A Review of Small Molecule Inhibitors and Functional Probes of Human Cathepsin L

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Dibyendu Dana CUNY Queens College

Sanjai Kumar CUNY Queens College

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Review A Review of Small Molecule Inhibitors and Functional Probes of Human Cathepsin L

1,2, , 1,2, Dibyendu Dana * † and Sanjai K. Pathak * 1 Chemistry and Biochemistry Department, Queens College of The City University of New York, 65-30 Kissena Blvd, Flushing, NY 11367, USA 2 Ph.D. Program in Biochemistry, The Graduate Center of the City University of New York (CUNY), 365 5th Ave, New York, NY 10016, USA * Correspondence: [email protected] (D.D.); [email protected] (S.K.P.); Tel.: +1-817-370-0490 (D.D.); +1-718-997-4120 (S.K.P.); Fax: +1-718-997-5531 (S.K.P.) Current Address: KemPharm Inc., 2200 Kraft Drive, Blacksburg, VA 24060, USA. † Received: 16 December 2019; Accepted: 4 February 2020; Published: 6 February 2020

Abstract: Human cathepsin L belongs to the cathepsin family of proteolytic enzymes with primarily an endopeptidase activity. Although its primary functions were originally thought to be only of a housekeeping enzyme that degraded intracellular and endocytosed proteins in lysosome, numerous recent studies suggest that it plays many critical and specific roles in diverse cellular settings. Not surprisingly, the dysregulated function of cathepsin L has manifested itself in several human diseases, making it an attractive target for drug development. Unfortunately, several redundant and isoform-specific functions have recently emerged, adding complexities to the drug discovery process. To address this, a series of chemical biology tools have been developed that helped define cathepsin L biology with exquisite precision in specific cellular contexts. This review elaborates on the recently developed small molecule inhibitors and probes of human cathepsin L, outlining their mechanisms of action, and describing their potential utilities in dissecting unknown function.

Keywords: human cathepsin L; cathepsin L probe; cathepsin L inhibitor; activity-based probes; clickable ABP; E-64; CA-074; KDP-1; cathepsin L ABP

1. Introduction Lysosomes play critical roles in human biology receiving, trafficking, processing, and degrading biological molecules from seminal cellular processes, such as endocytosis, phagocytosis, autophagy and secretion. Discovered by the ground-breaking work of de Duve, these single-membrane enclosed cytosolic organelle maintain an acidic (~4.5–5) pH environment, and house close to sixty proteolytic enzymes [1,2]. Among these are the eleven members of the cysteine cathepsin enzymes with a versatile expression and functional profile: Cathepsin L (L1), B, C, F, H, K, O, V (L2), X, S, and W[3]. These enzymes closely mimic the CA1 clan of the papain structure and catalytic cycle and mediate numerous crucial cellular events. For example, they participate in processes involving cell death, protein degradation, post-translational modifications of proteins, extracellular matrix (ECM) remodeling, autophagy, and immune signaling. Given that their functions are aberrantly dysregulated in several human diseases, many are considered prime targets for therapeutic development [4]. Several elegant reviews have recently emerged describing the importance of cysteine cathepsins in both normal physiology and human diseases [5–11]. The focus of this review is specifically on human cathepsin L, a ubiquitously expressed endopeptidase whose involvement in several human diseases has emerged in recent years. These include liver fibrosis, Type I and II diabetes, cardiac and bone, immune and kidney disorders [12–24] In addition, its role in a wide variety of highly invasive forms of cancer is

Molecules 2020, 25, 698; doi:10.3390/molecules25030698 www.mdpi.com/journal/molecules Molecules 2020,, 25,, 698 698 22 of of 40 41 variety of highly invasive forms of cancer is also now well established and has been a subject of elegantalso now reviews well established elsewhere and [25–27]. has been Unfortunately, a subject of elegantseveral reviewsoverlapping elsewhere and redundant [25–27]. Unfortunately, functions of cathepsinseveral overlapping L have also and emerged redundant [5,28–30]. functions It is oftherefore cathepsin critically L have important also emerged that [5 its,28 functional–30]. It is therefore biology incritically both normal important and thatdisease-specific its functional cell biology types in be both clearly normal annotated and disease-specific before significant cell types resources be clearly are directedannotated in before drug significantdiscovery resourcesendeavors. are directedIn this review, in drug discoverywe will briefly endeavors. outline In thisthe review,biogenesis we will of cathepsinbriefly outline L, describe the biogenesis its post-tra of cathepsinnslational L, processing describe its and post-translational trafficking, and processing highlight andkey trastructuralfficking, featuresand highlight required key for structural formation features of an required active and for mature formation cathepsin of an active L. A and brief mature summary cathepsin of cathepsin L. A brief L biologysummary and of cathepsinits role in Lhuman biology diseases and its is role provided in human next. diseases Finally, is provided a detailed next. and Finally,up-to-date a detailed report and on existingup-to-date cathepsin report onL-targeting existing cathepsinsmall molecule L-targeting inhibitors small and molecule functional inhibitors probes with and functionaltheir mechanistic probes detailswith their is described. mechanistic details is described. Human cathepsin cathepsin L L gene, gene, CTHL CTHL (Uniprot (Uniprot primar primaryy accession accession number: number: P07711), P07711), located located at the at 9q21-q22the 9q21-q22 position position of chromosome of chromosome 9, encodes 9, encodes for a total for aof total 333 amino of 333 acid amino peptide acid sequence peptide sequence (M.W. = 37,564(M.W. kDa)= 37,564 [31]. kDa) The [coding31]. The space coding includes space the includes regions the of regionsa N-terminal of a N-terminal signal peptide, signal two peptide, propeptides,two pro-peptides, and two mature and two peptides mature comprising peptides comprising of a heavy of (H) a heavychain and (H) a chain light and(L) chain a light (Figure (L) chain 1). After(Figure transcription,1). After transcription, the signal peptide the signal is co-translationally peptide is co-translationally removed in polysome removed and in the polysome resulting and 41 kDathe resultingpro-cathepsin 41 kDa L pro-cathepsin is translocated L is to translocated endoplasmic to endoplasmicreticulum where reticulum it undergoes where it undergoesN-linked glycosylationN-linked glycosylation with mannose with mannose rich sugars. rich sugars. The ma Thennose mannose sugars sugars on the on thepro-cathepsin pro-cathepsin L Lare are then then phosphorylated in cis Golgi by UDP-N-acetylglucosamine:N-acetylglucosaminephosphotransferaseUDP-N-acetylglucosamine:N-acetylglucosaminephosphotransferase enzyme [32]. [32]. The The mannose-6-pho mannose-6-phosphatesphate (M6P) (M6P) receptors receptors located located on on the the surface surface of of the the transtrans Golgi network recognize the the M6P-pro-cathepsin peptide peptide and and deliver deliver the the pro-cathepsin L L peptide to the lysosomelysosome via via the endolysosomal pathway. The The weakly acidic environment of endosomeendosome/lysosome/lysosome releasesreleases M6PM6P receptorsreceptors and and the the phosphate phosphate group group from from mannose mannose sugars sugars is removed is removed by aby lysosomal a lysosomal acid acidphosphatase phosphatase [33,34 ].[33,34]. Activation Activation to mature to mature cathepsin cathepsin L form thenL form occurs then by removaloccurs by of propeptidesremoval of propeptideseither by autocatalysis either by autocatalysi [35] or by aspartyls [35] or cathepsin by aspartyl D incathepsin the acidic D environmentin the acidic ofenvironment lysosome [36 of]. lysosomeThis leads [36]. to the This double leads chain to the form double of mature chain andform active of mature cathepsin and L,active comprising cathepsin of HL, andcomprising L domains, of Hconnected and L domains, by disulfide connected bridges, by (Figure disulfide2). Itbridges, is to be (F notedigure here 2). It that is severalto be noted isoforms here ofthat cathepsin several isoformsL have also of cathepsin been observed L have in specificalso been cell observed types due in tospecific alternative cell types splicing due ofto mRNAalternative transcripts splicing and of mRNAalternative transcripts translation and [alternative4,37–41]. translation [4,37–41].

