biomolecules

Review Review RecentRecent DevelopmentDevelopment ofof Aminoacyl-tRNAAminoacyl-tRNA Synthetase InhibitorsInhibitors forfor HumanHuman Diseases:Diseases: A Future Future Perspective Perspective

Soong-HyunSoong-Hyun Kim Kim, Seri, Seri Bae Bae and and Minsoo Minsoo Song Song * *

NewNew Drug Drug Development Development Center Center (NDDC),(NDDC), Daegu-GyeongbukDaegu-Gyeongbuk MedicalMedical Innovation Foundation (DGMIF), 8080 Cheombok-ro Cheombok-ro Dong-gu, Dong-gu, Daegu Daegu 41061, 41061, Korea;Korea; [email protected]@dgmif.re.kr (S.-H.K.); [email protected]@dgmif.re.kr (S.B.) (S.B.) * *Correspondence: Correspondence: [email protected] [email protected] Received: 30 October 2020; Accepted: 27 November 2020; Published: 1 December 2020


Received: 30 October 2020; Accepted: 27 November 2020; Published: 1 December 2020
 Abstract: Aminoacyl-tRNA synthetases (ARSs) are essential enzymes that ligate amino acids to Abstract:tRNAs andAminoacyl-tRNA translate the genetic synthetases code during (ARSs) protei are essentialn synthesis. enzymes Their that function ligate aminoin pathogen-derived acids to tRNAs andinfectious translate diseases the genetic has been code well during established, protein synthesis. which has Their led functionto the development in pathogen-derived of small molecule infectious diseasestherapeutics. has been The well applicability established, of whichARS inhibitors has led to for the other development human diseases, of small moleculesuch as fibrosis, therapeutics. has Therecently applicability been explored of ARS in inhibitors the clinical for othersetting. human There diseases, are active such studies as fibrosis, to find has small recently molecule been exploredtherapeutics in the for clinical cancers. setting. Studies There on central are active nervou studiess system to find (CNS) small disorders molecule are therapeutics burgeoning for as cancers. well. StudiesIn this onregard, central we nervous present systema concise (CNS) analysis disorders of the are recent burgeoning development as well. of InARS this inhibitors regard, we based present on asmall concise molecules analysis from of the the recent discovery development research of stage ARS inhibitorsto clinical basedstudies on as small well moleculesas a recent from patent the discoveryanalysis from research the medicinal stage to clinicalchemistry studies point as of wellview. as a recent patent analysis from the medicinal chemistry point of view. Keywords: aminoacyl-tRNA synthetase; small molecule inhibitors; human diseases Keywords: aminoacyl-tRNA synthetase; small molecule inhibitors; human diseases

1. Introduction 1. Introduction Aminoacyl-tRNA synthetases (ARSs) are a family of 20 essential enzymes that ligate amino acids to theirAminoacyl-tRNA corresponding synthetases tRNAs and (ARSs) translate are the a family genetic of code 20 essential during enzymesprotein synthesis that ligate [1]. amino The two- acids tostep their catalytic corresponding reaction tRNAs of ARSs and involves translate the the fo geneticrmation code of duringan enzyme-bound protein synthesis aminoacyl-adenylate [1]. The two-step catalytic(AMP-aa) reaction followed of ARSs by the involves formation the of formation an aminoacylated of an enzyme-bound tRNA (tRNA-aa) aminoacyl-adenylate by transferring the (AMP-aa) amino followedacid to the by thecorresponding formation of tRNA. an aminoacylated This catalytic tRNA reaction (tRNA-aa) of ARSs by transferringplays a pivotal the amino role in acid protein to the correspondingsynthesis, which tRNA. is essential This catalytic for the reaction growth ofand ARSs survival plays of a pivotalall cells role(Figure in protein 1). ARSs synthesis, have long which been is essentialstudied foras therapeutic the growth andtargets survival for pathogen-derived of all cells (Figure infectious1). ARSs have diseases, long beenpredominantly studied as therapeuticbecause of targetsconcerns for that pathogen-derived ARS catalytic site infectious inhibitors diseases, would predominantly block translation because in normal of concerns cells [2–4]. that In ARS this catalyticregard, sitevarious inhibitors seminal would reviews block have translation discussed in normal ARS inhibitors cells [2–4 ].for In the this dev regard,elopment various of antibiotics seminal reviews from havedifferent discussed perspectives ARS inhibitors [5–9]. for the development of antibiotics from different perspectives [5–9].

N O N NH2 tRNA N N N O- HO OH - O O O N NH2 O P O P O P O N O - N -O O N O HO OH N NH2 tRNA ATP N "AMP-aa" N protein "tRNA-aa" O OH synthesis O R + AMP H3N + - PPi H3N CO2 R ARS ARS amino acid ARS (aa) FigureFigure 1. 1.Two-step Two-step catalytic catalytic reactionreaction ofof aminoacyl-tRNAaminoacyl-tRNA synthetases (ARSs). ARSs ARSs use use an an amino amino acid acid (aa),(aa), ATP, ATP, and and tRNA tRNA as as substrates substrates to to produce produce anan aminoacylatedaminoacylated tRNA (tRNA-aa) that that leads leads to to protein protein synthesissynthesis in in the the ribosome. ribosome.

Biomolecules 2020, 10, x; doi: FOR PEER REVIEW www.mdpi.com/journal/biomolecules

Biomolecules 2020, 10, 1625; doi:10.3390/biom10121625 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, x FOR PEER REVIEW 2 of 24

Biomolecules 2020 10 However,, a, 1625significant amount of research targeting mammalian ARSs for the treatment2 of of 24 human diseases have recently emerged. For example, febrifugine derivatives, including halofuginone whichHowever, targets prolyl-tRNA a significant synthetase amount of (ProRS), research are targeting under investigation mammalian for ARSs the fortreatment the treatment of cancer, of humanfibrosis, diseases and inflammatory have recently diseases emerged. [10,11]. For example, Additionally, febrifugine threonyl-tRNA derivatives, synthetase including halofuginone(ThrRS) was whichidentified targets as a prolyl-tRNAtarget protein synthetase of antiangiogenic (ProRS), borrelidin are under [12], investigation and a peptide for thefragment treatment derived of cancer, from fibrosis,tryptophanyl-tRNA and inflammatory synthetase diseases (TrpRS) [10,11 was]. Additionally, studied as threonyl-tRNAa possible anti-angiogenic synthetase (ThrRS)agent [13]. was identifiedHowever, asPfizer a target has proteindropped of its antiangiogenic development borrelidin of the TrpRS [12], fragment and a peptide as an fragmentanti-angiogenic derived lead from a tryptophanyl-tRNAwhile ago. Accordingly, synthetase Kwon (TrpRS)et al. [14] was separate studiedly as provided a possible thorough anti-angiogenic reviews agent on the [13 physiology,]. However, Pfizerpathology, has droppedand structural its development aspects of ARSs, of the as TrpRS well fragmentas the development as an anti-angiogenic of ARS inhibitors lead a for while human ago. Accordingly,diseases, including Kwon infectious et al. [14] separatelydiseases, cancer, provided inflammatory thorough reviewsdiseases, on and the central physiology, nervous pathology, system and(CNS) structural disorders. aspects In this of review, ARSs, we as wellfocus as on the the development recent development of ARS of inhibitors ARS inhibitors for human ranging diseases, from includingdiscovery infectiousto clinical diseases,trials from cancer, medicinal inflammatory chemistry diseases, perspectives and centraland provide nervous a related system patent (CNS) disorders.analysis. In this review, we focus on the recent development of ARS inhibitors ranging from discovery to clinical trials from medicinal chemistry perspectives and provide a related patent analysis. 2. Structural Class of ARS Inhibitors 2. Structural Class of ARS Inhibitors 2.1. Benzoxaboroles 2.1. Benzoxaboroles Benzoxaboroles are a class of boron-containing compounds with a broad range of biological activities.Benzoxaboroles A subset of are benzoxaboroles a class of boron-containing have antimicrobial compounds activity withprimarily a broad due range to their of biologicalability to activities.inhibit leucyl-tRNA A subset synthetase of benzoxaboroles (LeuRS) havevia the antimicrobial oxaborole tRNA-trapping activity primarily (OBORT) due to mechanism, their ability towhich inhibit involves leucyl-tRNA the formation synthetase of a (LeuRS)stable tRNA via theLeu− oxaborolebenzoxaborole tRNA-trapping adduct in which (OBORT) the mechanism,boron atom whichinteracts involves with the the 2′ formation- and 3′-oxygen of a stable atoms tRNA of theLeu terminalbenzoxaborole 3′ tRNA adduct adenosi006Ee in which [15]. the boronIn a related atom − interactsstudy, researchers with the 20 -at and Anacor 30-oxygen Pharmaceuticals atoms of the terminal reported 30 tRNA an X-ray adenosi006Ee crystal [structure15]. In a relatedof Thermus study, researchersthermophilus atLeuRS Anacor complexed Pharmaceuticals with tRNA reportedLeu and anthe X-ray benzoxaborole-base crystal structured LeuRS of Thermus inhibitor thermophilus AN2690 LeuRSto form complexed a tRNA–AN2690 with tRNA adductLeu possessingand the benzoxaborole-based tetrahedral spiroborate LeuRS structure inhibitor 1 (Figure AN2690 2) to[16]. form The a tRNA–AN2690tetrahedral spiroborate adduct possessingadduct was tetrahedral stabilized by spiroborate the formation structure of covalent1 (Figure bonds2)[ 16 between]. The tetrahedral the Lewis spiroborateacidic boron adduct atom wasand stabilized the 2′- and by the3′-oxygen formation atoms of covalent of the bonds tRNA’s between 3′-terminal the Lewis adenosine acidic boron as a atomnucleophile and the [17,18] 20- and. The 30-oxygen oxaborole atoms scaffold of the can tRNA’s reversibly 30-terminal form covalent adenosine tetrahedral as a nucleophile complexes [17 with,18]. Thenucleophiles, oxaborole such scaff asold hydroxyl can reversibly groups, form because covalent of the tetrahedral presence of complexes the heterocyclic with nucleophiles, boron atom, such which as hydroxylhas an empty groups, p becauseorbital. ofTargets the presence for these of the compounds heterocyclic boroninclude atom, β-lactamases, which has ancyclic empty nucleotidep orbital. Targetsphosphodiesterase for these compounds 4 (PDE4), Rho-associated include β-lactamases, protein kinase cyclic (ROCK), nucleotide carbonic phosphodiesterase anhydrase, and 4 (PDE4), LeuRS Rho-associated[19]. protein kinase (ROCK), carbonic anhydrase, and LeuRS [19].

N H2N N N N O

tRNA O B O O F 1 Leu FigureFigure 2.2. AN2690-tRNA Leu adductadduct via via the the oxaborole oxaborole tRNA tRNA-trapping-trapping (OBORT) (OBORT) mechanism. mechanism.

A recent example of the benzoxaborole class of inhibitors is the nitrophenyl sulfonamide-based A recent example of the benzoxaborole class of inhibitors is the nitrophenyl sulfonamide-based benzoxaborole PT638; Si et al. investigated PT638 to determine its mode of action [15]. The finding that benzoxaborole PT638; Si et al. investigated PT638 to determine its mode of action [15]. The finding PT638 has potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) (MIC that PT638 has potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) [minimum inhibitory concentration] of 0.4 µg/mL) and does not inhibit Staphylococcus aureus LeuRS (MIC [minimum inhibitory concentration] of 0.4 μg/mL) and does not inhibit Staphylococcus aureus (Sa LeuRS) (IC50 [the half maximal inhibitory concentration] >100 µM) suggests the possibility that it LeuRS (Sa LeuRS) (IC50 [the half maximal inhibitory concentration] >100 μM) suggests the possibility hasthat targetsit has targets other than other LeuRS. than LeuRS. The selection The selection for resistance for resistance to PT638 to using PT638 MRSA using andMRSA whole-genome and whole- sequencing of three colonies that showed MICs of 3.125 µg/mL resulted in three mutations in the genome sequencing of three colonies that showed MICs≥ of ≥3.125 μg/mL resulted in three mutations LeuRSin the LeuRS gene, including gene, including D343Y, G302S,D343Y, andG302S, F233I, and all F233I, of which all of are which located are within located the within editing the domain editing of Sa Sa domainLeuRS, of therebySa LeuRS, confirming thereby confirmingLeuRS as Sa the LeuRS target as of the PT638. target A metabolismof PT638. A studymetabolism of PT638 study using of MRSA cell lysate and a SAR (structure-activity relationship) study of PT638 analogs revealed that Biomolecules 2020,, 10,, 1625x FOR PEER REVIEW 33 of of 24

PT638Biomolecules using 2020 MRSA, 10, x FOR cell PEER lysate REVIEW and a SAR (structure-activity relationship) study of PT6383 analogsof 24 PT638 might be a prodrug that can be reduced to its amine analog PT662 mainly by nitroreductase revealedPT638 usingthat PT638MRSA might cell lysate be a andprodrug a SAR that (structure-activity can be reduced relationship) to its amine study analog of PT662PT638 analogsmainly by NfsB in MRSA (Figure3). nitroreductaserevealed that NfsBPT638 in might MRSA be (Figure a prodrug 3). that can be reduced to its amine analog PT662 mainly by nitroreductase NfsB in MRSA (Figure 3).

Figure 3. Benzoxaborole PT638 is reduced by a nitroreductase to PT662. Figure 3. Benzoxaborole PT638 is reduced by a nitroreductase to PT662. Figure 3. Benzoxaborole PT638 is reduced by a nitroreductase to PT662. In a related study, GlaxoSmithKlineGlaxoSmithKline (GSK)(GSK) developeddeveloped aa seriesseries ofof 3-aminomethylbenzoxaborole3-aminomethylbenzoxaborole derivativesIn a related 4 and study,5 thatthat GlaxoSmithKline targettarget MycobacteriumMycobacterium (GSK) tuberculosis developed (Mtb)a series LeuRS of 3-aminomethylbenzoxaborole [[20].20]. The boron atom in the moleculesderivatives was 4 andcritical 5 that for target Mtb LeuRSMycobacterium activity tuberculosis since it forms (Mtb) a LeuRS bidentate [20]. covalent The boron adduct atom within the the molecules was critical for Mtb LeuRS activity since it forms a bidentateLeu covalent adduct with the terminal nucleotide of tRNA, Ade76, and trapstraps thethe 330′ end ofof tRNAtRNALeu inin the the editing site to inhibit terminal nucleotide of tRNA, Ade76, and traps the 3′ end of tRNALeu in the editing site to inhibit leucylation andand thusthus protein protein synthesis. synthesis. The The ( S()-aminomethylS)-aminomethyl side side chain chain at at C-3 C-3 in 4inand 4 and5 was 5 was critical critical for leucylation and thus protein synthesis. The (S)-aminomethyl side chain at C-3 in 4 and 5 was critical binding,for binding, and C-4and halogen C-4 halogen atoms (Clatoms and Br)(Cl significantlyand Br) significantly improved Mtb improvedLeuRS activity,Mtb LeuRS antitubercular activity, for binding, and C-4 halogen atoms (Cl and Br) significantly improved Mtb LeuRS activity, activityantitubercular against activityMtb H37Rv, against and Mtb selectivity H37Rv, and against selectivity other bacteria. against other However, bacteria. potential However, toxicity potential issues antitubercular activity against Mtb H37Rv, and selectivity against other bacteria. However, potential fromtoxicity the issues inhibition from of the mammalian inhibition cytoplasmicof mammali LeuRSan cytoplasmic and tolerance LeuRS issues and of tolerance5 in a once issues daily of dose 5 in ofa 50toxicity mg/kg issues in a mouse from acutethe inhibition tuberculosis of mammali (TB) infectionan cytoplasmic model causedLeuRS seriousand tolerance concerns issues for of its 5 use in a as a onceonce daily daily dose dose of of 50 50 mg/kgmg/kg inin aa mousemouse acute tuberculosistuberculosis (TB) (TB) infection infection model model caused caused serious serious TB drug in long-term therapy. Throughout a series of medicinal chemistry campaigns to examine the concernsconcerns for for its its use use as as a a TB TB drugdrug inin long-termlong-term therapy.therapy. Throughout Throughout a aseries series of of medicinal medicinal chemistry chemistry structure–activity relationship (SAR) of 3-aminomethylbenzoxaborole derivatives, compounds 6–10 campaignscampaigns to to examine examine thethe structure–activitystructure–activity relarelationshiptionship (SAR)(SAR) of of 3-aminomethylbenzoxaborole 3-aminomethylbenzoxaborole were found to improve both selectivity against human cytoplasmic LeuRS and HepG2 cell toxicity derivatives,derivatives, compounds compounds 66––1010 werewere foundfound toto improveimprove bothboth sele selectivityctivity against against hu humanman cytoplasmic cytoplasmic compared with 4 and 5, presumably by decreased lipophilicity and increased polarity in the molecule LeuRSLeuRS and and HepG2 HepG2 cell cell toxicity toxicity comparedcompared with 44 andand 55, ,presumably presumably by by decreased decreased lipophilicity lipophilicity and and (Figureincreasedincreased4)[ polarity21 polarity]. Triaging inin thethe lead moleculemolecule compounds (Figure(Figure through 4)4) [21].[21].in vivo TriagingTriagingpharmacokinetic leadlead compounds compounds (PK)analysis through through and in in evivoffi vivocacy assayspharmacokineticpharmacokinetic using acute (PK) (PK) and analysisanalysis chronic and mouseand efficacy TB infection assaassaysys usingusing models acuteacute identified and and chronic chronic8 (GSK656) mouse mouse asTB TB a infection preclinical infection candidatemodelsmodels identified identified for tuberculosis; 8 8(GSK656) (GSK656)8 successfully as as aa preclinicalpreclinical completed candidatecandidate its for first-time-in-humanfor tuberculosis; tuberculosis; 8 8successfully successfully (FTIH) study completed completed to evaluate its its thefirst-time-in-humanfirst-time-in-human safety, tolerability, (FTIH) (FTIH) pharmacokinetics, studystudy toto evaluate and food thethe esafety,safety,ffects of tolerability,tolerability, single (5, 15,pharmacokinetics, pharmacokinetics, and 25 mg) and and repeat and food food oral doseseffectseffects (5 of andof single single 15 mg,(5, (5, 15, 1415, and days,and 25 25 qd mg)mg) [quaque andand repeat die, once oraloral adosesdoses day]; (5 (5 NCT03075410) and and 15 15 mg, mg, 14 14 days, [22 days,]. Moreover,qd qd [quaque [quaque8 die,is currentlydie, once once ina day]a a day] phase ; NCT03075410); NCT03075410) 2 clinical trial [22].[22]. for Moreover, drug-sensitiveMoreover, 8 is pulmonarycurrentlycurrently inin tuberculosisa a phase phase 2 2clinical clinical to evaluate trial trial for for its drug-sensitive earlydrug-sensitive bacterial activity,pulmonarypulmonary safety, tuberculosis tuberculosis and tolerability to to evaluate evaluate (NCT03557281). itsits early bact Detailserialerial activity, activity, of the clinicalsafety, safety, an studies and dtolerability tolerability of 8 will (NCT03557281). be(NCT03557281). discussed in a laterDetailsDetails section. of of the the clinical clinical studies studies of of 88 willwill bebe discussed inin a a later later section. section.

