, we solved the X-ray structure of TR in complex with the diaryl sulfide compound RDS 777 (6-(sec-butoxy)-2-((3-chlorophenyl)thio)pyrimidin-4-amine), which impairs the parasite defense against the reactive oxygen species by inhibiting TR with high efficiency. The compound binds to the catalytic site and engages in hydrogen bonds the residues more involved in the catalysis, namely Glu466′, Cys57 and Cys52, thereby inhibiting the trypanothione binding. On the basis of the RDS 777–TR complex, we synthesized structurally related diaryl sulfide analogs as TR inhibitors able to compete for trypanothione binding to the enzyme and to kill the promastigote in the micromolar range. One of the most active among these compounds (RDS 562) was able to reduce the trypanothione concentration in cell of about 33% via TR inhibition. RDS 562 inhibits selectively Leishmania TR, while it does not inhibit the human homolog glutathione reductase.     

Structural analysis and molecular docking of trypanocidal aryloxy-quinones in trypanothione and glutathione reductases: a comparison with biochemical data

A set of aryloxy-quinones, previously synthesized and evaluated against Trypanosoma cruzi epimastigotes cultures, were found more potent and selective than nifurtimox. One of the possible mechanisms of the trypanocidal activity of these quinones could be inhibition of trypanothione reductase (TR). Considering that glutathione reductase (GR) is the equivalent of TR in humans, biochemical, kinetic, and molecular docking studies in TR and GR were envisaged and compared with the trypanocidal and cytotoxic data of a set of aryloxy-quinones. Biochemical assays indicated that three naphthoquinones (Nq-h, Nq-g, and Nq-d) selectively inhibit TR and the TR kinetic analyses indicated that Nq-h inhibit TR in a noncompetitive mechanism. Molecular dockings were performed in TR and GR in the following three putative binding sites: the catalytic site, the dimer interface, and the nicotinamide adenine dinucleotide phosphate-binding site. In TR and GR, the aryloxy-quinones were found to exhibit high affinity for a site near it cognate-binding site in a place in which the noncompetitive kinetics could be justified. Taking as examples the three compounds with TR specificity (TRS) (Nq-h, Nq-g, and Nq-d), the presence of a network of contacts with the quinonic ring sustained by the triad of Lys62, Met400′, Ser464′ residues, seems to contribute hardly to the TRS. Compound Nq-b, a naphthoquinone with nitrophenoxy substituent, proved to be the best scaffold for the design of trypanocidal compounds with low toxicity. However, the compound displayed only a poor and non-selective effect toward TR indicating that TR inhibition is not the main reason for the antiparasitic activity of the aryloxy-quinones.     

Thiol-induced reduction of antimony(V) into antimony(III): A comparative study with trypanothione, cysteinyl-glycine, cysteine and glutathione

Gluthathione (GSH) has been previously shown to promote the reduction of pentavalent antimony (Sb(V)) into the more toxic trivalent antimony (Sb(III)) in the antimonial drug, meglumine antimonate. However, this reaction occurred at acidic pH (pH 5) but not at the pH of the cytosol (pH 7.2) in which GSH is encountered. The aim of the present study was to further characterize the reaction between thiols and antimonial drugs, addressing the following aspects: (i) the reducing activity of cysteine (Cys) and cysteinyl-glycine (Cys-Gly), expected to be the predominant thiols in the acidic compartiments of mammalian cells; (ii) the reducing activity of trypanothione (T(SH)₂), the main intracelular thiol in Leishmania parasites; (iii) the influence of the state of complexation of Sb(V) on the rate of Sb(V) reduction. We report here that Cys, Cys-Gly and T(SH)₂ did promote the reduction of Sb(V) into Sb(III) at 37 °C. Strikingly, the initial rates of reduction of Sb(V) were much greater in the presence of Cys-Gly, Cys and T(SH)₂ than in the presence of GSH. These reactions occurred at both pH 5 and pH 7 but were favored at acidic pH. Moreover, our data shows that Sb(V) is reduced more slowly in the form of meglumine antimonate than in its non-complexed form, indicating that the complexation of Sb(V) tends to slow down the rate of its reduction. In conclusion, our data supports the hypothesis that Sb(V) is reduced in vivo by T(SH)₂ within Leishmania parasites and by Cys or Cys-Gly within the acidic compartments of mammalian cells.     