Figure 1. 1. TheThe primary primary sequence sequence of of the the full full length length huma humann inactive human cathepsin L: Blue = 1717 Amino Amino acid signal signal (prepro) (prepro) peptide, Purple = PropeptiPropeptidesdes/activation/activation peptides: peptides: 96 96 amino amino acid acid (Thr18–Glu113) (Thr18–Glu113) and 3 amino amino acid (Glu289–Asp291), Red = HeavyHeavy chain chain pe peptide,ptide, Pink = LightLight chai chainn peptide; peptide; * * Disulfide Disulfide bond pair residues, Cys135–Cys178,Cys135–Cys178, θƟ DisulfideDisulfide bond bond pair Cys169–Cys211, Ω DisulfideDisulfide bond bond pair Cys268–Cys322, ¥ TheThe site site of of N-linked N-linked glycosylation. glycosylation. The The two two key key catalytic catalytic residues residues of of the the active active site, site, Cys25 and His163 (numbering of ma matureture cathepsin L) residing in the heavy chain are underlined.

The propeptides act act as as an important regulatory regulatory on on/off/oﬀ switch as well as a folding catalyst in cathepsin activation. Not Not surprisingly, surprisingly, the the nature nature of of propeptides propeptides among among cysteine cysteine cathepsins cathepsins is is highly highly divergent byby both both chain chain lengths lengths and primaryand primary sequences. sequences. It is thought It is thatthought this uniquenessthat this uniqueness is functionally is functionallyrelevant given relevant its ubiquitous given presenceits ubiquitous in most presence tissues and in allows most fortissues the selective and allows suppression for the of selective enzyme suppressionactivity (hence of unintendedenzyme activity autoactivation) (hence unintend during theed transportautoactivation) to the endolysosomalduring the transport compartment. to the

MoleculesMolecules2020 2020, 25, 25,, 698 698 3 of3 of40 41

endolysosomal compartment. In cathepsin L, two inhibitory propeptides, one containing 96 amino In cathepsin L, two inhibitory propeptides, one containing 96 amino acid (Thr18–Glu113) and the acid (Thr18–Glu113) and the other containing 3 amino acid (Glu289–Asp291) exist. A crystal structure other containing 3 amino acid (Glu289–Asp291) exist. A crystal structure of human procathepsin L of human procathepsin L revealed that the 96 amino acid inhibitory propeptide chain spans in the revealed that the 96 amino acid inhibitory propeptide chain spans in the opposite directions of substrate opposite directions of substrate binding and forms several high-affinity non-covalent interactions bindingwith the and surrounding forms several residues high-a in ﬃactivenity non-covalentsite [42,43]. Interestingly, interactions this with opposite the surrounding direction binding residues ofin activeinhibitory site [ 42propeptide,43]. Interestingly, segment is this evolutionarily opposite direction conserved binding in other of inhibitorymembers of propeptide cysteine cathepsins, segment is evolutionarilyincluding in cathepsin conserved B. in other members of cysteine cathepsins, including in cathepsin B.

FigureFigure 2. 2. BiogenesisBiogenesis of human cathepsin cathepsin L. L. After After the the full full length length cathepsin cathepsin L mRNA L mRNA is transcribed, is transcribed, it itis is translated translated in in ribosomes. ribosomes. Following Following this, this, the the full-length full-length peptide peptide enters enters the the ribosomes-bound ribosomes-bound endoplasmicendoplasmic reticulum reticulum lumenlumen wherewhere signalsignal peptide is removed. removed. Pro-cathepsin Pro-cathepsin L L then then enters enters the the Golgi Golgi networknetwork where where it undergoesit undergoes N-linked N-linked glycosylation glycos atylation Asn108, at followed Asn108, by mannosefollowed phosphorylationby mannose andphosphorylation formation of appropriate and formation disulﬁde of appropriate linkages. In thedisulfide last step, linkages. modiﬁed In procathepsin the last step, L is modified shuttled to lysosomeprocathepsin by endolysosomal L is shuttled to pathways,lysosome by generating endolysosomal the double pathways, chain generating form of active the double and mature chain humanform cathepsinof active and L. mature human cathepsin L.