FigureFigure 4. 4. DevelopmentDevelopment of GSK656 GSK656 throug throughh hit-to-lead hit-to-lead optimization. optimization. Figure 4. Development of GSK656 through hit-to-lead optimization.

Biomolecules 2020, 10, 1625 4 of 24

2.2. Cladosporin CladosporinBiomolecules 2020, isolated10, x FOR PEER from REVIEWCladosporium cladosporioides and Aspergillus flavus was found4 of to24 have potent2.2. antimalarial Cladosporin activity against both liver- and blood-stage Plasmodium falciparum by targeting the parasite cytosolic lysyl-tRNA synthetase (Pf LysRS) and terminating protein biosynthesis [23]. Despite itsCladosporin high clearance isolated and from poor Cladosporium oral bioavailability cladosporioidesin and vivo Aspergillus[24,25], the flavus favorable was found selectivity to have and potent antimalarial activity against both liver- and blood-stage Plasmodium falciparum by targeting the potency shown by cladosporin against Pf LysRS over human LysRS spurred medicinal chemistry efforts parasite cytosolic lysyl-tRNA synthetase (Pf LysRS) and terminating protein biosynthesis [23]. Despite to furtherits high develop clearance it toand overcome poor oral its bioavailability metabolic limitations in vivo [24,25], as an the oral favorable drug. selectivity In this regard, and potency Rusch et al. analyzedshown cladosporin by cladosporin by incubatingagainst Pf LysRS it with over mouse human liver LysRS microsomes spurred medicinalin vitro andchemistry revealed efforts that to three majorfurther metabolically develop it weak to overcome points its might metabolic cause limitatio the metabolicns as an liabilitiesoral drug. ofIn cladosporinthis regard, Rusch (Figure et al.5)[ 26]. The hydroxylanalyzed cladosporin group, tetrahydropyran, by incubating it with and C-4mouse of liver isochromenone microsomes in were vitro potentially and revealed the that three three major metabolicallymajor metabolically weak points. weak Additionally,points might cause the the X-ray metabolic crystal liabilities structure of cladosporin of cladosporin (Figure and 5) [26].P. falciparum The LysRShydroxyl (Pf LysRS) group, suggested tetrahydropyran, an H-bond and with C-4 Glu332of isochromenone and the π /cationwere potentially ‘cage’ from the adjacent three major aromatic and chargedmetabolically residues, weak andpoints. the Additionally, interaction betweenthe X-ray thecrystal lactone structure ring of and cladosporin Arg559 throughand P. falciparum water as the LysRS (Pf LysRS) suggested an H-bond with Glu332 and the π/cation ‘cage’ from adjacent aromatic and major interactions [27]. Based on these basic findings, medicinal chemistry efforts were focused on the charged residues, and the interaction between the lactone ring and Arg559 through water as the major three metabolically weak points of cladosporin to generate the three major derivatives A (12), B (13), interactions [27]. Based on these basic findings, medicinal chemistry efforts were focused on the three and Cmetabolically(14) as representative weak points of examples. cladosporin Upon to generate screening the three these major libraries derivatives using A the (12 Transcreener), B (13), and C AMP assay(14 system) as representative to assess aminoacylation examples. Upon by screening LysRS [th23ese], thelibraries unsaturation using the ofTranscreener the isocoumarin AMP assay core and transformationsystem to assess of the aminoacylation metabolically by unstable LysRS tetrahydropyran[23], the unsaturation ring of to the cyclohexylisocoumarin ringcore yieldedand a selectivetransformation LysRS inhibitor of the metabolically with improved unstable metabolic tetrahydropyran stability at ring the to level the ofcyclohexyl hepatic ring extraction. yielded Ina line with aselective previous LysRS report inhibitor by Rusch with et improved al. [26] focusing metabolic on st metabolicability at the weak level points of hepatic of cladosporin, extraction. In Zhou line et al. confirmedwith a thatprevious the methylreport by tetrahydropyran Rusch et al. [26] focusing moiety ofon cladosporinmetabolic weak could points be of replaced cladosporin, by a Zhou more et stable methylcyclohexane,al. confirmed that the while methyl maintaining tetrahydropyran potency moiety in of the cladosporin ATP hydrolysis could be replaced assay (compounds by a more stable15 and methylcyclohexane, while maintaining potency in the ATP hydrolysis assay (compounds 15 and 16) 16)[28]. The methyl group in the cyclohexane ring was important for the hydrophobic interaction with [28]. The methyl group in the cyclohexane ring was important for the hydrophobic interaction with Ser344, resulting in increased potency of 4-fold compared to that of compound 15. The replacement Ser344, resulting in increased potency of 4-fold compared to that of compound 15. The replacement 3,4 withwith a lactam a lactam group group or or a a conjugated conjugated Δ∆3,4 doubledouble bond bond within within the isocou the isocoumarinmarin ring was ring tolerated, was tolerated, but but thethe twotwo phenolic hydroxyl hydroxyl groups groups were were critical critical for binding for binding to LysRS to LysRS(Figure (Figure5). 5).

FigureFigure 5. Optimization 5. Optimization of of cladosporin cladosporin to improv improvee pharmacokinetic pharmacokinetic properties properties [26,28]. [26 ,28]. Biomolecules 2020, 10, x 1625 FOR PEER REVIEW 5 5of of 24 24

Another medicinal chemistry campaign to develop a new antimalaria and anti-cryptosporidiosis drug Anothertargeting medicinal Pf LysRS1 chemistry and Cryptosporidium campaign to parvum develop (Cp) a new LysRS antimalaria was continued and anti-cryptosporidiosis by Baragaña et al. usingdrug targetingcladosporinPf LysRS1as a starting and Cryptosporidiumpoint [29]. Its low parvum druggability (Cp) LysRS due wasto high continued metabolic by instability Baragaña etwas al. optimizedusing cladosporin by replacing as a startingthe isocoumarin point [29 core]. Its with low th druggabilitye chromone duescaffold to high connected metabolic to a instability metabolically was stabilizedoptimized cyclohexane by replacing ring the isocoumarin with an amide core linker, with the as chromoneshown in scaFigureffold 6. connected Compound to a 21 metabolically was active againststabilized both cyclohexane Pf LysRS1 ring (IC with50 an= 0.015 amide μ linker,M, luciferase as shown ATP in Figure consumpt6. Compoundion assay)21 was and active whole-cell against bloodstreamboth Pf LysRS1 P. falciparum (IC50 = 0.015 3D7µ (ECM,50 luciferase [the half ATPmaximal consumption effective assay)concentration] and whole-cell = 0.27 μ bloodstreamM) and was selectiveP. falciparum compared3D7 (EC with50 [thehuman half (Hs) maximal LysRS e(ICffective50 = 1.8 concentration] μM). The pharmacokinetic= 0.27 µM) and properties was selective were optimizedcompared withby stabilizinghuman (Hs) theLysRS metabolically (IC50 = 1.8 labileµM). cyclohexyl The pharmacokinetic ring and blocking properties the werepoint optimized of potential by hydroxylationstabilizing the metabolicallyof the phenyl labile group cyclohexyl in the chromone ring and core. blocking As a the result, point compound of potential 21 hydroxylation showed good of systemicthe phenyl exposure group inwith the excellent chromone oral core. bioavailability As a result, compound(F = 100%) and21 showed a moderate good half-life systemic (t1/2 exposure = 2.5 h). Inwith vivo excellent efficacy oral was bioavailability evaluated using (F =a NODscidIL2R100%) and a moderateγnull mouse half-life (SCID) (t1 /model2 = 2.5 and h). Indoses vivo upeffi tocacy 40 mg/kgwas evaluated (po [per usingos, oral a NODscidIL2Radministration]),γnull and mouse it was (SCID) found modelthat a daily and doses oral dose up to of 40 ED mg90 /=kg 1.5 (po mg/kg [per (1.0–2.3os, oral administration]),mg/kg) (estimated and daily it was exposure found in that blood a daily AUC oralED90 dose [area of under ED90 = the1.5 curve mg/kg at (1.0–2.3 ED90] = mg11,000/kg) ng·h/mL/d(estimated (6900–14,000 daily exposure ng·h/mL/d)) in blood AUCreduced parasitemi[area undera by the 90%. curve A high at ED level] of= 11,000sequence ng identityh/mL/d ED90 90 · within(6900–14,000 the active ng h /sitemL /regiond)) reduced of Pf parasitemiaLysRS1 and by Cp 90%. LysRS A high (96% level identity) of sequence and an identity overall withinsequence the · identityactive site of region47.7% ofandPf similarityLysRS1 and of 64.6%Cp LysRS across (96% the identity) entire protein and an was overall observed sequence from identity the evaluation of 47.7% ofand compound similarity of21 64.6% against across Cryptosporidium the entire protein parvum was both observed in vitro from and the evaluationin vivo. Compounds of compound for21 cryptosporidiosisagainst Cryptosporidium treatment parvum mustboth alsoin have vitro goodand in ex vivoposure. Compounds in the gastrointestinal for cryptosporidiosis tract and treatment possibly alsomust some also havesystemic good exposure exposure since in the the gastrointestinal parasite is found tract predominantly and possibly also in the some gastrointestinal systemic exposure tract withsince some the parasite in the isbiliary found predominantlytract. In this regard, in the gastrointestinalcompound 21 was tract worthy with some of inconsideration the biliary tract. for cryptosporidiosisIn this regard, compound since 17%21 ofwas the worthy oral dose of considerationwas found in mouse for cryptosporidiosis stools, suggesting since that 17% some of the biliary oral excretiondose was had found occurred. in mouse Compound stools, suggesting 21 showed that efficacy some biliary in vivo excretion in twohad different occurred. Cryptosporidium Compound mouse21 showed models, efficacy NODin vivoSCIDin gamma two di ffanderent INF-Cryptosporidiumγ-knockout, atmouse 20 mg/kg models, (po). NOD However, SCID gammathe toxicity and concernINF-γ-knockout, of 21 at 50 at mg/kg 20 mg in/kg mice (po). (po) However, at which the the toxicity blood concentration concern of 21 inat mice 50 mg reached/kg in micethe EC (po)50 for at HepG2which the cells blood limited concentration further development. in mice reached It is thepresum EC50edfor that HepG2 the toxicity cells limited is due further to the development.inhibition of mammalianIt is presumed LysRS. that the toxicity is due to the inhibition of mammalian LysRS.

FigureFigure 6. 6. OptimizationOptimization of of cladosporin cladosporin by by replacing replacing the the core core with with a a chromone chromone scaffold. scaffold.

Considering the the significant significant structural diffe differencesrences between cladosporin derivatives ( (1111––1919)) and thethe newly newly found found chromone chromone derivatives derivatives (20 (20 andand 2121),), Baragaña Baragaña et et al. al. analyzed analyzed the the binding binding modes modes of cladosporinof cladosporin and and 20 20byby studying studying their their X-ray X-ray co-crystal co-crystal structure structure with with PfPf LysRS1LysRS1 (Figure (Figure 7)7)[ [29].29]. Cladosporin bindsbinds to toPf PfLysRS1 LysRS1 within within theATP-binding the ATP-binding pocket pocket with the with isocoumarin the isocoumarin moiety occupying moiety occupyingthe same space the same as the space adenine as the ring adenine of ATP, ring and theof ATP, tetrahydropyran and the tetrahydropyran system taking system the same taking position the sameas the position ribose ring as the of ATP.ribose The ring two of phenolic ATP. The hydroxy two phenolic groups hydroxy of cladosporin groups form of cladosporin hydrogen bonds form hydrogenwith E332 bonds and the with NH ofE332 N339, and while the NH the carbonylof N339, interacts while the with carbonyl a highly interacts coordinated withconserved a highly coordinatedwater molecule. conserved Although water20 molecule.binds with AlthoughPf LysRS1 20 in binds theATP-binding with Pf LysRS1 pocket in the in ATP-binding a similar manner pocket to incladosporin, a similar manner the bicyclic to cladosporin, core is rotated the 30 ◦bicyclicanticlockwise core is withrotated respect 30° anticlockwise to cladosporin. with The respect chromone to cladosporin. The chromone core stacks between F342, H338, and R559. The ring carbonyl forms an Biomolecules 2020, 10, 1625 6 of 24 Biomolecules 2020, 10, x FOR PEER REVIEW 6 of 24

H-bondcore stacks to the between backbone F342, NH H338, of andN339, R559. mimicking The ring the carbonyl N1 of adenine forms an and H-bond the O1 to theOH backbone of cladosporin. NH of TheN339, amide mimicking carbonyl the H N1 bonds of adenine to a highly and the conserved O1 OH ofwater cladosporin. molecule Thecoordinated amide carbonyl by the side H bonds chain to of a D558highly and conserved R559. The water cyclohexyl molecule moiety coordinated projects into by the a pocket side chain formed of D558by the and side R559. chains The of R330, cyclohexyl F342, andmoiety S344 projects and the into backbone a pocket of formedL555 and by G556. the side This chains pocket of is R330, completed F342, and by the S344 substrate and the lysine backbone and ofis similarL555 and to G556.that occupied This pocket by the is completed tetrahydropyran by the substrate ring of cladosporin, lysine and is except similar that tothat the occupiedcyclohexyl by ring the probestetrahydropyran deeper into ring the of pocket cladosporin, [29]. except that the cyclohexyl ring probes deeper into the pocket [29].

Figure 7. Binding modes of cladosporin and 20 toto Pf LysRS1. Pf LysRS1:Lys:LysRS1:Lys:20 showing the binding mode of 20 (C atoms, gold) bound to the ATP sitesite ofof PfPf LysRS1 (PDB ID code 6AGT) superimposed upon Pf LysRS1:Lys:cladosporin (cladosporin C atoms, slate; PDB ID code 4PG3). The image was reproduced from reference 2929 byby BaragañaBaragaña etet al.al. and adapted for repurposing.