Insights about resveratrol analogs against trypanothione reductase of Leishmania braziliensis: Molecular modeling,computational docking and in vitro antileishmanial studies

In this work, we combined molecular modeling, computational docking and in vitro analysis to explore the antileishmanial effect of some resveratrol analogs (ResAn), focusing on their pro-oxidant effect. The molecular target was the trypanothione reductase of Leishmania braziliensis (LbTryR), an essential component of the antioxidant defenses in trypanosomatid parasites. Three-dimensional structures of LbTryR were modeled and molecular docking studies of ResAn1-5 compounds showed the following affinity: ResAn1?>?ResAn2?>?ResAn4?>?ResAn5?>?ResAn3. Positive correlation was observed between these compounds’ affinity to the LbTryR and the IC₅₀ values against Leishmania sp (ResAn1?<?ResAn2?<?ResAn4), which allows for TryR being considered an important target for them. As the compound ResAn1 showed the best antileishmanial activity, and docking studies showed its high affinity for NADP binding site (NS) of TryR, plus having been able to induce ROS production in L. braziliensis promastigotes treated, ResAn1 probably occupies NS interfering in the electron transfer processes responsible for the catalytic reaction. The in silico prediction of ADMET properties suggests that ResAn1 may be a promising drug candidate with properties to cross biological membranes and high gastrointestinal absorption, not violating Lipinski’s rules. Ultimately, the antileishmanial effect of ResAn can be associated with a pro-oxidant effect which, in turn, can be exploited as an antimicrobial agent.

Communicated by Ramaswamy H. Sarma     

Leishmania infantum: provision of reducing equivalents to the mitochondrial tryparedoxin/tryparedoxin peroxidase system

Within the mitochondrion of Leishmania infantum, hydroperoxide metabolism relies on the activity of tryparedoxin-dependent peroxidases (TXNPxs). Tryparedoxins (TXNs) are thioredoxin-related oxidoreductases, which in vitro are reduced by the trypanothione reductase/trypanothione [TR/T(SH)₂] redox couple. Still, there is no evidence that this actually occurs in the mitochondrion. This communication addresses the question of how the mitochondrial TXN/TXNPx system is reduced. First, using a digitonin fractionation assay, we show that TR activity is absent from the L. infantum mitochondrion. The possibility that this organelle possesses alternative electron sources for TXN/TXNPx is then investigated. Biochemical assays performed with purified recombinant enzymes, revealed that TR and T(SH)₂ can be replaced, albeit less efficiently, by the dihydrolipoamide dehydrogenase/lipoamide redox system as TXN/TXNPx electron donor. This result challenges the classical view that T(SH)₂ is the only reductant for TXNs and add new prospects regarding the involvement of 2-oxo acid dehydrogenase complexes in L. infantum mitochondrial hydroperoxide metabolism.     

Rational design of peptide-based inhibitors of trypanothione reductase as potential antitrypanosomal drugs

Summary The rational design of ligands for the substrate-binding site of a homology-modelled trypanothione reductase (TR) was performed. Peptides were designed to be selective for TR over human glutathione reductase (GR). The design process capitalized on the proposed differences between the activesites of TR and human GR, subsequently confirmed by the TR crystal structure. Enzyme kinetics confirmed that forT. cruzi TR benzoyl-Leu-Arg-Arg-ß-naphthylamide was an inhibitor (K_i 13.8µM) linearly competitive with the native substrate, trypanothione disulphide, and did not inhibit glutathione reductase.     