TheThe dominantdominant pathwaypathway ofof regulationregulation of activa activatedted and and mature mature cathepsin cathepsin LL is is by by endogenous endogenous proteinprotein inhibitors, inhibitors, cystatins, cystatins, that that like like propeptide propepti competede compete with with the physiological the physiological substrates substrates for binding for tobinding the enzyme to the activeenzyme site active (Table site1)[ (Table5,44]. 1) Interestingly, [5,44]. Interestingly, protein protein inhibitory inhibitory agents agents of cathepsin of cathepsin L have alsoL have been also reported been reported in other in organisms.other organisms. For example, For example, Kotsyfakis Kotsyfakis M. etM. al.et al. reported reported the the existence existence of twoof two cathepsin cathepsin L inhibitory L inhibitory proteins proteins in the in carrier the carr ofier the of main the vectormain vector of Lyme of disease-carryingLyme disease-carrying parasite, parasite, Ixodus scapularis. Named so because of its abilities to specifically inhibit cathepsin L (IC50 = Ixodus scapularis. Named so because of its abilities to speciﬁcally inhibit cathepsin L (IC50 = 4.68 nM; Ki 4.68 nM; Ki = 95 pM) activity, sialostatin L abrogates the protective proteolytic activity of host cells at = 95 pM) activity, sialostatin L abrogates the protective proteolytic activity of host cells at the infestation the infestation sites, thereby promoting the tick’s survival [45]. In addition, it also possess a potent sites, thereby promoting the tick’s survival [45]. In addition, it also possess a potent anti-inﬂammatory anti-inflammatory and immunosuppressive activity by inhibiting cytotoxic killer T cells [46]. and immunosuppressive activity by inhibiting cytotoxic killer T cells [46].

Table 1. Reported physiological inhibitory ligands of human cathepsin L with their inhibition Table 1. Reported physiological inhibitory ligands of human cathepsin L with their inhibition constants. constants.

Inhibitory Ligand Inhibition Constant (Ki) Inhibitory Ligand Inhibition Constant (Ki) RecombinantRecombinant Cathepsin Cathepsin L PropeptideL Propeptide 0.088 0.088 nM nM [47] [47 ](pH (pH == 5.5)5.5) CystatinCystatin A A 1.3 1.3 nM nM [48,49] [48,49] CystatinCystatin B B 0.23 0.23 nM nM [48,49] [48,49] Cystatin C <0.005 nM [50] Cystatin C <0.005 nM [50] Cystatin D 18 nM [51] Cystatin D 18 nM [51] P41 of MHC Class II Molecule 2 pM [52] P41 of MHCL-Kinenogen Class II Molecule 1.7 pM 2 pM [53,54] [52] L-KinenogenCystatin F 0.31 1.7 pMnM [ 53[55],54 ] CystatinSialostatin F L 95 0.31 pM nM [45] [55 ] AntimicrobialSialostatin Peptide L LL-37 150 95 nM pM [56] [45] Antimicrobial Peptide LL-37 150 nM [56]

Molecules 2020, 25, 698 4 of 41

2. Functional Biology of Cathepsin L and Its Role in Human Diseases Different forms of cathepsin L enzyme have been isolated in distinct cell types and their intracellular and extracellular environment [4]. At specific cellular locations, they seem to play pivotal roles in a wide range of functional activities. In addition to the degradation of cellular proteins by the endolysosomal pathway, cathepsin L also participates in the autophagic degradation pathway; it specifically degrades two autophagosomal markers, LC3-II and GABARAP-II [57]. The specialized podocyte cells in the kidney play a critical role in the retention of various proteins required to be maintained in blood plasma [58]. It was shown that the cytoplasmic variant of active cathepsin L can degrade two key proteins, GTPase dynamin and synaptopodin, required for podocyte’s proper glomerular filtration function [23,59,60]. Consistently, a higher podocyte cathepsin L expression was found in a variety of proteinuric kidney diseases, and its functional inhibition (e.g., by creating cathepsin L resistant dynamin) led to reversing of the symptoms [23,61]. Interestingly, a nuclear isoform of cathepsin L is associated with another unique function in kidney cells; it is involved in the proteolytic processing of Cux1, a cell cycle regulatory transcription factor that promotes cell proliferation and polycystic kidney disease (PKD) [62,63]. Indeed, in mouse model of PKD, a lower level of nuclear cathepsin L was corelated with an increased level of Cux1 protein in cysts [62]. Another type of cells where cathepsin L seems to play a major role in maintaining homeostasis is cardiomyocytes [17,64–67]. Early investigation by Stypmann et al. on a year old Ctsl / mice showed − − that they developed many key traits of dilated cardiomyopathy, such as interstitial myocardial fibrosis, cardiac chamber dilation, and impaired contraction. In addition, the newborn Ctsl / mice acquired − − increased number of acidic organelles, although with altered morphology and function. These studies indicate that the inhibition of cathepsin L activity in these cells is detrimental to the proper functioning of cardiac cells. This was further corroborated by an overexpression study of cathepsin L in mice cardiomyocytes that exhibited cardioprotective effect by inhibiting the Akt signaling pathway [68]. The function of secreted extracellular cathepsin L in cardiac remodeling and repair has also been studied extensively since it is known that many proteins of extracellular matrix (ECM) are also the physiological substrate of cathepsin L; these include laminin, collagen type I, IV and XVIII, and fibronectin [69–71]. Ischemic heart disease is major risk factor for diabetic patients, unable to maintain their sugar levels efficiently. During cardiac repair, endothelial progenitor cells containing elevated levels of cathepsin L home on ischemic cells, and initiate the process of neovascularization [72–74]. In uncontrolled diabetic condition however, cathepsin L function in ECM space during neovascularization is significantly impaired; this hampers the ischemic repair process [15,75]. Controlled release of trypsinogen from pancreatic acinar cells and its subsequent activation in duodenum is a critically important event and must be tightly regulated. Dysregulated release of activated trypsin results in an acute pancreatitis condition. While cathepsin B efficiently cleaves trypsinogen to produce enhanced levels of active trypsin promoting pancreatitis, cathepsin L acts a regulatory brake, and keeps the activation process under control. And it does so by cleaving trypsinogen at a distinct site, 3 amino acids C terminal to cathepsin B, thereby inactivating trypsinogen itself [76]. Consistently, Ctsl / mice showed increased trypsin activity in pancreas and surprisingly − − reduced pancreatitis due to diminished inflammation; this effect was attributed due to enhanced apoptosis of acinar cells (vs necrosis). In neuro- and immuno-biology, cathepsin L has important documented roles as well [77–80]. For example, regulated secretory vesicles of the neuroendocrine system, such as chromaffin granules, house many mature function-ready enkephalin opioid peptides. Upon appropriate signaling stimuli, the activated enkephalin neuropeptides are secreted and released imparting their analgesic and immune-cell specific functions. Studies by Yasothornsrikul et al. revealed that cathepsin L is the proteolytic processing enzyme that renders active metenkephalin from proenkephalin [81]. In another studies, the severity of symptoms due to antigen-induced arthritis (AAI) was significantly reduced in Ctsl / mice due to a weakened T helper cell population in thymus [82]. − − Molecules 2020, 25, 698 5 of 41