2.3. Borrelidin Borrelidin Borrelidin is known as a potent inhibitor of threonyl-tRNA synthetase (ThrRS) (ThrRS) for for malaria. malaria. However, its lack of selectivity against human ThrRSThrRS and high toxicity to human cells limit further development as a drug candidate. In this regard, Novoa et al. derived a series of borrelidin analogs that circumvent these potential problems by showing higher selectivity and lower cytotoxicity from minor structural changes in the carboxylic acid side chain [30]. [30]. In In particular, particular, compound BC196 BC196 ( (2323),), which released rotational constraints by adopting linear carboxylic acid, and and compound compound BC220 BC220 ( (2424),), which converted terminal carboxylic acid to pyridyl ethyl ester on the cylcopentane ring, showed improved selectivityselectivity against againstP.falciparum P. falciparumover humanover human HEK293 HEK293 cells by 13,579-andcells by 13,579-and 4048-fold, respectively. 4048-fold, respectively.Considering thatConsidering borrelidin that exhibited borrelidin 355-fold exhibited activity 355-fold difference activity between differenceP. falciparum between P.and falciparum human andHEK293 human cells, HEK293 selectivity cells, was selectivity improved was by improved 11–38-fold. by From 11–38-fold.in vivo Fromefficacy in vivo studies efficacy using studies a Plasmodium using ayoelii Plasmodium17XL-infected yoelii 17XL-infected mouse model, mouse23 and model,24 showed 23 and 100% 24 survivalshowed over100% 20 survival days after over drug 20 days treatment after drugat 6 mg treatment/kg per day.at 6 Inmg/kg particular, per day.24 showedIn particular, complete 24 showed clearance complete of parasites clearance in the of blood parasites at 6 mgin /thekg perblood day, at 80%6 mg/kg survival, per andday, ~3% 80% parasitemia survival, and at 0.25~3% mg parasitemia/kg per day. at It0.25 is also mg/kg interesting per day. that It 24is alsowas interestingnot cleaved that to borrelidin 24 was bynotesterase, cleaved astodetermined borrelidin inbytime este courserase, as experiments determined with in time infected course red experimentsblood cells (RBCs) with infected over 27 red h, suggesting blood cells that (RBCs) the biologicalover 27 h, esuggestingffect of 24 isthat not the due biological to its conversion effect of 24 to isborrelidin not due to by its RBCs conversion or plasmodial to borrelidin esterases by (FigureRBCs or8). plasmodial esterases (Figure 8). Biomolecules 2020, 10, x FOR PEER REVIEW 7 of 24 Biomolecules 2020, 10, 1625 7 of 24 Biomolecules 2020, 10, x FOR PEER REVIEW 7 of 24

Figure 8. Optimization of borrelidin selectivity for Plasmodium falciparum over human threonyl-tRNA FiguresynthetaseFigure 8. 8.Optimization (ThrRS).Optimization of of borrelidin borrelidin selectivityselectivity for Plasmodium falciparum falciparum overover human human threonyl-tRNA threonyl-tRNA synthetasesynthetase (ThrRS). (ThrRS). 2.4. Benzothiazoles 2.4. Benzothiazoles 2.4.Unlike Benzothiazoles other LysRS inhibitors that target the active site of pathogen-derived LysRS, Kim et al. reportedUnlikeUnlike a new other other class LysRS LysRS of small inhibitors inhibitors molecule thatthat targettargetinhibitors the acti active, includingve site site of of YH16899, pathogen-derived pathogen-derived that bind LysRS, LysRS,to human Kim Kim et LysRS al. et al. reportedwithoutreported affecting a newa new class theclass catalytic of of small small activity molecule molecule of inhibitors,LysRSinhibitors [31], . including Physiologically, YH16899, YH16899, it isthat thatknown bind bind thatto to human LysRS human LysRSbinds LysRS to withoutthewithout 67-kDa aff ectingaffecting laminin the the catalyticreceptor catalytic activity (67LR), activity of aof LysRS targetLysRS [31 [31]for]. Physiologically,.antimetastatic Physiologically, ittherapeutics it is is known known that that [32], LysRS LysRS in bindsthe binds plasma to to the 67-kDamembranethe 67-kDa laminin and laminin enhances receptor receptor (67LR), cell migration (67LR), a target athat for target antimetastaticcauses for cancerantimetastatic therapeuticsmetastasis. therapeutics Thus, [32], init is the[32], thought plasma in the that membrane plasma directly andinhibitingmembrane enhances the and cellinteraction enhances migration cellbetween that migration causes LysRS cancerthat and causes metastasis. 67LR cancer by metastasis.a Thus, small it ismolecule Thus, thought it is could thatthought directly suppress that inhibitingdirectly cancer themetastasisinhibiting interaction and the betweenderive interaction a new LysRS between class and of 67LR antimetastaticLysRS by and a small 67LR cancer molecule by atherapy. small could molecule In suppress this regard, could cancer 25suppress showed metastasis cancer specific and deriveinhibitionmetastasis a new of and classthe derive ofbinding antimetastatic a new between class of cancer theantimetastatic LysRS therapy. an Incancerd this67LR therapy. regard, pair based25 Inshowed this on regard, in specific vitro 25 showed inhibitionpull-down specific of and the bindingimmunoprecipitationinhibition between of the the binding LysRS (IP) assays andbetween 67LR and the pair decreased LysRS based onan thedin 67LR vitroamount pairpull-down ofbased membranous andon in immunoprecipitation vitro 67LR pull-down using LysRS-and (IP) assaysoverexpressingimmunoprecipitation and decreased H226 thesquamous (IP) amount assays oflung and membranous carcinomadecreased 67LRthecells amount using(Figure LysRS-overexpressingof 9).membranous The in vivo 67LR efficacy H226using squamousof LysRS- 25 was overexpressing H226 squamous lung carcinoma cells (Figure 9). The in vivo efficacy of 25 was lungdemonstrated carcinoma using cells three (Figure different9). The in invivo vivo mouseefficacy models, of 25 includingwas demonstrated (i) a mouse using breast three cancer di ffmodelerent demonstrated using three different in vivo mouse models, including (i) a mouse breast cancer model in(4T1 vivo cells,mouse 100 models,mpk [mg including per kilogram] (i) a mouse and breast300 mpk, cancer po, model~60% inhibition (4T1 cells, 100of tumor mpk [mg metastasis), per kilogram] (ii) a (4T1 cells, 100 mpk [mg per kilogram] and 300 mpk, po, ~60% inhibition of tumor metastasis), (ii) a andTg (MMTV-PyVT) 300 mpk, po, ~60% model inhibition (100 mpk, of po, tumor ~70% metastasis), reduction (ii)of pulmonary a Tg (MMTV-PyVT) nodule formation), model (100 and mpk, (iii) po, a Tg (MMTV-PyVT) model (100 mpk, po, ~70% reduction of pulmonary nodule formation), and (iii) a ~70%cancer reduction cell colonization of pulmonary model nodule (A549 cells formation), expressi andng red (iii) fluorescence a cancer cell colonizationprotein (RFP), model ~50% (A549 reduction cells cancer cell colonization model (A549 cells expressing red fluorescence protein (RFP), ~50% reduction expressing red fluorescence protein (RFP), ~50% reduction of tumor metastasis in the brain and bone of oftumor tumor metastasis metastasis in in the the brain brain and and bone bone tumorstumors over 77 weeks).weeks). Overall, Overall, Kim Kim et et al. al. showed showed that that the the tumorsprotein–proteinprotein–protein over 7 weeks). interaction interaction Overall, of of KimLyLysRS-67LRsRS-67LR et al. showed isis a promising that the protein–protein approachapproach for for the interactionthe development development of LysRS-67LR of ofnew new istherapeutics atherapeutics promising for approachfor human human diseases. for diseases. the development of new therapeutics for human diseases.

FigureFigure 9.9. 9.YH16899, YH16899, a aprotein–protein protein–protein interaction interaction inhibi inhibitortortor ofof LysRSLysRS and and 67-kDa 67-kDa laminin laminin receptor receptorreceptor (67LR).(67LR).(67LR). 2.5. Halofuginone 2.5.2.5. Halofuginone Halofuginone Halofuginone (HF, 26), a well-known antifibrotic agent in preclinical and clinical studies, HalofuginoneHalofuginone (HF, (HF, 26 26),), aa well-knownwell-known antifibroticantifibrotic agentagent inin preclinicalpreclinical and and clinical clinical studies, studies, inhibitsinhibits prolyl-tRNA prolyl-tRNA synthetase synthetase (Pro (ProRS)RS) activity activity [10]. [10 ].Mechanistically, Mechanistically, 26 competitively26 competitively binds bindsto the to inhibits prolyl-tRNA synthetase (ProRS) activity [10]. Mechanistically, 26 competitively binds to the theproline-binding proline-binding pocket pocket of ofthe the catalytic catalytic site site of ofProRS ProRS and and causes causes drug drug resistance resistance due due to tothe the proline-binding pocket of the catalytic site of ProRS and causes drug resistance due to the accumulationaccumulation of of proline proline [ 33[33].]. ItIt is anticipated that that the the antifibrotic antifibrotic effect effect of 26 of would26 would be diminished be diminished in accumulation of proline [33]. It is anticipated that the antifibrotic effect of 26 would be diminished in in fibroticfibrotic tissue because because a a higher higher concentration concentration of of proline proline is isoften often observed observed in infibrotic fibrotic tissue tissue than than in in fibrotic tissue because a higher concentration of proline is often observed in fibrotic tissue than in nonfibroticnonfibrotic tissue tissue [34 ].[34]. In thisIn this regard, regard, Shibata Shibata et al. et from al. from Takeda Takeda Pharmaceuticals Pharmaceuticals developed developed T-3833261 T- nonfibrotic tissue [34]. In this regard, Shibata et al. from Takeda Pharmaceuticals developed T- (27), a potent ATP competitive inhibitor of ProRS that does not bind to the proline-binding site [11]. The Biomolecules 2020,, 1010,, 1625x FOR PEER REVIEW 88 of of 24

3833261 (27), a potent ATP competitive inhibitor of ProRS that does not bind to the proline-binding antifibroticsite [11]. The activity antifibrotic of 27 wasactivity evaluated of 27 was both evaluatedin vitro and bothin in vivo vitro. From and in the vivo. transforming From the growthtransforming factor betagrowth (TGF- factorβ)-induced beta (TGF- fibroticβ)-induced assay, 27 fibroticinhibited assay, the expression 27 inhibited of fibrotic the expression markers, of including fibroticα markers,-smooth muscleincluding actin α-smooth (α-SMA) muscle and typeactin I(α collagen-SMA) and (Col1a), type I by collagen reducing (Col1a), mothers by reducing against decapentaplegicmothers against homologdecapentaplegic 3 (Smad3) homolog expression 3 (Smad3) in human expression skin fibroblasts. in human skin These fibroblasts. results were These directly results correlated were directly with thecorrelatedin vivo withTGF- theβ-induced in vivo TGF- mouseβ-induced model. Themouse topical model. application The topical of application27 reduced of the 27 expression reduced the of fibroticexpression genes of suchfibrotic as genes Col1a1, such Col1a2, as Col1a1, and α -SMACol1a2, (Figure and α -SMA10). (Figure 10).

Figure 10. AntifibroticAntifibrotic prolyl-tRNA synthetase (Pro (ProRS)RS) inhibitors halofuginone and T-3833261.

In a related study, itit waswas presumedpresumed thatthat thethe proline-richproline-rich nature of profibroticprofibrotic proteinsproteins such as collagens may be attributed to glutamyl-prolyl-tRNAglutamyl-prolyl-tRNA synthetase (GluProRS)-mediated translational regulation duringduring cardiaccardiac fibrosis. fibrosis. Several Several studies studies with with indirect indirect evidence evidence support support the the regulatory regulatory role role of GluProRSof GluProRS as aas target a target gene gene for cardiac for cardiac fibrosis. fibrosis. For example, For example, hypoactive hypoactive mutations mutations in the ProRS in the domain ProRS ofdomain GluProRS of GluProRS lead to hypomyelinating lead to hypomyelinating leukodystrophy leukodystrophy without causing without any causing known cardiacany known dysfunction cardiac indysfunction patients, implyingin patients, that implying reduced that ProRS reduced enzymatic ProRS activity enzymatic may notactivity adversely may not aff ectadversely normal affect heart functionnormal heart while function reducing while proline-rich reducing protein proline-rich synthesis protein [35]. In synthesis this regard, [35]. Akashi In this Therapeutics regard, Akashi used 26Therapeuticsto treat Duchenne used 26 muscular to treat Duchenne dystrophy muscular (DMD), analogous dystrophy to (DMD), reducing analogous fibrosis by to 26reducingand increasing fibrosis muscleby 26 and strength increasing [36–39 muscle]. Recently, strength Wu et[36–39]. al. examined Recently, GluProRS-mediated Wu et al. examined regulatory GluProRS-mediated mechanisms ofregulatory profibrotic mechanisms protein synthesis of profibrotic at the translational protein synthesis level and at identified the translational increased level GluProRS and identified in failing humanincreased and GluProRS mouse hearts in failing [40]. human Increased and GluProRS mouse hear contributedts [40]. Increased to the elevated GluProRS translation contributed of proline to the (Pro)-richelevated translation (PRR) mRNAs, of proline and (Pro)-rich genetic knockout (PRR) mR ofNAs, the glutamyl-prolyl-tRNA and genetic knockout of synthetase the glutamyl-prolyl- gene (Eprs) reducedtRNA synthetase fibrosis in gene different (Eprs heart) reduced failure fibrosis mouse in models. different Additionally, heart failure the mouse selective models. GluProRS Additionally, inhibitor 26thereduced selective the GluProRS translation inhibitor of PRR 26 mRNAs, reduced such the astranslation collagens of and PRR other mRNAs, novel such PRR genes,as collagens including and latentother novel TGF-β PRR-binding genes, protein including 2 (LTBP2) latent andTGF- sulfataseβ-binding 1 (SULF1).protein 2 (LTBP2) and sulfatase 1 (SULF1).

2.6. Sulfonamido Propanamides and 2-Aminopyrimidines Aminoacyl-tRNA synthetase-inter synthetase-interactingacting multifunctional multifunctional protein protein 2 2 (AIMP2), a a scaffold scaffold in the multi-tRNA synthetase complex (MSC), dissociates from the MSC and influencesinfluences the activity of the β α α p53, TGF-β, tumor necrosis factor- α (TNF-(TNF-α),), and and Wingless/int-1 Wingless/int-1 (WNT) si signalinggnaling pathways pathways [41,42]. [41,42]. A splicingsplicing variant of AIMP2AIMP2 lacking exon 2 (AIMP2-DX2)(AIMP2-DX2) correlates positively with cancer by compromising the tumor-suppressive function of native AIMP2AIMP2 throughthrough competitivecompetitive interactionsinteractions with p53 fuse-bindingfuse-binding protein (FBP) and TNF receptor-associated factor 2 (TRAF2) [[43].43]. Lim Lim et et al. al. showed that AIMP2-DX2 binds with heat shock protein 70 (HSP70) and is stabilized by blocking the seven in absentia homolog 1 (Siah1)-dependent(Siah1)-dependent ubiquitination of AIMP2-DX2,AIMP2-DX2, which leads to cancer progression in vivovivo [[44].44]. RepresentativeRepresentative AIMP2-DX2 inhibitors (28~3030)) are are described described in in Figure Figure 11.11. Based on this modemode ofof actionaction ofof AIMP2-DX2,AIMP2-DX2, thethe protein–proteinprotein–protein interactioninteraction inhibitorinhibitor BC-DXI-495BC-DXI-495 ((28)) waswas foundfound toto specificallyspecifically inhibitinhibit thethe AIMP2-DX2AIMP2-DX2 ++ HSP70 interaction and suppress cancer cell growth in vitro (IC = 4.2 µM, luciferase assay; EC = 14.2 µM, cell viability assay, A549 cells) and growth in vitro (IC5050 = 4.2 μM, luciferase assay; EC50 = 14.2 μM, cell viability assay, A549 cells) and AIMP2-DX2-induced tumor tumor growth growth inin vivo vivo (~50%(~50% decrease, decrease, 50 50 mpk, mpk, ip ip [intraperitoneal [intraperitoneal injection], injection], 2 2weeks, weeks, H460 H460 stably stably expressing expressing AIMP2-DX2 AIMP2-DX2 WT WT [wild [wild type], type], BALB/cSLC BALB/cSLC mice).mice). Consecutively, Consecutively, Sivaraman etet al. al. reported reported BC-DXI-843 BC-DXI-843 (29), which(29), which was optimized was optimized from BC-DXI-495 from BC-DXI-495 [45]; 29 significantly [45]; 29 improved in vitro activity in luciferase and cell viability assays (IC = 0.92 µM, luciferase assay; significantly improved in vitro activity in luciferase and cell viability assays50 (IC50 = 0.92 μM, luciferase assay; EC50 = 1.2 μM, cell viability assay, A549 cells). The in vivo efficacy of 29 in the H460 xenograft Biomolecules 2020, 10, 1625 9 of 24

Biomolecules 2020, 10, x FOR PEER REVIEW 9 of 24 Biomolecules 2020, 10, x FOR PEER REVIEW 9 of 24 EC50 = 1.2 µM, cell viability assay, A549 cells). The in vivo efficacy of 29 in the H460 xenograft mouse mouse model was comparable with that of 28 by inhibiting ~60% tumor growth (50 mpk, bid [bis in modelmouse was model comparable was compar withable that with of 28 thatby inhibitingof 28 by inhibiting ~60% tumor ~60% growth tumor (50 growth mpk, (50 bid mpk, [bis in bid die, [bis twice in die, twice a day], ip, 2 weeks). In the same context, Lee et al. separately developed the 2- adie, day], twice ip, 2 weeks).a day], Inip, the 2 sameweeks). context, In the Lee same et al. separatelycontext, Lee developed et al. separately the 2-aminophenylpyrimidine developed the 2- aminophenylpyrimidine derivative 30 for AIMP2-DX2 interaction inhibition with >80% tumor derivativeaminophenylpyrimidine30 for AIMP2-DX2 derivative interaction 30 for inhibition AIMP2-DX2 with >interaction80% tumor inhibition growth inhibition with >80% in antumor H460 growth inhibition in an H460 xenograft mouse model [46]. xenograftgrowth inhibition mouse model in an [H46046]. xenograft mouse model [46].