Synthesis and biological evaluation of 2-methyl-1H-benzimidazole-5-carbohydrazides derivatives as modifiers of redox homeostasis of Trypanosoma cruzi

Twelve novel benzimidazole derivatives were synthesized and their in vitro activities against epimastigotes of Trypanosoma cruzi were evaluated. Two derivatives (6 and 7), which have 4-hydroxy-3-methoxyphenyl moiety in their structures, proved to be the most active in inhibiting the parasite growth. Compound 6 showed a trypanocidal activity higher than benznidazole (IC₅₀ = 5 µM and 7.5 µM, respectively) and less than nifurtimox (IC₅₀ = 3.6 µM). In addition, the ability of 6 and 7 to modify the redox homeostasis in T cruzi epimastigote was studied; cysteine and glutathione increased in parasites exposed to both compounds, whereas trypanothione only increased with 7 treatment. These results suggest that the decrease in viability of T. cruzi may be attributed to the change in cellular redox balance caused by compound 6 or 7. Furthermore, compounds 6 and 7 showed CC₅₀ values of 160.64 and 160.66 µM when tested in mouse macrophage cell line J774 and selectivity indexes (macrophage/parasite) of 32 and 20.1, respectively.     

Structure-based virtual screening,molecular docking,ADMET and molecular simulations to develop benzoxaborole analogs as potential inhibitor against Leishmania donovani trypanothione reductase

Visceral leishmaniasis (VL) is the most fatal form of leishmaniasis and it affects 70 countries worldwide. Increasing drug resistant for antileishmanial drugs such as miltefosine, sodium stibogluconate and pentamidine has been reported in the VL endemic region. Amphotericin B has shown potential antileishmanial activity in different formulations but its cost of treatment and associated nephrotoxicity have limited its use by affected people living in the endemic zone. To control the VL infection in the affected countries, it is necessary to develop new antileishmanial compounds with high efficacy and negligible toxicity. Computer aided programs such as binding free energy estimation; ADMET prediction and molecular dynamics simulation can be used to investigate novel antileishmanial molecules in shorter duration. To develop antileishmanial lead molecule, we performed standard precision (SP) docking for 1160 benzoxaborole analogs along with reference inhibitors against trypanothione reductase of Leishmania parasite. Furthermore, extra precision (XP) docking, ADMET prediction, prime MM-GBSA was conducted over 115 ligands, showing better docking score than reference inhibitors to get potential antileishmanial compounds. Simultaneously, area under the curve (AUC) was estimated using ROC plot to validate the SP and XP docking protocol. Later on, two benzoxaborole analogs with best MM-GBSA ΔG-bind were subjected to molecular simulation and docking confirmation to ensure the ligand interaction with TR. The presented drug discovery based on computational study confirms that BOB27 can be used as a potential drug candidate and warrants further experimental investigation to fight against VL in endemic areas.     

Polyamine derivatives as inhibitors of trypanothione reductase and assessment of their trypanocidal activities

Trypanothione reductase (TR) occurs exclusively in trypanosomes and leishmania, which are the etiological agents of many diseases. TR plays a vital role in the antioxidant defenses of these parasites and inhibitors of TR have potential as antitrypanosomal agents. We describe the syntheses of several spermine and spermidine derivatives and the inhibiting effects of these compounds on T. cruzi TR. All of the inhibiting compounds displayed competitive inhibition of TR-mediated reduction of trypanothione disulfide. The three most effective compounds studied were N⁴,N⁸-bis(3-phenylpropyl)spermine (12), N⁴,N⁸-bis(2-naphthylmethyl)spermine (14), and N¹,N⁸-bis(2-naphthylmethyl)spermidine (21), with K_i values of 3.5, 5.5 and 9.5 μM, respectively. Compounds 12, 14, and 21 were found to be potent trypanocides in vitro with IC₅₀ values ranging from 0.19 to 0.83 μM against four T. brucei ssp. strains. However, these compounds did not prolong the lives of mice infected with trypanosomes. This work indicates that certain polyamine derivatives which target a unique pathway in Trypanosomatidae have potential as antitrypanosomal agents.     