The expression of cathepsin L is highly dysregulated in several human diseases, including diabetes, AAI, abdominal aortic aneurysm, liver ﬁbrosis, and cancers [13,82–85]. Its involvement in numerous forms of highly invasive cancers is particularly noteworthy and is covered in great detail in recent reviews [25,26,86–88]. Early studies by Gottesman revealed that extracellular cathepsin L levels can increase up to 200-fold in transformed cells [89]. In the ECM environment, they can rapidly degrade several structural components and signiﬁcantly increase the migratory/invasive potential of these cells, promoting metastasis [86,87]. Not surprisingly, cathepsin L inhibition by small molecule is considered a viable strategy for the development of novel anti-cancer agents.

3. Small Molecule Inhibitors Over the years, several classes of physiological and synthetic inhibitors have been discovered targeting cathepsin L. Herein, the focus is on non-physiological inhibitors that could broadly be classified as reversible and irreversible inhibitors. Reversible and irreversible inhibitor could be distinguished by means of the mechanistic approaches they utilize for enzyme inactivation. Reversible inhibitors ‘generally’ engage with the target protein using non-covalent interaction; however, in certain cases, they form a quasi-covalent bond which eventually disengages from the active site, say, upon dilution. Irreversible inhibitors, on the other hand, permanently modify the protein of interest via the formation of a stable covalent bond. While both reversible and irreversible inhibitors have their pros and cons, it is generally believed that reversible inhibitors are preferred candidates in drug discovery and irreversible inhibitors not so due to their adverse immune responses [90]. There are, however, several examples of successful drugs being used in clinics that work by an irreversible inhibitory mechanism [91]. Irreversible inhibitors are also widely utilized in functional biology (e.g., in the development of activity-based probes). The majority of synthetic cathepsin L inhibitors have recognizable peptide sequences, often derived from its physiological substrates, that bind to the active site of the protein and often contain strategically placed electrophilic warheads that trap the nucleophilic Cys25 residue (Figure3) for activity. Knowledge of enzyme structure and its associated substrate specificity thus has played an important role in designing selective inhibitors of cathepsins [92,93]. In a seminal study, Choe et al. studied substrate specificity using a highly diversified positional scanning synthetic combinatorial library comprised of 160,000 fluorogenic tetrapeptides; this allowed to differentiate individual enzymes0 binding propensity based on their distinct amino acid preferences [93]. By capitalizing on this strategy, they successfully developed a selective substrate and substrate-based inhibitor of cathepsin K. The other important contribution to our understanding of subsite binding preferences of cathepsin L enzyme stems from several timely crystal structure studies that helped reveal key difference in the structural landscape of enzyme subsites [94–96]. In an important study, Shenoy et al. solved the crystal structure of ligand bound cathepsin L and documented the structural compositions of ligand binding sites (Figure4)[ 96]. Their analysis of Z-Phe-Tyr (O-tert-Butyl)-C(O)C(H)O bound cathepsin L revealed that (a) S1 subsite is relatively wide and unrestricted and composed of Asp162, Ser24, and Cys25, (b) S10 subsite is guided by Leu144, Trp189, Ala138 and Gly139 where Trp189 associates with Trp193 and Phe143 and forms an aromatic cluster that accommodates the tert-butyl group (c) side chains of Leu69 and Met70 help form the S2 subsite that engages in non-polar interactions with the phenyl side chain, and finally, (d) the carboxybenzyl group finds interaction with the Gly68 residue of S3 subsite (Figure4). However, it turns out that di fferent ligands may find slightly altered binding interactions with the enzyme subsites, depending on their structural features. For example, Shenoy et al. showed that although Z-Phe-Tyr (O-tert-Butyl)-DMK binds to the same subsites of cathepsin L as observed for the Z-Phe-Tyr (O-tert-Butyl)-C(O)C(H)O ligand and finds some alternative interactions with residues from the active site pocket. This finding has been corroborated by other studies as well that suggest that structural features of ligands can influence the subsite composition of the cathepsin L enzyme [94,95,97,98]. Such information has been duly capitalized to develop different classes of inhibitors that are discussed next. In the following section, we discuss the types of cathepsin L-targeting chemotypes that have been utilized for development of small molecule inhibitors. Molecules 2020, 25, 698 6 of 40