FigureFigure 11. 11. Aminoacyl-tRNA Aminoacyl-tRNAAminoacyl-tRNA synthetase-interactingsynthetase-interacting synthetase-interacting multifunctional multifunctional proteinprotein protein 2 2 lacking lacking 2 exon lackingexon 2 2 (AIMP2- (AIMP2- exon 2 (AIMP2-DX2)DX2)DX2) interaction interaction interaction inhibitors. inhibitors. inhibitors.

2.7.2.7. Bicylic Bicylic Azetidines Azetidines Lastly,Lastly, KatoKato Kato et etet al. al.al. reportedreported bicyclicbicyclic azetidines such as 3131 andand 3232 (Figure(Figure 12)1212)) as asas po potentpotenttent inhibitors inhibitorsinhibitors ofof Pf Pf phenylalanyl-tRNA phenylalanyl-tRNA synthetase synthetase (PheRS) (PheRS) (EC50 == 999 andand and 55 5 nM,nM, nM, respectively,respectively, respectively, againstagainst a a multi-drug multi-drug resistantresistant strain, strain, PfPf Dd2)Dd2)Dd2) forfor for thethe the treatmenttreatment treatment of of malaria malaria [47].[47]. [47 Lead].Lead Lead optimizationoptimization optimization campaigncampaign campaign allowed allowed allowed the the µ theimprovementimprovement improvement of of inin of vitrovitroin vitro potenciespotenciespotencies ofof 31 of against31 against PlasmodiumPlasmodium cynomolgicynomolgi cynomolgi (EC(EC5050(EC = = 3.34/2.86 3.34/2.8650 = 3.34 μ μM/2.86M for for small Msmall for smallformform [SF, form[SF, hypnozoite-like]/largehypnozoite-like]/large [SF, hypnozoite-like] form/formlarge [LF, form schizont], [LF, schizont], respectively),respectively), respectively), P.P. falciparumfalciparumP. falciparum liver-stageliver-stageliver-stage (EC (EC5050 = = (EC1.311.3150 μ μM),=M),1.31 and and µP. P.M), falciparumfalciparum and P. transmission-stage,transmission-stage, falciparum transmission-stage, gametocyte IV–VIV–V gametocyte (EC(EC5050 == 663 IV–V663 nM) nM) (EC to to50 EC EC=5050 =663 = 0.933/1.04 0.933/1.04 nM) to ECμμMM50 (for (for= 0.933SF/LF), SF/LF),/1.04 0.34 0.34µM μμM,M, (for andand SF 0.16/0.16LF), μμ 0.34M inµ 32M,, respectively. and 0.16 µM IssuesIssues in 32 in,in respectively. 3131 suchsuch asas poor poor Issues solubility solubility in 31 (<1 such(<1 μ μM asM poorinin PBS),PBS), solubility highhigh clearance (clearance<1 µM in in PBS),in humahuma highnn and clearance mouse in liver human microsomes and mouse (Cl(Cl liverintint = microsomes= 142142 andand 248248 (Cl μintμL/min/mg,L/min/mg,= 142 and 248respectively),respectively),µL/min/mg, andand respectively), aa highhigh volumevolume and ofof a distributiondistribution high volume inof vivo distribution (12(12 L/kg)L/kg) inwerewere vivo optimizedoptimized(12 L/kg) by by were replacing replacing optimized the the byhydroxymethylhydroxymethyl replacing the ofof hydroxymethyl 3131 toto N,NN,N-dimethylaminomethyl-dimethylaminomethyl of 31 to N,N-dimethylaminomethyl group inin 3232 toto givegive group improvedimproved in 32 solubility tosolubility give improved (15 (15 μ μMM solubilityinin PBS), PBS), decreased decreased (15 µM in clearanceclearance PBS), decreased inin humanhuman clearance and mouse inhuman liver microsomesmicrosomes and mouse (Cl(Cl liverintint == microsomes3131 andand 21 21 μ μL/min/mg,L/min/mg, (Clint = 31 andrespectively),respectively), 21 µL/min and /andmg, aa respectively), betterbetter volumevolume and ofof distribution a better volume in vivo of distribution (24(24 L/kg),L/kg), resultingresultingin vivo in (24in a a Llonger longer/kg), resulting half-life half-life (32 in(32 a longerh)h) which which half-life can can be be (32suitable suitable h) which for for single-dosesingle-dose can be suitable oral fortreatment. single-dose In vivo oral efficacyefficacy treatment. studies studiesIn of vivoof 32 32 in einffi multiple multiplecacy studies stages stages of 32ofof malariain malaria multiple models models stages includingincluding of malaria PlasmodiumPlasmodium models including berghei CD-1Plasmodium andand P.P. falciparumfalciparum berghei CD-1 huRBChuRBC and NSG P.NSG falciparum mouse mouse models huRBCmodels NSGshowedshowed mouse complete complete models eliminationelimination showed complete ofof blood-blood- elimination and liver-stage of blood- parasitesparasites and liver-stage byby aa singlesingle parasites dosedose during during by a singlethe the study study dose duringperiod.period. theThis This study study study period. hashas servedserved This study asas anan example has served of asdeveloping an example aa curecure of developing forfor malariamalaria a as cureas well well for as as malaria the the potential potential as well astoto the givegive potential prophylaxisprophylaxis to give andand prophylaxis preventprevent diseasedisease and prevent transmissi diseaseon. Additionally, transmission. VinayakVinayak Additionally, etet al.al. reported Vinayakreported that etthat al. reportedbicyclicbicyclic azetidine thatazetidine bicyclic 3232 is azetidineis aa potentpotent32 inhibitorinhibitoris a potent of inhibitorCryptosporidium of Cryptosporidium parvumparvum PheRSPheRS parvum ((CpCp PheRS) PheRSPheRS)( Cpfor for PheRS)diarrheal diarrheal for diarrhealdiseasedisease inin disease youngyoung in childrenchildren young children [48].[48]. TheThe [48 ].in The vivoin vivoefficacyeffi cacyof 3232 of for32for for cryptosporidiosiscryptosporidiosis cryptosporidiosis waswas was shownshown shown in in immunosuppressedimmunosuppressed mouse mousemouse models. models.models. Overall,Overall,Overall, itit isis thought thought that thatthat bicyclic bicyclicbicyclic azetidines azetidinesazetidines suchsuch such asas as32 32 32possess possess possessa uniqueaa uniqueunique core corecore skeleton skeletonskeleton and warrantandand warrantwarrant further further examination examination to exploit toto their exploitexploit chemical theirtheir space chemicalchemical for applications spacespace forfor notapplicationsapplications only in infectious not not only only diseases,inin infectiousinfectious but diseases,diseases, also for other but also diseases. for other diseases.diseases.

Figure 12. BRD7929, a potent inhibitor of Pf PheRS for malaria. Figure 12.12. BRD7929, a potent inhibitor of Pf PheRS for malaria.

Biomolecules 2020, 10, 1625 10 of 24 Biomolecules 2020, 10, x FOR PEER REVIEW 10 of 24

3. Aminoacyl-tRNA Synthetase (ARS) Therapeutics in Patents As mentioned before, much research onon eukaryoticeukaryotic aminoacyl-tRNAaminoacyl-tRNA synthetases (eARSs) has recently been conducted to determine the differe differentnt roles of human ARSs other than being the “housekeeping”“housekeeping” enzyme for proteinprotein synthesissynthesis inin variousvarious humanhuman diseasesdiseases [[14].14]. Human ARSs have also become major targets in the treatment of proliferativeproliferative diseases, such as cancers, inflammatoryinflammatory diseases, autoimmune diseases, and fibrosis.fibrosis. In this section, we discuss patent patentss related to therapeutic development targetingtargeting both both prokaryotic prokaryotic and and eukaryotic eukaryotic ARSs. ARSs. Since Since Gadakh Gadakh et al. atet the al. Rega at the Institute Rega forInstitute Medical for ResearchMedical Research reported reported a patent reviewa patent on revi ARSew inhibitorson ARS inhibitors as antimicrobial as antimicrobial agents in agents 2012 [ 7in], we2012 specifically [7], we specifically analyzed patentsanalyzed from patents 2013 tofrom the present2013 to day.the present From the day. SciFinder From the search, SciFinder eight relevant search, patentseight relevant were identified, patents were and identified, we present and them we in presen the alphabeticalt them in the order alphabetical of ARS targets. order of ARS targets.

3.1. Glycyl-tRNA Synthetase (GlyRS) Inhibitors Professors Schimmel and Yang at the Scripps Research Institute disclosed four different different glycylsulfamoyladenosine derivatives (GlySAs, FigureFigure 1313)) as human human glycyl-tRNA glycyl-tRNA synthetase synthetase (GlyRS) (GlyRS) inhibitors for cancercancer therapytherapy byby loweringlowering neddylationneddylation [[49].49]. The The covalent attachment of NEDD8 (neuronal precursorprecursor cell-expressedcell-expressed developmentally developmentally downregulated downregulated gene gene 8) 8) to to the the core core cullin cullin protein protein of cullin-RINGof cullin-RING ligase ligase E3s (CRLs,E3s (CRLs, e.g., Ubc12), e.g., Ubc12), called neddylation, called neddylation, is a crucial is process a crucial for the process ubiquitination for the ofubiquitination multiple protein of multiple substrates protein mediated substrates by mediated GlyRS. GlyRS by GlyRS. is highly GlyRS expressed is highly in expressed most malignant in most cancers,malignant such cancers, as breast such cancer, as breast ovarian cancer, cancer, ovaria andn lungcancer, cancer. and Selectivelung cancer. binding Selective of GlySAs binding to theof catalyticGlySAs to domain the catalytic of human domain GlyRS of human inhibited GlyRS the attachmentinhibited the of attachment NEDD8, consequently of NEDD8, consequently reducing the levelreducing of neddylation the level of byneddylation more than by 50% more in variousthan 50% cancer in various cells. cancer The patent cells. providedThe patent the provided systematic the modesystematic of action mode (MOA) of action evaluation (MOA) of GlyRS evaluation in the neddylationof GlyRS in pathway the neddylation and the confirmation pathway ofand GlyRS the inhibitionconfirmation with of the GlyRS crystal inhibition structure wi ofth GlySA the crystal (33) binding structure to humanof GlySA GlyRS (33) (PDBbinding code: to 2ZT8).human GlySAs GlyRS were(PDB screenedcode: 2ZT8). with GlySAs the NCI were 60 cellscreened panel with assay the at NCI a single 60 cell dose panel of5–10 assay M, at anda single GlySA dose (33 of) showed5–10 M, aand range GlySA of growth(33) showed inhibition a range values of growth (GI50 =inhibition0.372–3.715 valuesµM) (GI in50 58= 0.372–3.715 cancer cell μ lines.M) in With 58 cancer the lung cell metastasislines. With model the lung induced metastasis by MDA-MB-231 model induced cells, 33bywas MDA-MB-231 administered cells, via intravenous33 was administered injection twice via aintravenous week at adose injection of 4 mgtwice/kg a ( nweek= 10). at Aftera dose 10 of days 4 mg/kg of injection (n = 10). of After GlyRS 10 inhibitors, days of injection33 reduced of GlyRS lung metastasesinhibitors, 33 in reduced the mouse lung model metastases with 69% in ethefficacy, mouse which model was with greater 69% than efficacy, that(4.4 which mg /waskg, 40%greater effi cacy)than ofthat MLN4924. (4.4 mg/kg, MLN4924, 40% efficacy) an inhibitor of MLN4924. of NEDD8-activating MLN4924, an enzyme inhibitor (NAE), of NEDD8-activating is under development enzyme for various(NAE), is types under of development tumors in a clinical for various setting type [50s]. of tumors in a clinical setting [50].

Figure 13.13. TheThe structurestructure of of glycylsulfamoyladenosine glycylsulfamoyladenosine derivatives derivatives (GlySAs) (GlySAs) as eukaryotic as eukaryotic glycyl-tRNA glycyl- synthetasetRNA synthetase (GlyRS) (GlyRS) inhibitors. inhibitors. 3.2. Leucyl-tRNA Synthetase (LeuRS) Inhibitors 3.2. Leucyl-tRNA Synthetase (LeuRS) Inhibitors Oxford Drug Design Ltd. reported a leucyl-tRNA synthetase (LeuRS) inhibitor from Oxford Drug Design Ltd. reported a leucyl-tRNA synthetase (LeuRS) inhibitor from 2-amino-N- 2-amino-N-(arylsulfinyl) acetamide compounds as an antibiotic agent [51]. They prepared a series (arylsulfinyl) acetamide compounds as an antibiotic agent [51]. They prepared a series of 46 of 46 compounds and their corresponding TFA (trifluoroacetic acid) salts, as shown in Figure 14, compounds and their corresponding TFA (trifluoroacetic acid) salts, as shown in Figure 14, of which of which the dissociation constants (Kd) were measured for Escherichia coli LeuRS by isothermal titration the dissociation constants (Kd) were measured for Escherichia coli LeuRS by isothermal titration calorimetry (ITC) and were found to range from 1.39 nM to 9130 nM. The antibacterial activity of the calorimetry (ITC) and were found to range from 1.39 nM to 9130 nM. The antibacterial activity of the 26 compounds was evaluated in seven bacterial strains (ATCC25922, EFFLUX, B1966, ATCC700603, 26 compounds was evaluated in seven bacterial strains (ATCC25922, EFFLUX, B1966, ATCC700603, ATCC27853, B1931, and ATCC49247) with MIC values from 0.25 to >64 mg/L, and compound 37 was ATCC27853, B1931, and ATCC49247) with MIC values from 0.25 to >64 mg/L, and compound 37 was the most potent compound with MIC values in all strains (0.5, 0.25, 1, 1, 32, 2, and 4 mg/L, Biomolecules 2020, 10, 1625 11 of 24 Biomolecules 2020, 10, x FOR PEER REVIEW 11 of 24 Biomolecules 2020,, 10,, xx FORFOR PEERPEER REVIEWREVIEW 11 of 24 the most potent compound with MIC values in all strains (0.5, 0.25, 1, 1, 32, 2, and 4 mg/L, respectively) respectively) comparable to those of controls, such as ciprofloxacin, ceftazidime, meropenem, and comparablerespectively) to comparable those of controls, to those such of controls, as ciprofloxacin, such as ceftazidime,ciprofloxacin, meropenem, ceftazidime, and meropenem, azithromycin. and azithromycin.respectively) comparable No compound to those showed of controls,any potential such cytotoxicas ciprofloxacin, effects against ceftazidime, the human meropenem, hepatic andcell Noazithromycin. compound No showed compound any potential showed any cytotoxic potential effects cytotoxic against effects the human against hepatic the human cell linehepatic HepG2 cell lineazithromycin. HepG2 (ATCC No compound HB-8065). showed any potential cytotoxic effects against the human hepatic cell (ATCCline HepG2 HB-8065). (ATCC HB-8065).