Design and synthesis of naphthalimide group-bearing thioglycosides as novel β-N-acetylhexosaminidases inhibitors

GH20 human β-N-acetylhexosaminidases (hsHex) and GH84 human O-GlcNAcase (hOGA) are involved in numerous pathological processes and emerged as promising targets for drug discovery. Based on the catalytic mechanism and structure of the catalytic domains of these β-N-acetylhexosaminidases, a series of novel naphthalimide moiety-bearing thioglycosides with different flexible linkers were designed, and their inhibitory potency against hsHexB and hOGA was evaluated. The strongest potency was found for compound 15j (K_i?=?0.91?µM against hsHexB; K_i?>?100?µM against hOGA) and compound 15b (K_i?=?3.76?µM against hOGA; K_i?=?30.42?µM against hsHexB), which also exhibited significant selectivity between these two enzymes. Besides, inhibitors 15j and 15b exhibited an inverse binding patterns in docking studies. The determined structure–activity relationship as well as the established binding models provide the direction for further structure optimizations and the development of specific β-N-acetylhexosaminidase inhibitors.     

Isolation of a new bioactive cinnamic acid derivative from the whole plant of Viola betonicifolia

A new cinnamic acid derivative was isolated from the whole plant of Viola betonicifolia as off white needle. On the basis of various modern spectroscopic techniques including HREI–MS and 1D and 2D NMR, its structure was elucidated as 2,4-dihydroxy, 5-methoxy-cinnamic acid. It showed marked inhibition against DPPH (diphenyl-2-picryl hydrazyl) free radicals with IC₅₀ = 124?±?5.76 µM. The antioxidant property of the compound was compared with α-tocopherole and vitamin C having IC₅₀ values 96?±?0.46 and 90?±?0.56 µM, respectively. In case of antiglycation assay, the compound exhibited moderate activity (IC₅₀ = 355?±?7.56 µM) similar to standard compound, rutin (IC₅₀ = 294?±?0.56 µM). However, it was non-toxic to PC-3 cell line. It is concluded that 2,4-dihydroxy, 5-methoxy-cinnamic acid has antiglycation potential which was further augmented by its antioxidant activity and thus offered an ideal natural therapeutic option for the effective management of diabetes.     

Synthesis and antitumor activity of novel 2, 3-didithiocarbamate substituted naphthoquinones as inhibitors of pyruvate kinase M2 isoform

The M2 isoform of pyruvate kinase (PKM2) is a potential antitumor therapeutic target. In this study, we designed and synthesised a series of 2, 3-didithiocarbamate substituted naphthoquinones as PKM2 inhibitors based on the lead compound 3k that we previously reported. Among them, compound 3f (IC₅₀?=?1.05?±?0.17 µM) and 3h (IC₅₀?=?0.96?±?0.18 µM) exhibited potent inhibition of PKM2, and their inhibitory activities are superior to compound 3k (IC₅₀?=?2.95?±?0.53 µM) and the known PKM2 inhibitor shikonin (IC₅₀?=?8.82?±?2.62 µM). In addition, we evaluated in vitro antiproliferative effects of target compounds using MTS assay. Most target compounds exhibited dose-dependent cytotoxicity with IC₅₀ values in nanomolar concentrations against HCT116, MCF7, Hela, H1299 and B16 cells. These small molecule PKM2 inhibitors not only provide candidate compounds for cancer therapy, but also offer a tool to probe the biological effects of PKM2 inhibition on cancer cells.     

Oxidative Stress as a Defense Mechanism Against Parasitic Infections

Many parasites – including the causative agents of malaria, Chagas' disease and schistosomiasis – are more susceptible to reactive oxygen species (ROS) than their hosts are. This is manifested by one or more of the following criteria: 1. Susceptibility of the parasite to ROS in vitro; 2. macrophage-based defense mechanisms against the parasite in vivo; 3. successful therapy using agents which lead to oxidative stress; 4. selection advantage (with respect to parasite infections) of human populations whose antioxidant capacity is impaired by a gene defect or by strong oxidants in their staple food.

Our laboratory is involved in developing inhibitors against antioxidant enzymes thus mimicking natural experiments. Since glutathione reductase is a protein of known atomic structure the methods of drug design by receptor fit (DDRF) can be applied for this enzyme. Another promising target enzyme is trypanothione reductase which was found so far only in trypanosomatids, and specifically, not in their hosts. Consequently the trypanothione pathway may be a general target in the design of drugs against diseases caused by trypanosomes and leishmanias.     