Molecules 2020, 25, 698 6 of 40 3.1. Epoxysuccinates 3.1. EpoxysuccinatesEpoxysuccinate inhibitors have historically played a crucial role in deciphering the cysteine proteaseEpoxysuccinate biology. This inhibitors class of compounds have historically contains played an epoxide a crucial ring role as an in electrophilic deciphering warhead the cysteine that trapsprotease the biology. active Thissite classcatalytic of compounds cysteine containsresidue anof epoxidethe protein. ring as E-64 an electrophilic (L-trans-Epoxysuccinyl- warhead that leucylamido(4-guanidino)butane)traps the active site catalytic cysteine(Entry 1;residu Tablee of2), theperhaps protein. the E-64most (L-trans-Epoxysuccinyl-studied commercially availableleucylamido(4-guanidino)butane) universal cathepsin inhibitor (Entry of 1;this Table class, 2), showed perhaps only the a marginalmost studied selectivity commercially towards cathepsinavailable Luniversal compared cathepsin to cathepsin inhibitor B and of a moderatethis class, 24 showed-fold selectivity only a overmarginal cathepsin selectivity H, as reportedtowards bycathepsin Barrett L et compared al. [99]. Althoughto cathepsin they B and developed a moderate several 24-fold synthetic selectivity analogs over cathepsinof E-64 with H, as improved reported potency,by Barrett they et al.lacked [99]. desirable Although selectivity they developed toward secathepsinveral synthetic L. They analogs proposed of theE-64 probable with improved binding orientationpotency, they of E-64lacked that desirable follows selectivitya non-substrate-like toward cathepsin orientation, L. They i.e., itproposed occupies theonly probable non-prime binding sites (Figure 3) at the enzyme pocket. Moleculesorientation2020 ,of25 ,E-64 698 that follows a non-substrate-like orientation, i.e., it occupies only non-prime6 sites of 41 (Figure 3) at the enzyme pocket. a. b. O R2 a. b. R1 O Phe Eps Pyr (z) R R1 2 Phe Eps Pyr (z) Cbz Phe Arg AMC (y) Cat L S Cbz Phe Arg AMC Agm (y) Eps Leu (x) Cat L SR1 OH SH Agm Eps Leu (x) R1 OH SH Cat L S R2 Cat L S R S3 S2 S1 S1' S2' R & R = Different substituents2 1 2 S3 S2 S1 S1' S2' R & R = Different substituents Figure 3. (a) Proposed binding mode of (x) E-64, (y) a putative cathepsin1 L 2substrate, and (z) CLIK148 to the active site of cathepsin L. (b) Inactivation mechanism of cathepsin L by epoxy-containing Figure 3. (a) Proposed bindingbinding modemode of of ( x(x)) E-64, E-64, ( y(y)) a a putative putative cathepsin cathepsin L L substrate, substrate, and and (z )(z CLIK148) CLIK148 to inhibitors. theto the active active site ofsite cathepsin of cathepsin L. (b) L. Inactivation (b) Inactivation mechanism mechanism of cathepsin of ca Lthepsin by epoxy-containing L by epoxy-containing inhibitors. inhibitors.

Figure 4.4.Schematic Schematic representation representation of Z-Phe-Tyrof Z-Phe-Tyr (O-tert-Butyl)-C(O)C(H)O (O-tert-Butyl)-C(O)C(H)O bound withinbound the within cathepsin the L subsites. The key subsite-forming amino acid residues with their corresponding numbers are shown cathepsinFigure 4. LSchematic subsites. The representation key subsite-forming of Z-Phe-Tyr amino acid(O-tert-Butyl)-C(O)C(H)O residues with their corresponding bound within numbers the in blue whereas the catalytic diad forming residues are depicted in red. (The authors used PDB:3OF8 arecathepsin shown L in subsites. blue whereas The key the subsite-forming catalytic diad forming amino acid residues residues are depictedwith thei rin corresponding red. (The authors numbers used file to construct the figure [96]). PDB:3OF8are shown filein blue to construct whereas thethe figurecatalytic [96]). diad forming residues are depicted in red. (The authors used 3.1. EpoxysuccinatesPDB:3OF8 file to construct the figure [96]). In another study, Gour-Salin et al. reported a number of epoxysuccinyl amino acid benzyl esters in whichEpoxysuccinateIn another they systematically study, inhibitorsGour-Salin varied et have al.the reported amino historically acid a number attached played of epoxysuccinylto a the crucial epoxide role aminoring. in This acid deciphering was benzyl intended esters the tocysteinein whichinvestigate they protease systematicallyits effect biology. in determining varied This the class amino selectivity of acid compounds attachedtoward tocathepsin containsthe epoxide L anor ring. epoxideS [100]. This wasTheir ring intended results as an surprisinglyelectrophilicto investigate indicate warhead its effect that that inthe determining trapsspecificity the active of selectivitythese site analogs catalytic toward did not cysteine cathepsin follow residue the L trend,or ofS [100]. generally the protein. Their observed results E-64 for(L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane)surprisingly substrate, indicate possibly that due the tospecificity E-64 like of thesebinding analogs orientation did (Entry not atfollow 1; Tablethe theenzyme2), perhapstrend, pocket. generally the most Among observed studied all commerciallyfor substrate, availablepossibly universaldue to E-64 cathepsin like binding inhibitor orientation of this class, at showed the enzyme only a pocket. marginal Among selectivity all towards cathepsin L compared to cathepsin B and a moderate 24-fold selectivity over cathepsin H, as reported by Barrett et al. [99]. Although they developed several synthetic analogs of E-64 with improved potency, they lacked desirable selectivity toward cathepsin L. They proposed the probable binding orientation of E-64 that follows a non-substrate-like orientation, i.e., it occupies only non-prime sites (Figure3) at the enzyme pocket. In another study, Gour-Salin et al. reported a number of epoxysuccinyl amino acid benzyl esters in which they systematically varied the amino acid attached to the epoxide ring. This was intended to investigate its effect in determining selectivity toward cathepsin L or S [100]. Their results surprisingly indicate that the specificity of these analogs did not follow the trend, generally observed for substrate, possibly due to E-64 like binding orientation at the enzyme pocket. Among all synthesized compounds, Molecules 2020, 25, 698 7 of 41

HO-Eps-Arg-OBzl (Entry 2, Table2) showed a signiﬁcant 89-fold selectivity for cathepsin L over cathepsin S. Notably in a seminal study, Katanuma et al. developed several cathepsin L-selective E-64 analogs (Cathepsin L Inhibitor by Katunuma: CLIK) with the help of computational modeling based on the stereo-structure [101]. Three of the developed CLIK inhibitors were hydrolytically stable and showed highly selective inhibition for hepatic cathepsin L in vivo. Further, they elucidated the inhibition mechanism of this class of compounds based on the crystal structure of papain-CLIK148 (Entry 3, Table2) complex [ 102]. This crystal structure revealed that CLIK148, unlike E-64, binds to both prime and non-prime sites of the active site pocket. Notably, the speciﬁcity toward cathepsin L was attributed to the existence of phenylalanine residue at the S2 site, and a hydrophobic interaction mediated by N-terminal pyridine ring (Figure3).