Figure 14. Synthesis of 2-amino-N-(arylsulfinyl) acetamide compounds and the structure of the Figure 14.14. SynthesisSynthesis of of 2-amino- N-(arylsulfinyl)-(arylsulfinyl) acetamide acetamide compounds compounds and and the the structure of thethe prokaryoticFigure 14. SynthesisLeuRS inhibitor of 2-amino- 37. N-(arylsulfinyl) acetamide compounds and the structure of the prokaryotic LeuRSLeuRS inhibitorinhibitor 3737... Jirgensons et al. at the Latvian Institute of Organic Synthesis developed N-aminoacyl- Jirgensons et et al. al. at the atLatvian the Inst Latvianitute of Institute Organic ofSynthesis Organic developed Synthesis N-aminoacyl- developed arylsulfonamideJirgensons etderivatives al. at the as Latvianaminoacyl-tRNA Institute syof nthetaseOrganic (ARS) Synthesis inhibitors developed for the Ntreatment-aminoacyl- of Narylsulfonamide-aminoacyl-arylsulfonamide derivatives as derivatives aminoacyl-tRNA as aminoacyl-tRNA synthetase (ARS) synthetase inhibitors (ARS) for inhibitorsthe treatment for the of bacterialarylsulfonamide infections derivatives [52,53]. asArylsulfonyl aminoacyl-tRNA chlorides synthetase were (ARS) converted inhibitors to forthe the corresponding treatment of treatmentbacterial ofinfections bacterial infections[52,53]. Arylsulfonyl [52,53]. Arylsulfonyl chlorides chlorides were wereconverted converted to tothe the corresponding arylsulfonamides,bacterial infections in [52,53].which theArylsulfonyl terminal amine chlorides was coupledwere converted with amino to acidsthe tocorresponding provide 52 arylsulfonamides, inin which which the the terminal terminal amine amine was coupled was coupled with amino with acids amino to provide acids to 52 compoundsprovide 52 compoundsarylsulfonamides, of N-aminoacyl-arylsulfonamides, in which the terminal amine as shown was coupledin Scheme with 1. All amino compounds acids wereto provide tested for52 ofcompoundsN-aminoacyl-arylsulfonamides, of N-aminoacyl-arylsulfonamides, as shown in as Scheme shown 1in. Scheme All compounds 1. All compounds were tested were for testedin vitro for incompounds vitro enzyme of N -aminoacyl-arylsulfonamides,inhibition assays against leucyl as shown-, valyl- in and Scheme isoleucyl-tRNA 1. All compounds synthetases were tested (LeuRS, for enzymein vitro enzyme inhibition inhibition assays againstassays leucyl-,against leucyl valyl--, and valyl- isoleucyl-tRNA and isoleucyl-tRNA synthetases synthetases (LeuRS, (LeuRS, ValRS, ValRS, and IleRS, respectively) from E coli and S. aureus and showed IC50 values from 0.0034 to >50 andValRS, IleRS, and respectively) IleRS, respectively) from E. from coli and E coliS. aureusand S.and aureus showed and showed IC50 values IC50 fromvalues 0.0034 from to 0.0034>50 µ toM >50 for μValRS,M for andE. coli IleRS, LeuRS respectively) and from 0.26 from to E>50 coli μ Mand for S. S. aureus aureus and LeuRS. showed However, IC50 values none fromof the 0.0034 compounds to >50 E.μM coli forLeuRS E. coli and LeuRS from and 0.26 from to > 500.26µM to for>50S. μ aureusM for S.LeuRS. aureus However, LeuRS. However, none of the none compounds of the compounds presented presentedμM for E. coliany LeuRS inhibitory and fromactivities 0.26 toat >50 concentrations μM for S. aureus of 50 LeuRS. μM or However, below against none of IleRS the compoundsand ValRS anypresented inhibitory any activities inhibitory at concentrationsactivities at concentrations of 50 µM or below of 50 against μM or IleRS below and against ValRS bacterialIleRS and enzymes ValRS bacterialpresented enzymes any inhibitory from either activities E. coli at or concentrations S. aureus. Therefore, of 50 μtheM scopeor below of substituents against IleRS on theand phenyl ValRS frombacterial either enzymesE. coli or fromS. aureus either. Therefore, E. coli or S. the aureus scope. Therefore, of substituents the scope on the of phenyl substituents ring was on investigated the phenyl ringbacterial was enzymesinvestigated from further. either ItE. wascoli orfound S. aureus that .the Therefore, compounds the scope bearing of substituentsa meta-substituted on the phenylphenyl further.ring was It investigated was found that further. the compounds It was found bearing that the a meta-substituted compounds bearing phenyl a meta-substituted ring as well as a leucinephenyl ring aswas well investigated as a leucine further. moiety It presented was found a significant that the compounds increase in bearingthe inhibition a meta-substituted of LeuRS from phenyl E. coli moietyring as well presented as a leucine a significant moiety presented increase in a significant the inhibition increase of LeuRS in thefrom inhibitionE. coli ofand LeuRSS. aureus from E.with coli and S. aureus with submicromolar activity (Figure 15) (e.g., IC50 values of compound 42: 0.002 μM for submicromolarand S. aureus with activity submicromolar (Figure 15) activity (e.g., IC (Figure50 values 15) of (e.g., compound IC50 values42: 0.002of compoundµM for E. 42 coli: 0.002LeuRS μM and for E.and coli S. LeuRS aureus andwith 0.33 submicromolar μM for S. aureus activity LeuRS). (Figure 15) (e.g., IC50 values of compound 42: 0.002 μM for 0.33E. coliµ MLeuRSLeuRS for S. andand aureus 0.330.33LeuRS). μM for S. aureus LeuRS).LeuRS).

Scheme 1. SynthesisSynthesis of of N-aminoacyl-arylsulfonamides.-aminoacyl-arylsulfonamides. Scheme 1. Synthesis of N-aminoacyl-arylsulfonamides.

Figure 15. Representative examples of N-aminoacyl-arylsulfona-aminoacyl-arylsulfonamidesmides and their inhi inhibitorybitory activities Figure 15. Representative examples of N-aminoacyl-arylsulfonamides and their inhibitory activities againstFigure 15.LeuRS Representative from Escherichia examples coli and of StaphylococcusN-aminoacyl-arylsulfona aureus.. mides and their inhibitory activities against LeuRS from Escherichia coli andand Staphylococcus aureus.. 3.3. Methionyl-tRNA Synthetase (MetRS) Inhibitors 3.3. Methionyl-tRNA Synthetase (MetRS) Inhibitors 3.3. Methionyl-tRNA Synthetase (MetRS) Inhibitors The Buckner group at the University of Washington found that compounds bearing The Buckner group at the University of Washington found that compounds bearing N- N-containingThe Buckner heteroaromatic group at bicyclic the University rings inhibit of methionyl-tRNAWashington found synthetase that compounds (MetRS) for antibacterialbearing N- containingThe Buckner heteroaromatic group bicyclicat the Universityrings inhibit of me Washingtonthionyl-tRNA found synthetase that compounds(MetRS) for antibacterialbearing N- treatmentcontaining[ 54heteroaromatic,55]. A total of bicyclic 249 compounds rings inhibit with me fourthionyl-tRNA major different synthetase scaffolds (MetRS) were synthesizedfor antibacterial and treatmentcontaining [54,55]. heteroaromatic A total of bicyclic 249 compounds rings inhibit with me fourthionyl-tRNA major different synthetase scaffolds (MetRS) were synthesized for antibacterial and treatment [54,55]. A total of 249 compounds with four major different scaffolds were synthesized and testedtreatment in 13 [54,55]. different A total in vitro of 249 assays compounds against MetRS with four (Trypanosoma major different brucei scaffolds) ATP depletion were synthesized (A), MetRS and (S. tested in 13 different in vitro assays against MetRS (Trypanosoma brucei) ATP depletion (A), MetRS (S. aureustested )in ATP 13 different depletion in (B), vitro MetRS assays (Brudella against) MetRS ATP depletion (Trypanosoma (C), MetRSbrucei) (ATPGiardia depletion) aminoacylation (A), MetRS (D), (S. aureus) ATP depletion (B), MetRS (Brudella) ATP depletion (C), MetRS (Giardia) aminoacylation (D), Biomolecules 2020, 10, 1625 12 of 24 Biomolecules 2020, 10, x FOR PEER REVIEW 12 of 24

Biomolecules 2020, 10, x FOR PEER REVIEW 12 of 24 T.tested brucei in (E), 13 diTrypanosomafferent in vitro cruziassays (F), Giardia against intestinalis MetRS (Trypanosoma (ATCC50508) brucei (G),) Leishmania ATP depletion amazonensis (A), MetRS (H), P.(T.S. falciparumbrucei aureus (E),) ATP Trypanosoma(3D7) depletion (I), S. (B),aureuscruzi MetRS (F) (ATCC29213), Giardia (Brudella intestinalis) (J), ATP S. depletion aureus(ATCC50508) MRSA (C), MetRS (G),(ATCC43300) Leishmania (Giardia )(K), amazonensis aminoacylation Enterococcus (H), faecalis(D),P. falciparumT. brucei(ATCC29212) (E),(3D7)Trypanosoma (I), (L), S. and aureus cruziEnterococcus (ATCC29213)(F), Giardia faecium intestinalis(J), (ATCC51559)S. aureus(ATCC50508) MRSA (M). (ATCC43300) Two (G), compoundsLeishmania (K), Enterococcus amazonensisbearing the same(H)faecalis, P. scaffold falciparum(ATCC29212) of pyridoimidazole,(3D7) (L), (I), andS. aureus Enterococcus 45(ATCC29213) and 46 faecium, showed (J), (ATCC51559) S.excellent aureus MRSApotency (M). (ATCC43300) Twoin bacterial compounds MetRS (K), Enterococcus bearing inhibition the (ICfaecalissame50), scaffoldbacterial(ATCC29212) of growth pyridoimidazole, (L), inhibition and Enterococcus (EC 45 and50), faeciumand 46, showedMIC(ATCC51559) values excellent in most (M). potency Twoof the compoundsin in bacterial vitro cell MetRS bearing assays inhibition the (Figure same 16). sca(ICff50old), bacterial of pyridoimidazole, growth inhibition45 and (EC46,50 showed), and MIC excellent values potency in most in of bacterial the in vitro MetRS cell inhibition assays (Figure (IC50), bacterial16). growth inhibition (EC50), and MIC values in most of the in vitro cell assays (Figure 16).

Figure 16. Four major scaffolds of bacterial methionyl-tRNA synthetase (MetRS) inhibitors and the biologicalFigureFigure 16.16. activities Four majormajor of 45 scascaffolds andffolds 46 against ofof bacterialbacterial 13 different methionyl-tRNAmethionyl-tRNA bacterial cell synthetasesynthetase lines. (MetRS)(MetRS) inhibitorsinhibitors andand thethe biologicalbiological activitiesactivities ofof 4545 andand 4646 againstagainst 1313 didifferentfferent bacterialbacterialcell celllines. lines. The Martin group at the University of Texas at Austin reported T. brucei methionyl-tRNA synthetaseTheThe MartinMartin (MetRS) group group inhibitors at theat the University for University the treatment of Texas of Texas at Austinof African at reportedAustin trypanosomiasis reportedT. brucei methionyl-tRNAT. brucei and sleepingmethionyl-tRNA synthetase sickness [56,57].(MetRS)synthetase Two inhibitors (MetRS) major for classes inhibitors the treatment of compounds for ofthe African treatment incorpor trypanosomiasis ofating African tetracyclic trypanosomiasis and sleeping tetrahydro-beta-carboline sickness and [56sleeping,57]. Two sickness and major 4- amino-2-piperidoneclasses[56,57]. ofTwo compounds major classes were incorporating preparedof compounds tetracyclicto selectively incorpor tetrahydro-beta-carboline atinginhibit tetracyclic the MetRS tetrahydro-beta-carboline of andT. brucei 4-amino-2-piperidone. The inhibitory and 4- activitieswereamino-2-piperidone prepared of three to selectively tetracyclic were prepared inhibit carboline the to MetRS selectivelycompounds of T. brucei inhibit (47.– The49 the) were inhibitory MetRS tested of activities T.for bruceithe of T.. three Thebrucei tetracyclicinhibitory MetRS aminoacylationcarbolineactivities compoundsof three assay, tetracyclic (showing47–49) carboline were submicromolar tested compounds for the ICT.50 bruceivalues(47–49 MetRS) aswere depicted aminoacylationtested in for Figure the T. assay,17. brucei One showing MetRSof the tetracyclicsubmicromolaraminoacylation compounds ICassay,50 values showingexhibited as depicted submicromolarno off-target in Figure acti 17ICvity.50 One values when of the asscreened tetracyclicdepicted against in compounds Figure a panel 17. exhibited Oneof 45 of CNS the no proteins.otetracyclicff-target activity compounds when screenedexhibited against no off-target a panel acti of 45vity CNS when proteins. screened against a panel of 45 CNS proteins.

R1 Me Me H O R O Cl 1 H R1 Me Me H N O R1OH O O Cl H H R2 Cl

N N OHR2 O H H H R2 47Cl 48 49 Cl NH Me N R T. brucei MetRS H H 2 97447 297 48 586 49 Cl NH Me aminoacylation, IC50 (nM) T. brucei MetRS 974 297 586 aminoacylation, IC (nM) Figure 17. Structures of three tetracyclic carbolines 50 ( 47–49) and their inhibitory activities against the aminoacylationFigure 17. Structures of Trypanosoma of three tetracyclic brucei MetRS. carbolines (47–49) and their inhibitory activities against the aminoacylation of Trypanosoma brucei MetRS. Additionally, thethe internal internal C-C C-C bond bond of piperidine of piperidine was removed was removed to yield 37to 4-amino-2-piperidones,yield 37 4-amino-2- including 50–61, which were screened for T. brucei growth inhibition at a single dose (1 µM or 10 µM). piperidones,Additionally, including the 50internal–61, which C-C were bond screened of piperidine for T. brucei was growth removed inhibition to yield at a 37single 4-amino-2- dose (1 Eleven compounds (50–60) showed decreased activity in the T. brucei growth inhibition assay by μpiperidones,M or 10 μM). including Eleven 50 compounds–61, which were (50– 60screened) showed for T.decreased brucei growth activity inhibition in the atT. abrucei single growth dose (1 showing more than 80% inhibition at 10 µM; 61 with a methoxy on the C-5 of the indole ring showed inhibitionμM or 10 assay μM). by Eleven showing compounds more than ( 80%50–60 inhibition) showed at decreased 10 μM; 61 activitywith a methoxy in the T.on bruceithe C-5 growth of the better activity with ~60% inhibition at 1 µM (Table1). indoleinhibition ring assay showed by showingbetter activity more withthan ~60%80% inhibition inhibition at at 10 1 μMM; (Table 61 with 1). a methoxy on the C-5 of the indole ring showed better activity with ~60% inhibition at 1 μM (Table 1). BiomoleculesBiomolecules2020 2020, 10,, 10 1625, x FOR PEER REVIEW 1313 of of 24 24 BiomoleculesBiomoleculesBiomolecules 2020 2020 2020, ,10 10, ,10 ,x x ,FOR FORx FOR PEER PEER PEER REVIEW REVIEW REVIEW 1313 13 ofof of 2424 24 Biomolecules 2020, 10, x FOR PEER REVIEW 13 of 24 Biomolecules 2020, 10, x FOR PEER REVIEW 13 of 24 Biomolecules 2020, 10, x FOR PEER REVIEW 13 of 24 Biomolecules 2020, 10, x FORTable PEER 1. T. REVIEW brucei growth inhibition by 4-amino-2-piperidones 50–61. 13 of 24 TableTableTable 1. 1. T.1. T. T.brucei brucei brucei growth growth growth inhibition inhibition inhibition by by by 4-amino-2-piperidones 4-amino-2-piperidones 4-amino-2-piperidones 50 50 –50–6161–.61 . . BiomoleculesBiomolecules 2020 2020, 10, 10, x, FORxTable FORTable PEER PEER 1. T.1. REVIEW T. bruceiREVIEW bruceigrowth growth inhibition inhibition by by 4-amino-2-piperidones 4-amino-2-piperidones50 –5061–.61. 1313 of of 24 24 Biomolecules 2020, 10, x FORTable PEER 1. T. REVIEW brucei growth inhibition by 4-amino-2-piperidones 50–61. 13 of 24 BiomoleculesBiomolecules 2020 2020, 10, 10, x, FORx FORTable PEER PEER 1. REVIEW T. REVIEW brucei growth inhibition by 4-amino-2-piperidones 50–61. 1313 of of 24 24 Biomolecules 2020, 10, x FORTable PEER 1. REVIEW T. brucei growth inhibition by 4-amino-2-piperidones 50–61. 13 of 24 TableTable 1. 1. T. T. brucei brucei growth growth inhibition inhibition by by 4-amino-2-piperidones 4-amino-2-piperidones 50 50–61–61. . Table 1. T. brucei growth inhibition by 4-amino-2-piperidones 50–61. TableTable 1. 1.T. T. brucei brucei growth growth inhibition inhibition by by 4-amino-2-piperidones 4-amino-2-piperidones 50 50–61–61. . Table 1. T. brucei growth inhibition by 4-amino-2-piperidones 50–61.