New quinoxalinone inhibitors targeting secreted phospholipase A2 and α-glucosidase

Elevated blood glucose and increased activities of secreted phospholipase A2 (sPLA2) are strongly linked to coronary heart disease. In this report, our goal was to develop small heterocyclic compound that inhibit sPLA2. The title compounds were also tested against α-glucosidase and α-amylase. This array of enzymes was selected due to their implication in blood glucose regulation and diabetic cardiovascular complications. Therefore, two distinct series of quinoxalinone derivatives were synthesised; 3-[N′-(substituted-benzylidene)-hydrazino]-1H-quinoxalin-2-ones 3a–f and 1-(substituted-phenyl)-5H-[1,2,4]triazolo[4,3-a]quinoxalin-4-ones 4a–f. Four compounds showed promising enzyme inhibitory effect, compounds 3f and 4b–d potently inhibited the catalytic activities of all of the studied proinflammatory sPLA2. Compound 3e inhibited α-glucosidase (IC₅₀?=?9.99?±?0.18 µM); which is comparable to quercetin (IC₅₀?=?9.93?±?0.66 µM), a known inhibitor of this enzyme. Unfortunately, all compounds showed weak activity against α-amylase (IC₅₀?>?200 µM). Structure-based molecular modelling tools were utilised to rationalise the SAR compared to co-crystal structures with sPLA2-GX as well as α-glucosidase. This report introduces novel compounds with dual activities on biochemically unrelated enzymes mutually involved in diabetes and its complications.     

Febrifugine analogues as Leishmania donovani trypanothione reductase inhibitors: binding energy analysis assisted by molecular docking,ADMET and molecular dynamics simulation

Visceral leishmaniasis affects people from 70 countries worldwide, mostly from Indian, African and south American continent. The increasing resistance to antimonial, miltefosine and frequent toxicity of amphotericin B drives an urgent need to develop an antileishmanial drug with excellent efficacy and safety profile. In this study we have docked series of febrifugine analogues (n = 8813) against trypanothione reductase in three sequential docking modes. Extra precision docking resulted into 108 ligands showing better docking score as compared to two reference ligand. Furthermore, 108 febrifugine analogues and reference inhibitor clomipramine were subjected to ADMET, QikProp and molecular mechanics, the generalized born model and solvent accessibility study to ensure the toxicity caused by compounds and binding-free energy, respectively. Two best ligands (FFG7 and FFG2) qualifying above screening parameters were further subjected to molecular dynamics simulation. Conducting these studies, here we confirmed that 6-chloro-3-[3-(3-hydroxy-2-piperidyl)-2-oxo-propyl]-7-(4-pyridyl) quinazolin-4-one can be potential drug candidate to fight against Leishmania donovani parasites.     

New pyrazolopyrimidine derivatives as Leishmania amazonensis arginase inhibitors

Arginase performs the first enzymatic step in polyamine biosynthesis in Leishmania and represents a promising target for drug development. Polyamines in Leishmania are involved in trypanothione synthesis, which neutralize the oxidative burst of reactive oxygen species (ROS) and nitric oxide (NO) that are produced by host macrophages to kill the parasite. In an attempt to synthesize arginase inhibitors, six 1-phenyl-1H-pyrazolo[3,4-d]pyrimidine derivatives with different substituents at the 4-position of the phenyl group were synthesized. All compounds were initially tested at 100 µM concentration against Leishmania amazonensis ARG (LaARG), showing inhibitory activity ranging from 36 to 74%. Two compounds, 1 (R=H) and 6 (R=CF₃), showed arginase inhibition >70% and IC₅₀ values of 12 µM and 47 µM, respectively. Thus, the kinetics of LaARG inhibition were analyzed for compounds 1 and 6 and revealed that these compounds inhibit the enzyme by an uncompetitive mechanism, showing K_is values, and dissociation constants for ternary complex enzyme-substrate-inhibitor, of 8.5 ± 0.9 µM and 29 ± 5 µM, respectively. Additionally, the molecular docking studies proposed that these two uncompetitive inhibitors interact with different LaARG binding sites, where compound 1 forms more H-bond interactions with the enzyme than compound 6. These compounds showed low activity against L. amazonensis free amastigotes obtained from mice lesions when assayed with as much as 30 µM. The maximum growth inhibition reached was between 20 and 30% after 48 h of incubation. These results suggest that this system can be promising for the design of potential antileishmanial compounds.     