3.2. Peptidyldiazomethane and Peptidylchloromethane The other classes of alkylating agents that initially played an important role in deciphering the mechanistic aspects of cathepsin inhibition were peptidyldiazomethane (Entry 4, Table2) and peptidylchloromethane (Entry 5, Table2). Crawford et al. reported highly potent inhibitors from these two chemotypes that spans the active site and shows signiﬁcant selectivity improvement over other cysteine proteases [103]. However, these classes of inhibitors suﬀer from stability issues and have found limited utility in in vivo assays.

3.3. Peptidylhydroxylamine and Peptidylhydroxamates Peptidylhydroxylamines were first introduced as mechanism-based inhibitors of serine and cysteine proteinases [104–107]. Bromme et al. adapted this scaffold and prepared a library of N-peptidyl-O-acyl hydroxylamines which exhibited rapid and selective inactivation of several lysosomal cysteine proteinases [108]. This class of inhibitors occupied the active site of the enzyme and irreversibly inactivated cysteine cathepsins as the free enzyme activity was not recovered when the enzyme-inhibitor complex was exposed to exhaustive ultrafiltration (up to an enzyme/free inhibitor ratio of <1:0.05) or chromatography on Sephadex G-10. Among developed inhibitors, Z-Phe-Phe-NHO-MA (Entry 6, Table2) inhibited cathepsin L with most potency and showed significant selectivity, 58-fold and 436-fold, over cathepsin S and cathepsin B, respectively. Interestingly the stability and efficacy of this class of inhibitors were determined by the nature of substitutions on hydroxylamine oxygen as their electron-withdrawing tendencies showed a positive correlation with inactivation kinetics. Although this class of inhibitors exhibited desirable traits with respect to both potency and selectivity, they suffered majorly from aqueous stability issue; half-lives (t1/2) only in the range of 45209 min in aqueous solution. In another study, Bromme et al. developed a series of N-peptidyl-O-acyl hydroxamates with lysine in P1 position with improved inhibitory profile for cysteine proteases over their serine counterparts [109]. The maximum inhibition was observed by Z-Phe-Lys-NHO-NBz (Entry 8, Table2), with 7-fold selectivity over cathepsin S, and >100-fold selectivity over cathepsin B. The authors postulated that the active site Cys residue attacks the carbonyl of the hydroxamate and forms a tetrahedral intermediate, whereas the nitrogen of the hydroxamate, which primarily remains deprotonated (pKa < 5), presumably engages in an electrostatic interaction with the active site His159 residue (Figure5). Molecules 2020, 25, 698 8 of 40 Molecules 2020, 25, 698 8 of 40

Figure 5. Postulated active-site interactions between papain, a prototypical cysteine protease, and Figure 5. Postulated active-site interactions between papain, a prototypical cysteine protease, and peptidyl hydroxamate inhibitor. Active-site cysteine forms a covalent bond with the carbonyl group peptidyl hydroxamate inhibitor. Active-site cysteine forms a covalent bond with the carbonyl group of the inhibitor and the negatively charged nitrogen engages in electrostatic interaction with the of the inhibitor and the negatively charged nitrogen engages in electrostatic interaction with the positively charged histidine residue. positively charged histidine residue. In a follow-up study, Bromme et al. synthesized and tested a series of new inhibitors, with the In a follow-up study, Bromme et al. synthesized and tested a series of new inhibitors, with the general formula of Z-Phe-Gly-NHO-CO-Aa (Aa: amino acid), against papain class of enzymes [110]. general formula of Z-Phe-Gly-NHO-CO-Aa (Aa: amino acid), against papain class of enzymes [110]. This class of inhibitors covalently modified active-site Cys residue via sulfenamidation, like in their This class of inhibitors covalently modified active-site Cys residue via sulfenamidation, like in their N-peptidyl-O-acyl hydroxamate counterparts, as shown by the mass spectrometric analysis. These N-peptidyl-O-acyl hydroxamate counterparts, as shown by the mass spectrometric analysis. These inhibitors further provided the option of varying the leaving group that targets S2′ site; more inhibitors further provided the option of varying the leaving group that targets S2′ site; more i i hydrophobic substituents were preferred at this position as comparison of ki/Ki values of the hydrophobic substituents were preferred at this position as comparison of ki/Ki values of the 2 inactivation exhibited the following trend for Aa: Gly < Ala < Val < Leu < Phe < 4-NO2-Ph. The inactivation exhibited the following trend for Aa: Gly < Ala < Val < Leu < Phe < 4-NO2-Ph. The nitrophenyl analog (Entry 7, Table 2) which incur both hydrophobicity and electron-withdrawing nitrophenyl analog (Entry 7, Table 2) which incur both hydrophobicity and electron-withdrawing property exerted the maximum potency and selectivity for cathepsin L over other tested cysteine property exerted the maximum potency and selectivity for cathepsin L over other tested cysteine proteases. Molecules 2020, 25, 698proteases. 8 of 41

Table 2. Reported small molecule inhibitory agents of human cathepsin L, their mechanism of Table 2. Reported small molecule inhibitory agents of human cathepsin L, their mechanism of Table 2. Reportedinhibition, small molecule inhibitory inhibitory parameters agents and ofselectivity human cathepsinprofile, and L, their their demonstrated mechanism ofutilities. inhibition, inhibitory parameters and selectivity proﬁle, and their inhibition, inhibitory parameters and selectivity profile, and their demonstrated utilities. demonstrated utilities. Mechanism of Inhibitor Mechanism of Inhibitor Selectivity Inhibition # Chemical Structure Inhibitor Class Efficacy Selectivity Inhibition References # Chemical Structure Inhibitor Class Efficacy Selectivity# Inhibition ReferencesMechanism of # Chemical Structure Inhibitor Class EfficacyInhibitor EﬃFactorcacy # (Demonstrated References # Chemical Structure Inhibitor Class (Cathepsin L) Factor # Selectivity(Demonstrated Factor # Inhibition References (Cathepsin(Cathepsin L) L) Utilities) Utilities) (Demonstrated Utilities)