% Inhib. % Inhib. ID R R n % Inhib. ID 1R1 R22 n %% Inhib. Inhib. (10 µM) IDIDID RR1R 1 1 RR2R 2 2 n n n %(10 Inhib. μM) ID R1 R2 n (10(10%(10 μ μInhib.M) μM)M) ID R1 R2 n %(10 Inhib. μM) ID R1 R2 n %(10 Inhib. μM) ID R1 R2 n %%(10 Inhib. Inhib. μM) IDID RR1 1 RR2 2 n n %(10 Inhib. μM) 50 ID50 R1 R2 n %(10%(10 Inhib. μInhib.97 μM)M) 97 50ID50ID50 RR1 1 RR2 2 n n %(10 Inhib.979797 μ M) ID50 R1 R2 n (10(10 μ97 M)μM) 50 (10 μ97M) 50 97 5050 9797 50 97 505150 979397 51 515051 939793 93 5151 9393 51 93 51 93 5151 9393 5152 9398 52515252 51 1 98939898 93 52 5152 11 1 1 9398 98 52 1 98 52 1 98 52 1 98 525253 1 989892 53535253 1 1 92929892 525352 1 989298 53 5253 1 1 9892 92 53 1 92 53 92 535354 929298 54545354 98989298 535453 929892 5354 9298 54 54 98 98 54 98 5454 9898 5554 55 9498 94 54555554 98949498 5455 9894 55 94 55 55 94 94 5555 9494 5556 1 9493 56555656 55 11 1 93949393 94 5556 1 9493 56 1 93 56 1 93 56 5656 1 1 19393 93 56 1 93 565756 1 1 938593 575657 1 859385 5757 8585 57 85 57 85 5757 8585 57 57 85 85 575857 1 859585 585758 1 1 958595 5858 1 1 9595 58 1 95 58 1 95 58 1 95 5858 1 1 9595 5859 10 9599 58 59585959 58 010 01 199959999 95 95 5859 1 0 9599 5960 02 9996 60605960 22 02 96969996 595960 0 02 999996 596061 02 999698 61596161 6059 0 20 98999898 9699 59 (5-MeO596061 0 21 0(~60%999698 99 (5-MeO(5-MeO(5-MeO606061 121 12 (~60%(~60%(~60%969698 -indole)(5-MeO6061 21 @(~60% 19698 μ M -indole)-indole)-indole)(5-MeO60616160 2 12 @@ 1@ 1(~60% 9698 μ1μ9896M Mμ M 60 -indole)(5-MeO6061 2 1 2@(~60%96 198 μ M 96 -indole)(5-MeO6161 1 @(~60%98 198 μ M (5-MeO-indole)(5-MeO61 1 1 (~60%@(~60%98 1 μ M 3.4. Phenylalanyl-tRNA61 -indole)(5-MeO Synthetase (PheRS) Inhibitors 1 @(~60% 1 μM 98 3.4.3.4.3.4. Phenylalanyl-tRNA Phenylalanyl-tRNA Phenylalanyl-tRNA-indole)(5-MeO-indole)(5-MeO Synthetase Synthetase Synthetase (PheRS) (PheRS) (PheRS) Inhibitors Inhibitors Inhibitors 1 1 @(~60%@ 1(~60% 1μ Mμ M 3.4. Phenylalanyl-tRNA(5-MeO(5-MeO-indole) Synthetase (PheRS) Inhibitors 1 1 (~60%@ 1 μ M (~60% 3.4. Phenylalanyl-tRNAThe Kahne-indole)-indole) group Synthetaseat Harvard (PheRS) University Inhibitors develope d tRNA synthetase@ @1 1μinhibitors MμM of secondary 3.4.TheThe Phenylalanyl-tRNAThe KahneKahne-indole) Kahne-indole) groupgroup group atat Synthetase at HarvardHarvard Harvard University(PheRS)University University Inhibitors developedevelope develope dd tRNAdtRNA tRNA synthetasesynthetase synthetase@ 1 inhibitorsinhibitors μinhibitorsM @ 1ofofµ ofM secondarysecondary secondary 3.4.3.4.amines Phenylalanyl-tRNA Phenylalanyl-tRNAThe for Kahne killing group Gram-negative Synthetase Synthetaseat Harvard (PheRS) (PheRS)bacteria University Inhibitors Inhibitorsand treati develope ng tuberculosisd tRNA synthetase [58]. Although inhibitors the target of secondary enzyme aminesamines3.4.amines Phenylalanyl-tRNAThe for for for killing Kahnekilling killing Gram-negative Gram-negativegroup Gram-negative Synthetaseat Harvard bacteria bacteria (PheRS)bacteria University and and Inhibitorsand treati treati treati developengng ng tuberculosis tuberculosis tuberculosisd tRNA [58].synthetase [58]. [58]. Although Although Although inhibitors the the the target target target of secondaryenzyme enzyme enzyme 3.4.isamines3.4. notPhenylalanyl-tRNA Phenylalanyl-tRNAThe specifically for Kahne killing group mentionedGram-negative Synthetase Synthetaseat Harvard in their(PheRS) (PheRS)bacteria University patent, Inhibitors Inhibitorsand ph treatidevelopeenylalanyl-tRNA ng tuberculosisd tRNA synthetase [58]. Although (PheRS;inhibitors the FARS, target of secondary formerly enzyme is3.4. notisamines notPhenylalanyl-tRNA ThespecificallyThe specifically forKahne Kahne killing groupmentioned group mentionedGram-negative Synthetaseat at Harvard Harvard in intheir their(PheRS) bacteriaUniversity patent, University patent, Inhibitors andph ph enylalanyl-tRNAdevelope treatidevelopeenylalanyl-tRNA ng tuberculosisd dtRNA tRNA synthetase synthetase synthetase [58]. Although (PheRS; inhibitors (PheRS;inhibitors FARS,the FARS, oftarget of secondary formerlysecondary formerly enzyme 3.4.isisamines not Phenylalanyl-tRNAnot specificallyThe specifically for Kahne killing mentionedgroup mentionedGram-negative Synthetase at Harvard in in their (PheRS)their bacteria patent,University patent, Inhibitors and ph phenylalanyl-tRNA treatidevelopeenylalanyl-tRNAng tuberculosisd tRNA synthetase synthetase [58]. Although (PheRS; (PheRS;inhibitors the FARS, FARS, target of secondaryformerly formerly enzyme aminesisFRS) notThe The mightspecifically forKahne Kahne killing have group group mentionedGram-negativebeen at at exploredHarvard Harvard in their bacteriadueUniversity University patent, to theand ph developespecif treati developeenylalanyl-tRNAicng notetuberculosisd dtRNA tRNAabout synthetase synthetase synthetaseFRS [58]. in Although the inhibitors (PheRS;inhibitors table the of FARS, of target antimicrobialof secondary secondary formerly enzyme FRS)aminesFRS)FRS)aminesis not Themightmight mightspecificallyfor forKahne killing have killinghave have group beenGram-negativebeen mentionedGram-negativebeen exploredatexplored exploredHarvard in duetheirbacteriadue bacteriadueUniversity to patent,to to the theand theand specif specif ph treatidevelopespecif treatienylalanyl-tRNAicicng icng notenote tuberculosis note tuberculosisd tRNA aboutabout about synthetase FRS FRSsynthetase [58].FRS [58]. inin Although in theAlthoughthe the inhibitors table(PheRS;table table the of theof of targetantimicrobialFARS, antimicrobial of targetantimicrobial secondary enzyme formerly enzyme aminesisactivity.FRS) not mightspecifically for They killing have synthesized mentionedGram-negativebeen explored 458 in secondary their bacteriadue patent, to amines,theand ph specif treatienylalanyl-tRNA onengic tertiary notetuberculosis about cyclic synthetase FRS [58]. amine, in Although the 15 (PheRS; tabletertiary the of FARS, targetcyclicantimicrobial formerly enzymeamides, activity.aminesisactivity.isFRS)amines not notThe specifically might specificallyforThey Kahnefor They killing killing synthesizedhave synthesized group mentionedGram-negative mentionedGram-negativebeen at Harvardexplored458 458 in secondary in theirsecondary theirbacteria Universitybacteriadue patent, patent, to amines, and amines, theand ph ph treatidevelopedspecifenylalanyl-tRNA treati enylalanyl-tRNAone oneng icngtertiary tuberculosis tertiarynote tuberculosis tRNA aboutcyclic cyclic synthetasesynthetase synthetase [58].amine,FRS [58]. amine, Althoughin Although 15the inhibitors(PheRS; 15 tertiary(PheRS; tabletertiary the the FARS, of cyclictargetFARS, of targetcyclicantimicrobial secondary formerlyamides, enzyme formerly enzymeamides, aminesactivity.isactivity.FRS) not mightspecificallyfor They They killing havesynthesized synthesized Gram-negative mentionedbeen explored 458 458 secondaryin secondary theirbacteria due patent, to amines,and amines,the ph treatispecifenylalanyl-tRNA one onengic tertiary tuberculosis tertiarynote about cyclic cyclic synthetase [58]. FRSamine, amine, Althoughin 15the 15 (PheRS; tertiary tabletertiary the of targetFARS,cyclic cyclicantimicrobial enzyme amides,formerly amides, isFRS)activity.andis not not specificallyone mightspecifically Theytertiary have synthesized mentioned cyclic mentionedbeen carbamateexplored 458 in in theirsecondary their due compound. patent, patent, to amines,the ph ph specifenylalanyl-tRNA Theenylalanyl-tRNA one icantimicrobial tertiarynote about cyclic synthetase synthetase activitiesFRS amine, in the (PheRS; of15 (PheRS; tableeachtertiary FARS,compoundof FARS, cyclicantimicrobial formerly formerly amides, were andaminesisFRS)andFRS)andactivity. not oneone mightspecificallyone formight tertiarytertiary They killingtertiary have have synthesized cyclic cyclicGram-negativementioned beencyclic been carbamate carbamateexplored carbamateexplored 458 in theirsecondary bacteria due compound. compound.due compound. patent, to to andthe amines,the ph treatingspecif TheThespecifenylalanyl-tRNA The antimicrobialoneicantimicrobial icantimicrobial tuberculosisnote tertiarynote about about cyclic synthetaseactivities activitiesFRS[ 58activitiesFRS ].amine, in Althoughin the ofofthe (PheRS; of15 eachtableeach tableeachtertiary the compound compoundof FARS,compoundof target antimicrobial cyclicantimicrobial formerly enzyme amides, werewere were FRS)activity.measuredand one might Theytertiary via have synthesizedthe cyclic beenGram-negative carbamateexplored 458 secondary due compound.(E. tocoli amines,the and specif TheAcinetobacter one icantimicrobial tertiarynote about baumannii cyclic activitiesFRS amine, in(A. the 15ofbau tableeachtertiary)) MIC compoundof cyclicantimicrobialprotocol amides, were and measuredisFRS)activity.measuredactivity. notandFRS) specificallymightone might They Theyviatertiary viahave thehavesynthesized synthesizedthe mentioned Gram-negativebeencyclic beenGram-negative explored carbamateexplored 458 458 in secondary their secondary due( E.due compound. patent,( E. colito colito amines,theand amines,the phenylalanyl-tRNAand specif Acinetobacterspecif TheAcinetobacter one oneic icantimicrobial tertiarynote tertiarynote about baumanniiabout cyclicbaumannii cyclic synthetase FRS activities FRSamine, amine, in( A. in (the A. bau15the (PheRS; of15 bau tabletertiary)) tableeachtertiary ))MIC MICof FARS, compound of cyclicprotocolantimicrobial cyclicantimicrobialprotocol formerly amides, amides, and were and FRS)measuredactivity.measuredand mightone Theytertiaryvia viahave the synthesizedthe beencyclicGram-negative Gram-negative explored carbamate 458 secondary due ( E.compound.(E. tocoli coli the andamines, and specif Acinetobacter TheAcinetobacter oneic antimicrobialnote tertiary about baumannii baumannii cyclic FRS activities amine, in( A. (theA. bau 15of bautable )) eachtertiary ))MIC MICof compound antimicrobialprotocol cyclicprotocol amides, andwere and andmeasuredtuberculosisactivity. one Theytertiary via luminescence synthesizedthe cyclic Gram-negative carbamate 458assay. secondary Among compound.(E. coli amines, thesande The Acinetobactercompounds, one antimicrobial tertiary thebaumannii cyclic methyl activities amine, (substitutionA. of15bau eachtertiary)) MIC compound of cyclic protocolR6 and amides, orthowere and tuberculosisactivity.andtuberculosisandtuberculosis one one Theytertiary tertiary luminescence luminescence synthesizedluminescence cyclic cyclic carbamate carbamate assay. 458assay. assay. secondary Among Among compound.Among compound. thes amines,thes thesee compounds,The ecompounds, The compounds, one antimicrobial antimicrobial tertiary the the cyclicthe methyl methyl activitiesmethyl activities amine, substitution substitution substitution of15 of eachtertiary each compoundof ofcompound cyclicofR R6 R6and and6 and amides, ortho orthowere orthowere activity.andmeasuredtuberculosis one They tertiary via synthesizedluminescence the cyclic Gram-negative carbamate 458 assay. secondary Among compound.(E. coli amines, thesande The Acinetobacter compounds,one antimicrobial tertiary cyclicthebaumannii methyl activities amine, (substitutionA. 15 ofbau tertiary each)) MIC compound cyclicof protocolR6 andamides, orthowere and andmeasuredtuberculosissubstitutionand one one tertiary tertiary via ofluminescence the Rcyclic 5 cyclicon Gram-negative the carbamate carbamate secondary assay. compound.Among compound.(benzylicE. coli thesand amine Thee TheAcinetobactercompounds, antimicrobial significan antimicrobial tlythebaumannii increased activitiesmethyl activities (substitutionA. theof ofbau each antimicrobial each)) MICcompound compound of protocolR6 and activities were orthowere and substitutionandmeasuredsubstitutionsubstitutionmeasuredtuberculosis one tertiary via of viaof ofRluminescence theR 5 the R5oncyclic on 5 Gram-negativeon the Gram-negativethe the carbamatesecondary secondary secondary assay. benzylic compound.(Amongbenzylic E. (benzylicE. coli coli and aminethes amineand amine TheeAcinetobacter Acinetobactercompounds, significan significanantimicrobial significantly tlybaumannii tlythe baumanniiincreased increased increased activitiesmethyl ( A.the (thesubstitutionA. theofbau antimicrobial bau antimicrobialeach antimicrobial)))) MIC MICcompound ofprotocol protocolR activities6 activitiesand activities were orthoand and measuredtuberculosissubstitution via ofluminescence theR5 on Gram-negative the secondary assay. Among (benzylicE. coli thesand aminee Acinetobactercompounds, significan tlythebaumannii increased methyl (substitutionA. the bau antimicrobial)) MIC of protocolR6 and activities ortho and measuredsubstitutionmeasured via via ofthe theR 5 Gram-negativeon Gram-negative the secondary ( E. (benzylicE. coli coli and and amine Acinetobacter Acinetobacter significan baumannii tlybaumannii increased ( A.(A. thebau bau antimicrobial)) ))MIC MIC protocol protocol6 activities and and measuredtuberculosistuberculosissubstitution via luminescence ofluminescencethe R 5 Gram-negativeon the secondary assay. assay. Among (AmongE. benzylic coli thesand thes aminee eAcinetobactercompounds, compounds, significan thebaumannii tlythe methyl increased methyl (substitutionA. substitution thebau antimicrobial)) MIC of of protocolR R6 and and activities ortho orthoand tuberculosissubstitution ofluminescence R5 on the secondary assay. Among benzylic thes aminee compounds, significan tlythe increased methyl substitution the antimicrobial of R6 and activities ortho tuberculosissubstitutionsubstitutiontuberculosis ofluminescence ofluminescence R R5 on5 on the the secondary secondary assay. assay. Among Amongbenzylic benzylic thes thes amine aminee ecompounds, compounds, significan significan tlythe tlythe increased methyl increased methyl substitution thesubstitution the antimicrobial antimicrobial of of R 6R and6 activitiesand activities ortho ortho tuberculosissubstitution luminescence of R5 on the secondary assay. Among benzylic thes aminee compounds, significan thetly methyl increased substitution the antimicrobial of R6 and activities ortho substitutionsubstitution of of R 5R on5 on the the secondary secondary benzylic benzylic amine amine significan significantlytly increased increased the the antimicrobial antimicrobial activities activities substitution of R5 on the secondary benzylic amine significantly increased the antimicrobial activities Biomolecules 2020, 10, 1625 14 of 24