Design and synthesis of new fused carbazole-imidazole derivatives as anti-diabetic agents: In vitro α-glucosidase inhibition,kinetic, and in silico studies

Twenty three fused carbazole–imidazoles 6a–w were designed, synthesized, and screened as new α-glucosidase inhibitors. All the synthesized fused carbazole-imidazoles 6a-w were found to be more active than acarbose (IC₅₀?=?750.0?±?1.5?µM) against yeast α-glucosidase with IC₅₀ values in the range of 74.0?±?0.7–298.3?±?0.9?µM. Kinetic study of the most potent compound 6v demonstrated that this compound is a competitive inhibitor for α-glucosidase (K_i value?=?75?µM). Furthermore, the in silico studies of the most potent compounds 6v and 6o confirmed that these compounds interacted with the key residues in the active site of α-glucosidase.     

Synthesis and biological evaluation of some thiazole derivatives as new cholinesterase inhibitors

In the present study, some thiazole derivatives were synthesized via the ring closure reaction of 1-[2-(2-oxobenzo[d]thiazol-3(2H)-yl)acetyl]thiosemicarbazide with various phenacyl bromides. The chemical structures of the compounds were elucidated by ¹H NMR, ¹³C NMR and mass spectral data and elemental analyses. Each derivative was evaluated for its ability to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) using a modification of Ellman’s spectrophotometric method. The compounds were also investigated for their cytotoxic properties using MTT assay. The most potent AChE inhibitor was found as compound 4e (IC₅₀?=?25.5?±?2.12 µg/mL) followed by compounds 4i (IC₅₀?=?38.50?±?2.12 µg/mL), 4c (IC₅₀?=?58.42?±?3.14 µg/mL) and 4g (IC₅₀?=?68?±?2.12 µg/mL) when compared with eserine (IC₅₀?=?0.025?±?0.01 µg/mL). Effective compounds on AChE exhibited weak inhibition on BuChE (IC₅₀ > 80 µg/mL). MTT assay indicated that the cytotoxic dose (IC₅₀?=?71.67?±?7.63 µg/mL) of compound 4e was higher than its effective dose.     

Synthesis and bioevaluation study of novel N-methylpicolinamide and thienopyrimidine derivatives as selectivity c-Met kinase inhibitors

Four series of N-methylpicolinamide moiety and thienopyrimidine moiety bearing pyridazinone were designed and synthesized and evaluated for the IC₅₀ values against three cancer cell lines (A549, HepG2 and MCF-7) and some selected compounds were further evaluated for the activity against c-Met, Flt-3, VEGFR-2, c-Kit and EGFR kinases. Three compounds (35, 39 and 43) showed more active than positive control Foretinib against A549, HepG2 and MCF-7 cell lines. The most promising compound 43 showed superior activity against A549, HepG2 and MCF-7, with the IC₅₀ values of 0.58?±?0.15?µM, 0.47?±?0.06?µM and 0.74?±?0.12?µM, which were 3.73–5.39-fold more activity than Foretinib, respectively. The experiments of enzyme-based showed that 43 restrain the c-Met selectively, with the IC₅₀ values of 16?nM, which showed equal activity to Foretinib (14?nM) and better than the compound 5 (90?nM). Moreover, AO and Annexin V/PI staining and docking studies were carried out.     

Design,synthesis and biological evaluation of novel L-isoserine tripeptide derivatives as aminopeptidase N inhibitors

Aminopeptidase N (APN/CD13) is one of the essential proteins for tumour invasion, angiogenesis and metastasis as it is over-expressed on the surface of different tumour cells. Based on our previous work that L-isoserine dipeptide derivatives were potent APN inhibitors, we designed and synthesized L-isoserine tripeptide derivatives as APN inhibitors. Among these compounds, one compound 16l (IC₅₀?=?2.51?±?0.2 µM) showed similar inhibitory effect compared with control compound Bestatin (IC₅₀?=?6.25?±?0.4 µM) and it could be used as novel lead compound for the APN inhibitors development as anticancer agents in the future.