2nd Order 2nd Order Cat B: 1.1 Irreversible Barrett et al., 2nd −Order1 −1 Cat B: 1.1 Irreversible Barrett et al., 1 Epoxy Succinyl (M−2nd1 s−1) Order (MCat1 B:s 11.1) IrreversibleCat B: 1.1 Barrett et al.,Irreversible 1 1 EpoxyEpoxy Succinyl Succinyl (M−1 s−1) Cat− H:− 24 (In vitro) 1982 [99] Barrett et al., 1982 [99] 96250 Cat H: 24 (In vitro) 1982 [99] 96250 96250 Cat H: 24 (In vitro)

3 2nd Order (103 3 1 2nd Order2nd (10 Order3 (10 M− Irreversible Gour-Salin et 2nd Order−1 −1 (10 Irreversible Gour-Salin et 2 Epoxy Succinyl M−1 s−1) 1 Cat S: 89 Irreversible Gour-Salin etIrreversible Gour-Salin et al., 1994 2 2 EpoxyEpoxy Succinyl Succinyl M−1 s−1) s− ) Cat S: 89 (InCat vitro) S: 89 al., 1994 [111] 73 (In vitro) al., 1994 [111](In vitro) [111] 73 73

Cat B: NI Irreversible −6 6 Cat B: NI IrreversibleCat B: NI 10−6 M (100%10− M (100%Cat B: NI Irreversible Katunuma etIrreversible Katunuma et al.,1999 3 3 EpoxyEpoxy Succinyl Succinyl 10−6 M (100% Cat S: 3 (InCat vitro S: 3 Katunuma et 3 Molecules 2020, 25, 698 Epoxy Succinyl inhibition)inhibition) Cat S: 3 (In vitro al.,19999 of 40 [101](In vitro cellular) [101] Molecules 2020, 25, 698 inhibition) Cat K: NI Catcellular) K: NI al.,19999 of 40 [101] Cat K: NI cellular)

2nd Order Peptidyldiazometha 2nd Order Cat1 B: 411 Irreversible Crawford et al., Peptidyldiazometha −12nd−1 Order (MCat sB: 41) IrreversibleCat B: 41 Crawford et al.,Irreversible Crawford et al., 1988 4 44 Peptidyldiazomethane(M(M−1 s s−1)) − − nene 1128000Calpain:Calpain: NI NI Calpain:(In(In vitro) vitro) NI 19881988 [103] [103] (In vitro) [103] 11280001128000

Peptidylchlorometha Peptidylchlorometha 2nd Order Peptidylchloromethanene 2nd Order Cat1 B: 1131 IrreversibleCat B: 113 Crawford et al.,Irreversible Crawford et al., 1988 5 ne (M−12nd s−1) Order (MCat− B:s− 113) Irreversible Crawford et al., 5 5 (chloromethylketone (M−1 s−1) Calpain: <200 (In vitro) 1988 [103] (chloromethylketone(chloromethylketone)21500000 21500000Calpain: <200 Calpain:(In vitro)< 200 1988 [103] (In vitro) [103] ) 21500000 )

2nd Order Cat B: 436 N-peptidyl-O-acyl 2nd Order Cat B: 436 Irreversible Bromme et al., N-peptidyl-O-acyl −1 −1 Irreversible Bromme et al., 66 (M(M−1 s s−1)) CatCat S: S: 58 58 hydroxylamineshydroxylamines (In(In vitro) vitro) 19891989 [108] [108] 12220001222000 CatCat H: H: ND ND

N-peptidyl-O- 2nd Order Cat B: 59 N-peptidyl-O- 2nd Order Cat B: 59 Irreversible Bromme et al., −1 −1 Irreversible Bromme et al., 77 carbamoylcarbamoyl amino amino (M(M−1 s s−1)) CatCat S: S: 10 10 (In(In vitro) vitro) 19931993 [110] [110] acidacid hydroxamates hydroxamates 931800931800 Papain:Papain: 184 184

Cat S: 7 2nd Order Cat S: 7 N-peptidyl-O-acyl 2nd Order Cat B: 101 Irreversible Bromme et al., 8 N-peptidyl-O-acyl (M−1 s−1) Cat B: 101 Irreversible Bromme et al., 8 hydroxamates (M−1 s−1) Cat H: 4655 (In vitro) 1993 [109] hydroxamates 3538000 Cat H: 4655 (In vitro) 1993 [109] 3538000 a SPSP a: :II II

2nd Order Peptidyl 2nd Order Cat S: 7 Irreversible Krantz, 1994 Peptidyl −1 −1 Cat S: 7 Irreversible Krantz, 1994 99 (M(M−1 s s−1)) (acyloxy)methanes(acyloxy)methanes CatCat B: B: 4 4 (In(In vitro) vitro) [112][112] 1070000010700000

Peptidyl 2nd Order Peptidyl 2nd Order Irreversible Torkar et al., −1 −1 Irreversible Torkar et al., 1010 acyloxymethylacyloxymethyl (M(M−1 s s−1)) CatCat B: B: 11 11 (In(In vitro) vitro) 20132013 [113] [113] ketoneketone 875000875000

2nd Order 2nd Order −1 −1 (M(M−1 s s−1)) CatCat B B ~ ~ 2(L) 2(L) IrreversibleIrreversible MartichonokMartichonok et et 1111 PeptidylPeptidyl aziridine aziridine (L)(L) ~ ~ 20000 20000 CatCat B B ~ ~ 2(D) 2(D) (In(In vitro) vitro) al.,al., 1995 1995 [114] [114] (D) ~ 110000 (D) ~ 110000