FRS) might have been explored due to the specific note about FRS in the table of antimicrobial activity. They synthesized 458 secondary amines, one tertiary cyclic amine, 15 tertiary cyclic amides, and one tertiary cyclic carbamate compound. The antimicrobial activities of each compound were measured viaBiomolecules the Gram-negative 2020, 10, x FOR PEER (E. coli REVIEWand Acinetobacter baumannii (A. bau)) MIC protocol and tuberculosis14 of 24 luminescence assay. Among these compounds, the methyl substitution of R and ortho substitution Biomolecules 2020, 10, x FOR PEER REVIEW 6 14 of 24 (Figure 18). In particular, the para- or meta-methyl substitution of R8 provided the most potent of R5 on the secondary benzylic amine significantly increased the antimicrobial activities (Figure 18). antimicrobial compounds, 66 and 67, with MIC values of 33 μM (E. coli WT), less than 1 μM (E. coli (FigureIn particular, 18). In the particular, para- or meta-methylthe para- or substitutionmeta-methyl ofsubstitution R8 provided of theR8 provided most potent theantimicrobial most potent Δ-TolC), and 33 μM (A. bau) against Gram-negative bacteria as well as Mycobacterium tuberculosis antimicrobialcompounds, 66 compounds,and 67, with 66 MICand 67, values with of MIC 33 µ valuesM(E. coliof 33WT), μM less(E. coli than WT), 1 µM( lessE. than coli ∆1 -TolC),μM (E. andcoli (MTB) activity of 10 μM against H37Rv WT. Furthermore, they showed that combination treatment Δ33-TolC),µM(A. and bau )33 against μM (A. Gram-negative bau) against Gram-negative bacteria as well bacteria as Mycobacterium as well as tuberculosis Mycobacterium(MTB) tuberculosis activity of of compound 62 (R1-6 = -H, R7 = -1-cyclohexene, HCl salt) and AN3365 (an LeuRS inhibitor) 10(MTB)µM againstactivity H37Rvof 10 μM WT. against Furthermore, H37Rv WT. they Furthermore, showed that they combination showed that treatment combination of compound treatment62 dramatically decreased the viability of bacterial cells, suggesting that the combination therapy could (Rof 1-compound6 = -H, R7 = 62-1-cyclohexene, (R1-6 = -H, R HCl7 = salt)-1-cyclohexene, and AN3365 HCl (an LeuRS salt) and inhibitor) AN3365 dramatically (an LeuRS decreased inhibitor) the attenuate antibiotic resistance, thus expanding the lifespan of antibiotics. viabilitydramatically of bacterial decreased cells, the suggesting viability thatof bacterial the combination cells, suggesting therapy that could the attenuate combination antibiotic therapy resistance, could attenuatethus expanding antibiotic the lifespanresistance, of thus antibiotics. expanding the lifespan of antibiotics.

Figure 18. Structures of the secondary benzylamines with antimicrobial activities via prokaryotic phenylalanyl-tRNA synthetase (PheRS) inhibition. Figure 18. Structures of the secondary benzylamines wi withth antimicrobial activities via prokaryotic phenylalanyl-tRNA synthetasesynthetase (PheRS) inhibition. 3.5. Prolyl-tRNA Synthetase (ProRS) Inhibitors 3.5. Prolyl-tRNA Synthetase (ProRS) Inhibitors 3.5. Prolyl-tRNADaewoong SynthetasePharmaceutical (ProRS) Co. Inhibitors unveiled heterocyclic compounds to inhibit abnormal human prolyl-tRNADaewoong synthetase Pharmaceutical (ProRS) Co. activity unveiled for heterocyclicthe treatment compounds of cancers, to inhibitinflammatory abnormal diseases, human Daewoong Pharmaceutical Co. unveiled heterocyclic compounds to inhibit abnormal human prolyl-tRNAautoimmune synthetasediseases, and (ProRS) fibrosis activity [59]. forA thetotal treatment of 230 ofnitrogen-containing cancers, inflammatory heteroaromatic diseases, prolyl-tRNA synthetase (ProRS) activity for the treatment of cancers, inflammatory diseases, autoimmunecompounds with diseases, various and substitutions fibrosis [59]. were A total synthesi of 230 nitrogen-containingzed and categorized heteroaromatic into three groups compounds in terms autoimmune diseases, and fibrosis [59]. A total of 230 nitrogen-containing heteroaromatic withof their various biological substitutions activities wereto inhibit synthesized the human and ProRS categorized enzyme, into as three shown groups in Figure in terms 19. The of their IC50 compounds with various substitutions were synthesized and categorized into three groups in terms biologicalvalues of 26 activities compounds to inhibit (class the A) human were 100 ProRS nM enzyme,or less, the as shownvalues inof Figure89 compounds 19. The IC (class50 values B) ranged of 26 of their biological activities to inhibit the human ProRS enzyme, as shown in Figure 19. The IC50 compoundsfrom 100 to (class500 nM, A) and were the 100 values nM or of less, 115 the compou valuesnds of 89(class compounds C) were (class500 nM B) or ranged higher. from Compounds 100 to 500 values of 26 compounds (class A) were 100 nM or less, the values of 89 compounds (class B) ranged nM,in class and A the shared values disubstituted of 115 compounds imidazolyl (class C)aromatic were 500 bicyclic nM or rings, higher. yielding Compounds potent in classhuman A sharedProRS from 100 to 500 nM, and the values of 115 compounds (class C) were 500 nM or higher. Compounds disubstitutedinhibition. imidazolyl aromatic bicyclic rings, yielding potent human ProRS inhibition. in class A shared disubstituted imidazolyl aromatic bicyclic rings, yielding potent human ProRS inhibition.

Figure 19.19. Human prolyl-tRNA synthetase (ProRS) inhibitorsinhibitors for antiproliferativeantiproliferative diseases and the most potent imidazolyl aromaticaromatic bicyclebicycle compoundscompounds (class(class A).A). Figure 19. Human prolyl-tRNA synthetase (ProRS) inhibitors for antiproliferative diseases and the 3.6. Multi-tRNAmost potent imidazolyl Synthetase aromatic Inhibitors bicycle compounds (class A). 3.6. Multi-tRNA Synthetase Inhibitors The Qi group at the University of Florida prepared 23 sulfamide adenosine derivatives as 3.6. Multi-tRNAThe Qi group Synthetase at the InhibitorsUniversity of Florida prepared 23 sulfamide adenosine derivatives as aminoacyl-tRNA synthetase inhibitors for the treatment of proliferative diseases such as cancers, aminoacyl-tRNA synthetase inhibitors for the treatment of proliferative diseases such as cancers, inflammatoryThe Qi group diseases, at andthe University autoimmune of diseases Florida [60pr].epared As described 23 sulfamide in Figure adenosine 20, a protected derivatives adenosyl as inflammatory diseases, and autoimmune diseases [60]. As described in Figure 20, a protected aminoacyl-tRNAamine was extended synthetase as the protected inhibitors adenosyl for the sulfamide,treatment of whichproliferative the terminal diseases amine such was as coupledcancers, adenosyl amine was extended as the protected adenosyl sulfamide, of which the terminal amine was withinflammatory the different diseases, kinds of and amino autoimmune acids to complete diseases the [60]. synthesis As described of the aminoacyl in Figure sulfamide 20, a adenosineprotected coupled with the different kinds of amino acids to complete the synthesis of the aminoacyl sulfamide adenosylcompounds amine (aa-SA). was extended The cell viability as the protected assay of theaden aa-SAsosyl sulfamide, was performed of which in prostate the terminal cancer amine cell lines, was adenosine compounds (aa-SA). The cell viability assay of the aa-SAs was performed in prostate coupled with the different kinds of amino acids to complete the synthesis of the aminoacyl sulfamide and IC50 values of 0.0015–27 µM for LNCaP and 0.0011–16 µM for PC3 were observed. cancer cell lines, and IC50 values of 0.0015–27 μM for LNCaP and 0.0011–16 μM for PC3 were adenosine compounds (aa-SA). The cell viability assay of the aa-SAs was performed in prostate observed. cancer cell lines, and IC50 values of 0.0015–27 μM for LNCaP and 0.0011–16 μM for PC3 were observed. Biomolecules 2020, 10, 1625 15 of 24 Biomolecules 2020, 10, x FOR PEER REVIEW 15 of 24

Figure 20. SyntheticSynthetic scheme scheme of of sulfamide sulfamide adenosine adenosine derivati derivativesves (aa-SAs) (aa-SAs) and and their their biological biological activities against prostate cancer cell lines.

For L-Gln-lactam-SA ( (8989),), a a panel panel of of other cancer cell lines was tested for cell viability to give cytotoxic activitiesactivities (IC (IC50)50 of) 0.19–25.24of 0.19–25.24µM (TableμM (Table2). Additionally, 2). Additionally, apoptotic apoptotic staining and staining cell cycle and/arrest cell cycle/arrestassays were assays performed were onperformed LNCaP andon LNCaP PC3 cells and for PC3 all cells aa-SA for compounds. all aa-SA compounds. aa-SA-induced aa-SA-induced apoptosis apoptosisand a shift and of the a shift cell of cycle the tocell the cycle sub to G0 the/G1 sub and G0/G1 S phases and were S phases observed were (Tableobserved2). (Table 2).

Table 2. Cytotoxicity of L-Gln-lactam-SA ( 89) against a panel of cancer cell lines.

IC50 (μICM)50 (µM) AA PanelPanel of of Cancer Cancer Cell Cell Lines Lines L-Gln-lactam-SAL-Gln-lactam-SA (89) (89) MOLT-4MOLT-4 (human (humanacute lymphoblastic acute lymphoblastic leukemia) leukemia) 2.44 2.44 HL60 (human (humanacute promyelocytic acute promyelocytic leukemia) leukemia) 0.46 0.46 MDA231 (breast cancer) 11.03 MDA231A549 (breast (lungcancer) cancer) 11.03 7.93 HCT116A549 (lung cancer) (colon cancer) 7.93 1.48 HCT116HeLa (colon (cervical cancer) cancer) 1.48 25.24 MCF-7HeLa (cervical (breast cancer) cancer) 25.24 0.19 MCF-7 (breast cancer) 0.19 4. Aminoacyl-tRNA Synthetase (ARS) Therapeutics in Clinical Trials 4. Aminoacyl-tRNA Synthetase (ARS) Therapeutics in Clinical Trials Since FDA approval of the natural product mupirocin (an IleRS inhibitor) to treat bacterial infections,Since FDA prokaryotic approval ARS of hasthe attractednatural product much attention mupirocin as one(an ofIleRS the inhibitor) most promising to treat targets bacterial for infections,developing prokaryotic antimicrobial ARS therapeutics has attracted in much recent atte years.ntion Nevertheless,as one of the AN2690most promising (a synthetic targets small for developingmolecule, tavaborole) antimicrobial is the therapeutics only FDA-approved in recent drug years. for nailNevertheless, onychomycosis AN2690 to inhibit (a synthetic fungal LeuRS.small molecule,In this section, tavaborole) we discuss is smallthe only molecule FDA-approved therapeutics drug that for are undergoingnail onychomycosis clinical trials to inhibit as prokaryotic fungal LeuRS.or eukaryotic In this aminoacyl-tRNAsection, we discuss synthetase small molecule (ARS) th inhibitors.erapeutics A that recent are underg reviewoing published clinical in trialsNature as prokaryoticReviews Drug or Discoveryeukaryotic(2019) aminoacyl-tRNA by the Kim groupsynthetase at the (ARS) Medical inhibitors. Bioconvergence A recent review Research published Center indescribed Nature Reviews the ARS Drug inhibitors Discovery that are(2019) currently by the being Kim evaluatedgroup at the in clinicalMedical trials Bioconvergence [14]. Thus, this Research review Centerwill provide described the latest the ARS updates inhibitors on the that clinically are curren activetly compounds being evaluated based uponin clinical their trials reported [14]. clinical Thus, thisoutcome. review The will Cortellis provide search the (2020-10-08)latest updates with on the the keywords clinically “tRNA active synthetasecompounds inhibitor” based upon and statustheir reportedof “phase clinical 1 clinical”, outcome. “phase The 2 clinical”,Cortellis search “phase (2020-10-08) 3 clinical”, andwith “clinical” the keywords revealed “tRNA that synthetase five small inhibitor”molecule therapeutics,and status of including “phase 1 DWN12088, clinical”, “phase CRS3123, 2 clinical”, GSK3036656, “phase mupirocin 3 clinical”, bioadhesive and “clinical” gel, revealedand DWP17011, that five are beingsmall activelymolecule evaluated therapeu intics, clinical including studies (TableDWN12088,3). Since CRS3123, mupirocin GSK3036656, has already mupirocinbeen marketed bioadhesive for bacterial gel, skinand infectionDWP17011 and, are information being actively on DWP17011 evaluated is in fairly clinical limited, studies clinical (Table trials 3). Sinceof the remainingmupirocin threehas compoundsalready been will marketed be discussed for inbacterial this section. skin infection and information on DWP17011 is fairly limited, clinical trials of the remaining three compounds will be discussed in this section. Biomolecules 2020, 10, x FOR PEER REVIEW 16 of 24

Biomolecules 2020Table, 10 ,3. 1625 A list of small molecules in clinical trials for the development of ARS inhibitors. 16 of 24 Clinical Trial # Drug Name. Company Target Indications Status (Country) Table 3. A list of small molecules in clinical trials for the development of ARS inhibitors. IPF; ACTRN12619001239156 DWN12088 Daewoong Pharma eProRS Phase I Scleroderma Clinical(AU) Trial # Drug Name. Company Target Indications Status NCT01551004(Country) (US) CRS3123 Crestone pMetRS CDI; HPI Phase I IPF; ACTRN12619001239156 DWN12088 Daewoong Pharma eProRS Phase I NCT02106338 (US) Scleroderma NCT03075410(AU) (UK) GSK3036656 GSK pLeuRS MTBI Phase I NCT01551004 (US) CRS3123 Crestone pMetRS CDI; HPI Phase I NCT02106338NCT03557281 (US) (SA) Mupirocin Laboratorios Ojer NCT03075410 (UK) GSK3036656 GSKpIleRS pLeuRS BSI; MTBIImpetigo Phase Phase I III Not specified (SP) bioadhesive gel Pharma NCT03557281 (SA) Mupirocin Laboratorios Ojer BSI; DWP17011 Daewoong Pharma eProRSpIleRS Fibrosis Phase Phase III I Not specifiedspecified (SP) (KR) bioadhesive gel Pharma Impetigo (IPF:DWP17011 idiopathic pulmonary DaewoongPharma fibrosis, CDI: eProRS Clostridium Fibrosis difficile infection, Phase I HPI: Helicobacter Not specified (KR)pylori (IPF:infection, idiopathic MTBI: pulmonary Mycobacterium fibrosis, tuberculosis CDI: Clostridium infection, difficile BSI:infection, bacterial HPI: skinHelicobacter infection). pylori infection, MTBI: Mycobacterium tuberculosis infection, BSI: bacterial skin infection). 4.1. DWN12088, Eukaryotic Prolyl-tRNA Synthetase (eProRS) Inhibitor 4.1. DWN12088, Eukaryotic Prolyl-tRNA Synthetase (eProRS) Inhibitor Excessive deposition of collagen is pathologically considered to be the main cause of fibrosis, and prolineExcessive residues deposition are the of collagenmajor constituents is pathologically of collagen. considered Therefore, to be an the eProRS main causeinhibitor of fibrosis, would anddownregulate proline residues collagen are synthesis the major and constituents would be of collagen.a potential Therefore, therapeutic an eProRSagent against inhibitor fibrosis. would downregulateDaewoong Pharmaceutical collagen synthesis Co. andLtd. would developed be a potential DWN12088 therapeutic (91) (Figure agent against 21), one fibrosis. of the Daewoong class A Pharmaceuticalcompounds (eProRS Co. Ltd. IC50 developed = 78 nM) DWN12088covered in (the91) aforementioned (Figure 21), one ofpatent the class (WO A compounds2018147626 (eProRSA1), for ICthe50 treatment= 78 nM) coveredof idiopathic in the pulmonary aforementioned fibrosis patent (IPF). (WO In 2018147626IPF patient-derived A1), for the primary treatment lung of fibroblasts, idiopathic pulmonarytreatment with fibrosis 91 (IPF).reduced In IPFthe patient-derivedsynthesis of collagen primary and lung α-smooth fibroblasts, muscle treatment actin with(α-SMA)91 reduced under thetransforming synthesis ofgrowth collagen factor- and βα -smooth(TGF-β) muscle induction. actin In (α -SMA)the mouse under model transforming of bleomycin-induced growth factor-β (TGF-pulmonaryβ) induction. fibrosis, In oral the administration mouse model of of bleomycin-induced 91 for 2 weeks significantly pulmonary reduced fibrosis, cardiac oral administration fibrosis (ED50 of= 0.491 formg/kg, 2 weeks in vitro) significantly and the reduced collagen cardiac amount fibrosis in lung (ED tissues50 = 0.4 and mg /bronchoalveolarkg, in vitro) and thelavage collagen fluid amount(BALF) [61]. in lung Remarkably, tissues and 91 bronchoalveolarprovided considerable lavage repair fluid of (BALF) lung function [61]. Remarkably, in this mouse91 model,provided as considerablemeasured by repairwhole-body of lung plethysmograph. function in this mouse GLP (g model,ood laboratory as measured practice) by whole-body toxicity studies plethysmograph. of 91 with GLPa 4-week (good repeated laboratory dose practice) in rats toxicity and monkeys studies of were91 with conducted a 4-week to repeated examine dose a wide in rats safety and monkeys margin. wereFurthermore, conducted 91to was examine designated a wide an safety orphan margin. drug for Furthermore, IPF by the 91FDAwas in designatedAugust 2019 an [62], orphan and drug a phase for IPFI clinical by the FDAtrial in August(a randomized, 2019 [62], and parallel-a a phase Issignment, clinical trial (adouble-blind, randomized, parallel-assignment,placebo-controlled; double-blind,ACTRN12619001239156) placebo-controlled; began in Australia ACTRN12619001239156) in September 2019. began The in goal Australia of the study in September was to assess 2019. Thethe safety, goal of tolerability, the study was and to pharmacokinetic assess the safety, properties tolerability, of single- and pharmacokinetic and multiple-ascending properties doses of single- of 91 andcompared multiple-ascending with placebo dosesfollowing of 91 oralcompared administration with placebo in healthy following volunteers oral administration (expected n in= 80) healthy [63]. volunteersThe study (expectedwas conductedn = 80) [63in ].two The studyparts: waspart conducted A (single-ascending in two parts: dose, part Asix (single-ascending cohorts, eight dose,subjects/cohort) six cohorts, and eight part subjects B (multiple-ascending/cohort) and part dose, B (multiple-ascending four cohorts, eight subjects/cohort). dose, four cohorts, In part eight A, subjectstwo subjects/cohort). in each In partcohort A, received two subjects placebo, in each and cohort six subjects received received placebo, a single and sixdose subjects (100 mg, received 200 mg, a single500 mg, dose or 800 (100 mg) mg, of 200 91 mg,. In 500part mg, B, two or 800 subjects mg) of in91 each. In part cohort B, two were subjects given inplacebo, each cohort and six were subjects given placebo,received andmultiple six subjects doses (25 received mg, 75 multiple mg, 150doses mg, or (25 300 mg, mg) 75 every mg, 150 12 mg,h for or 14 300 days. mg) The every safety 12 h and for 14tolerability days. The of safety 91 patients and tolerability were evaluated of 91 patients in each were cohort evaluated prior to in dose each cohortescalation. prior Blood to dose samples escalation. for BloodPK and samples PD (pharmacodynamics) for PK and PD (pharmacodynamics) analysis were collected analysis at werecertain collected time points at certain from time the pointspre-dose from of the91 through pre-dose 72 of h91 post-dose.through 72 The h post-dose. preliminary The results preliminary showed results that human showed ProRS that human was significantly ProRS was significantlyoverexpressed overexpressed compared with compared normal withlung normaltissue from lung tissueIPF patients from IPF [61]. patients This phase [61]. ThisI study phase was I studyexpected was to expected be completed to be completed in October in October2020, and 2020, more and detailed more detailed clinical clinical results results are expected are expected to be to bereported reported soon. soon.

Figure 21. Chemical structures of DWN12088 and CRS3123. Biomolecules 2020, 10, 1625 17 of 24

4.2. CRS3123, Prokaryotic Methionyl-tRNA Synthetase (pMetRS) Inhibitor Crestone, under license from Cardiovascular Systems, developed the antibiotic CRS3123 (92) (Figure 21) (formerly REP3123), a prokaryotic methionyl-tRNA synthetase (pMetRS) inhibitor, for the oral treatment of Clostridium difficile infection (CDI) and Helicobacter pylori infection (HPI) [64]. Notably, 92 prevents protein synthesis by inhibiting C. difficile MetRS with high specificity (Ki = 0.020 nM) and high selectivity (>1000-fold over human MetRS). The in vitro efficacy of 92 was estimated against numerous clinical isolates of C. difficile and epidemic strains BI/NAP1/027 (n = 108), showing MIC values of 0.25–1 mg/L[65]. While 92 did not exhibit activity against most Gram-negative bacteria, it had high potency (MIC90 < 1 mg/L) against clinically important Gram-positive bacteria, including S. aureus, Streptococcus pyogenes, E. faecalis, and E. faecium, which are resistant to current antimicrobial agents. In a clindamycin-induced hamster model of CDI, 92 demonstrated superior efficacy to vancomycin, reducing the production of toxins and spores from C. difficile [66,67]. Consequently, 92 was suggested as a promising candidate for the treatment of CDI with special features, including (i) a novel mode of action with maintaining activity against pre-existing resistances; (ii) high selectivity and potency against a narrow spectrum of C. difficile, while not affecting normal intestinal microbiota; and (3) the inhibition of toxin production and spore formation from C. difficile, alleviating the severity and spread of the disease [68].

4.2.1. Phase I Trial of a Single Dose of CRS3123 (NCT01551004) In May 2012, the first-time-in-human (FTIH), randomized, double-blind, placebo-controlled, dose-escalation phase I clinical study (NCT01551004) was initiated in the US to assess the safety and pharmacokinetics of 92 after a single oral dose in healthy adults. Five cohorts of eight subjects received 92 or placebo each in a 3:1 ratio [69]; 92 was supplied in 100 and 200 mg capsules, and cohorts A through E received a single oral dose of 100 mg, 200 mg, 400 mg, 800 mg, and 1200 mg, respectively. The clinical data reported by Crestone Inc. in 2017 showed that no serious adverse events or immediate allergic reactions were observed during the study [70]. In the 92-treated groups, hemoglobin reduction, headache, and abnormal urine analysis were the most frequent adverse events, which were mild to moderate and dose-independent. The increase in the absorption rate of 92 was less than dose proportional, and the relative bioavailability of the 1200 mg dose group was lower than that of the 800 mg dose group. The phase I study showed the safety and tolerability of 92 within the dose ranges and led to further investigation of CRS3123 for the treatment of CDI.

4.2.2. Phase I Trial to Determine the Safety and Pharmacokinetics of CRS3123 (NCT02106338) An additional single center, randomized, placebo-controlled, double-blind, multiple-ascending dose phase I study (NCT02106338) was pursued to evaluate the safety and tolerability of escalating doses of 92 by oral administration to healthy adults [71]. In this study, thirty healthy subjects aged 18–45 years were randomized into three cohorts, receiving either oral doses of 200, 400, and 600 mg 92 (eight subjects per cohort) or placebo (two subjects per cohort) every 12 h for 10 days. The safety and tolerability and the plasma, urine, and fecal concentrations and systemic exposure of 92 were determined after multiple oral doses for 10 days. The total duration of this study was 46 weeks. Recently, Crestone Inc. reported the results of this clinical study, showing that 92 was generally safe and well-tolerated without serious adverse events (SAEs), severe treatment-emergent adverse events (TEAEs), and any clinically significant cardiac symptoms [68]. Pharmacokinetically, 92 was absorbed rapidly (Tmax = 1–2 h), and the half-life in plasma was approximately 5–6 h without accumulation after multiple dosing. The increase in plasma concentrations was less than proportional to the increase in dose. Importantly, the high oral dose of 92 was not fully absorbed, and fecal concentrations of all dosages were considerably above the target MIC90 value of 1 mg/L for pharmacodynamic success. Moreover, 92 was inactive against important commensal anaerobes, such as Bacteroides, bifidobacteria, and clostridia, showing favorable microbiome data over other current medications for CDI. The results Biomolecules 2020, 10, 1625 18 of 24 of the two phase I clinical studies described above assured further clinical evaluation of 92 patients with CDI. According to the Cortellis report (Clarivate Analytics, 2020), a phase II clinical study to evaluate 92 in CDI is being planned, and preclinical evaluation for gastric H. pylori infection (HPI) with or without a proton pump inhibitor is underway [64].

4.3. GSK3036656, Prokaryotic Leucyl-tRNA Synthetase (pLeuRS) Inhibitor Prokaryotic leucyl-tRNA synthetase (pLeuRS) plays a crucial role in bacterial protein synthesis and has become one of the major targets for the development of antimicrobial therapeutics. In particular, boron-containing compounds have been investigated as LeuRS inhibitors by GlaxoSmithKline. GSK has been developing GSK3036656 (8) (Figure4) (other names, GSK656 and GSK070) as a pLeuRS inhibitor for the treatment of M. tuberculosis infection (MTBI), in-licensed from Anacor Pharmaceuticals. Notably, 8 inhibited Mtb LeuRS (IC50 = 0.20 µM), showing in vitro antitubercular activity (Mtb H37Rv, MIC = 0.02 µM) with high selectivity over human mitochondrial and cytoplasmic LeuRS (IC50 of > 300 µM and 132 µM, respectively) [21]. Moreover, 8 exhibited remarkable PK profiles in mice (AUCPO [area under the curve of oral administration] = 2.94 h µg/mL with ~100% BA [bioavailability], Cl · [clearance] = 8.5 mL/min/kg, t1/2 = 3.6 h) and in vivo efficacy (ED99 = 0.4 mg/kg) for MTB treatment in acute and chronic TB infection mouse models with superior tolerability (well-tolerated at 300 mg/kg). The preclinical data of 8 showed that it had a tolerable therapeutic window based on a number of preclinical safety and risk assessment studies, as disclosed at the 26th ECCMID in Amsterdam, the Netherlands, in April 2016 [72]. The human efficacious dose was predicted in the range of 0.5–2.7 mg/kg/day, which is lower than that of other antitubercular agents.

4.3.1. First-Time-in-Human (FTIH) Safety and Pharmacokinetics (PK) Study of GSK3036656 in Healthy Subjects (NCT03075410) In March 2017, a randomized, single-group interventional, double-blind, placebo-controlled, phase I, first-time-in-human (FTIH) study (NCT03075410) to evaluate the safety, tolerability, and pharmacokinetics of single and repeated doses of 8 in healthy participants (expected n = 58) was launched in the UK [73]. The study was conducted in two parts, including part A (single dose, two cohorts, nine subjects/cohort) and part B (repeat dose, four cohorts, ten subjects/cohort). In part A, single doses (5 mg, 15 mg, 25 mg, or placebo) of 8 were administered orally. In part B, repeat doses (5 mg, 15 mg, or placebo) of 8 were administered orally once a day for 14 days. The increase in the absorption rate was proportional to the increase in both the single dose and 14-day repeat doses [22]. Based on the PK parameters Cmax and AUC, accumulation with repeated administration increased approximately 2- to 3-fold, and food intake did not influence the PK parameters. Notably, 8 was not easily metabolized in plasma, but approximately 90% of 8 and 10% of deboronated metabolites by oxidation were detected in urine. Based on drug-related materials detected in urine, the absorbed amounts of 8 from single (25 mg) and repeat (15 mg) dosing were at least 50% and 78%, respectively. From this phase I clinical study completed in August 2017, the safety and tolerability of 8 was acquired after single and multiple doses with no reports of serious adverse events. Importantly, the results from the clinical laboratory, vital signs, electrocardiogram (ECG), and telemetry presented no cardiotoxicity concerns. Clinical trial simulations (CTS) were performed to predict the dose range of 8 that produces the highest possible early bactericidal activity (EBA0–14) for the phase II clinical trial, and 10 to 15 mg was suggested to be the optimal dosage of 8 for TB treatment.

4.3.2. An Early Bactericidal Activity, Safety, and Tolerability Study of GSK3036656 in Subjects with Drug-sensitive Pulmonary Tuberculosis (NCT03557281) In March 2019, an open-label, randomized, sequentially assigned, dose-escalation, phase IIa study (NCT03557281) was initiated to evaluate the bactericidal activity, safety, and tolerability of 8 compared to RIFAFOUR e-275 (Sanofi-Aventis) in patients (expected n = 80) with drug-sensitive pulmonary tuberculosis in South Africa [74]. This study was designed to investigate the early bactericidal activity Biomolecules 2020, 10, 1625 19 of 24

(EBA), safety, and tolerability of 8 in four cohorts of subjects with rifampicin-susceptible tuberculosis. The primary objective of this dose-escalation study was to estimate the antituberculosis effect of 8 by counting serial colony-forming units (CFUs) of MTB in sputum over 14 days. Subjects in each cohort will be treated with either 8 or a standard-of-care (RIFAFOUR e-275) regimen in a 3:1 ratio. The duration of this study for an individual subject was planned to be 5 weeks: 1 week of screening, 2 weeks of treatment, and another 2 weeks of follow-up visit. The reporting of proof-of-concept data was delayed to the first half of 2021 due to the current COVID-19 outbreak [72].

5. Conclusions Nie et al. summarized the roles of ARSs in various immune diseases, such as autoimmune diseases, infectious diseases, and tumor immunity, and described their role as important regulators and signaling molecules in the development of immune cells [75]. Antisynthetase syndrome (ASSD) is a heterogeneous group of autoimmune diseases, including interstitial lung disease (ILD), myositis, mechanic’s hands, Raynaud’s phenomenon, and arthritis. ASSD is often caused by specific anti-ARS autoantibodies [76], and the specificity of anti-ARS autoantibodies is related to the clinical features, disease severity, and survival of ASSD patients [77,78]. ARSs are also dysregulated in other autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, immune thrombocytopenia, and systemic lupus erythematosus [79–82]. For example, Wang et al. analyzed the interaction of indoleamine-2,3-dioxygenase (IDO)-expressing dendritic cells (DCs) and TrpRS-expressing CD4(+)T cells in Graves’ disease (GD) patients. In general, IDO and tryptophanyl-tRNA synthetase (TrpRS) are responsible for the metabolism and utilization of tryptophan, respectively, and play important roles in immune regulation [83]. From their analysis, the ratio of serum kynurenine to tryptophan was increased in GD, TrpRS expression from CD4(+) T cells was increased, and there was resistance to IDO-mediated immunosuppression from DCs [84]. ARSs are also closely related to tumor immunity, that is, tumor cells can regulate the functions of immune cells by secreting ARSs, or tumor-related immune cells can secrete ARSs and affect tumor development. For example, Wellman et al. suggested that the direct correlation of ThrRS levels with disease stage in ovarian cancer patients is derived from ThrRS-regulated angiogenesis and immune cell responses [85]. Additionally, Adam et al. discovered that cancer cells could upregulate TrpRS in two different ways to adapt to the nutritional stress caused by tryptophan degradation. First, tryptophan depletion caused by the high expression of indoleamine-2,3-dioxygenase-1 (IDO1) and tryptophan-2,3-dioxygenase (TDO2) in LN229 glioblastoma cells upregulates TrpRS expression through the activation of the general control nonderepressible-2 (GCN2) kinase and phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), followed by the activation of activating transcription factor 4 (ATF4). Second, tumor-infiltrating T cells jointly induce the expression of IDO1 and TrpRS in breast cancer, colon carcinoma, and B-cell lymphoma by secreting IFN-γ [86]. As far as we understand, no small molecule directly related to the autoimmune diseases caused by ARSs is known. Given the high limitations of current immuno-oncology therapies in the clinic, ARS-regulating small molecules that are related to tumor immunity can also be an interesting area to explore. The important functions of tRNA synthetases in neurological and neuromuscular disorders have been explored recently as well [87]. ARSs have been studied as therapeutic targets predominantly for pathogen-derived infectious diseases because of concerns that ARS catalytic site inhibitors would block translation in normal cells. However, their indispensable role in other human diseases has been explored, and some ARS inhibitors are undergoing clinical studies for fibrosis as an example. Given the vast number of studies reported on the role of ARS in tumors, it is reasonable to expect that more clinical studies will emerge for cancer therapeutics in the near future. The role of ARS in central nervous system disorders has recently been revealed. As such, the relatively unexplored area of ARS as a target for human diseases other than infectious diseases is being explored by burgeoning biological investigations. Upon these advancements in the newly found roles of ARSs in human diseases, approaches using small molecules targeting ARSs may drive innovative next-generation therapeutic agents. Biomolecules 2020, 10, 1625 20 of 24

Author Contributions: Conceptualization and design: M.S. and S.-H.K.; writing-original draft preparation: M.S., S.-H.K. and S.B. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by NRF-2017M3A9G4052951. Acknowledgments: We give special thanks to all ARS team members throughout medicinal chemistry, DMPK, and structure biology at NDDC, DGMIF, for their hard work. The authors also give sincere thanks to the reviewers and the editors for their essential comments to improve our manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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