2nd2nd Order Order −1 −1 N-acylatedN-acylated (M(M−1 min min−1)) CatCat B: B: 13 13 IrreversibleIrreversible Schirmeister,Schirmeister, 1212 aziridinesaziridines (type (type I) I) (S,S+R,R):(S,S+R,R): Papain:Papain: 22 22 (In(In vitro) vitro) 19991999 [115] [115] 32273227

2nd2nd Order Order −1 −1 N-unsubstitutedN-unsubstituted (M(M−1 min min−1)) CatCat B: B: 10(R,R) 10(R,R) IrreversibleIrreversible Schirmeister,Schirmeister, 1313 aziridinesaziridines (type (type II) II) (R,R):(R,R): 16261 16261 CatCat B: B: 76(S,S) 76(S,S) (In(In vitro) vitro) 19991999 [115] [115] (S,S):(S,S): 3130 3130

2nd Order N-acylated 2nd Order N-acylated −1 −1 (M(M−1 min min−1)) CatCat B: B: 3(R,R) 3(R,R) IrreversibleIrreversible Schirmeister,Schirmeister, 1414 bispeptidylbispeptidyl (R,R):(R,R): 5896 5896 CatCat B: B: 3(S,S) 3(S,S) (In(In vitro) vitro) 19991999 [115] [115] aziridinesaziridines (type (type III) III) (S,S):(S,S): 1210 1210

Molecules 2020, 25, 698 9 of 40 Molecules 2020, 25, 698 9 of 40 Molecules 2020,, 2525,, 698698 9 of 40 Molecules 2020, 25, 698 9 of 40

2nd Order Peptidyldiazometha 2nd Order Cat B: 41 Irreversible Crawford et al., Peptidyldiazometha 2nd −Order1 −1 Cat B: 41 Irreversible Crawford et al., 4 Peptidyldiazometha 2nd(M Order−1 s−1) Cat B: 41 Irreversible Crawford et al., 4 Peptidyldiazomethane 2nd(M Order−1 s−1) Calpain:Cat B: 41 NI Irreversible(In vitro) Crawford1988 [103] et al., 4 Peptidyldiazomethane 2nd(M− 1Order s−1 ) Calpain:Cat B: 41 NI Irreversible(In vitro) Crawford1988 [103] et al., 4 Peptidyldiazomethane (M1128000−1 s−1) Calpain:Cat B: 41 NI Irreversible(In vitro) Crawford1988 [103] et al., 4 ne (M1128000−1 s −1) Calpain: NI (In vitro) 1988 [103] 4 ne 1128000(M s ) Calpain: NI (In vitro) 1988 [103] Molecules 2020, 25, 698 ne 1128000 Calpain: NI (In vitro) 1988 [103] 9 of 41 1128000

Peptidylchlorometha Peptidylchlorometha 2nd Order Peptidylchloromethane 2nd Order Cat B: 113 Irreversible Crawford et al., Peptidylchloromethane 2nd −Order1 −1 Cat B: 113 Irreversible Crawford et al., 5 ne 2nd(M Order−1 s−1) TableCat 2. Cont B: 113. Irreversible Crawford et al., 5 Peptidylchlorometha(chloromethylketonene 2nd(M Order−1 s−1) Calpain:Cat B: 113 <200 Irreversible(In vitro) Crawford1988 [103] et al., 5 (chloromethylketonene 2nd(M− 1Order s−1 ) Calpain:Cat B: 113 <200 Irreversible(In vitro) Crawford1988 [103] et al., 5 (chloromethylketonene 21500000(M−1 s−1) Calpain:Cat B: 113 <200 Irreversible(In vitro) Crawford1988 [103] et al., 5 (chloromethylketone) 21500000(M −1 s −1) Calpain: <200 (In vitro) 1988 [103] 5 (chloromethylketone) 21500000(M s ) Calpain: <200 (In vitro) 1988 [103]Mechanism of (chloromethylketone) 21500000Inhibitor Calpain: Eﬃcacy <200 (In vitro) 1988 [103] # Chemical Structure Inhibitor) Class 21500000 Selectivity Factor # Inhibition References ) (Cathepsin L) (Demonstrated Utilities) 2nd Order Cat B: 436 N-peptidyl-O-acyl 2nd Order Cat B: 436 Irreversible Bromme et al., N-peptidyl-O-acyl 2nd −Order1 −1 Cat B: 436 Irreversible Bromme et al., 6 N-peptidyl-O-acyl 2nd(M Order−1 s−1) CatCat B: S: 436 58 Irreversible Bromme et al., 6 N-peptidyl-O-acylhydroxylamines 2nd(M Order−1 s−1) CatCat B: S: 436 58 Irreversible(In vitro) Bromme1989 [108] et al., 6 N-peptidyl-O-acylhydroxylamines 2nd(M− 1Order s−1) Cat1 B:S: 436581 IrreversibleCat(In vitro) B: 436 Bromme1989 [108] et al., 6 N-peptidyl-O-acylhydroxylaminesN-peptidyl-O-acyl (M1222000−2nd1 s−1) Order (MCatCat H: sS: ND58) Irreversible(In vitro) Bromme1989 [108] et al., Irreversible Bromme et al., 1989 6 6 hydroxylamines (M1222000−1 s−1) CatCat− H: S:− ND58 (InCat vitro) S: 58 1989 [108] hydroxylamineshydroxylamines 1222000 1222000Cat H: ND (In vitro) 1989 [108] (In vitro) [108] 1222000 Cat H: ND Cat H: ND

N-peptidyl-O- 2nd Order Cat B: 59 N-peptidyl-O- 2nd Order Cat B: 59 Irreversible Bromme et al., N-peptidyl-O- 2nd −Order1 −1 Cat B: 59 Irreversible Bromme et al., 7 carbamoylN-peptidyl-O- amino 2nd(M Order−1 s−1) CatCat B:S: 5910 Irreversible Bromme et al., 7 carbamoylN-peptidyl-O-carbamoylN-peptidyl-O- amino 2nd(M Order−1 s−1) CatCat B:S: 5910 Irreversible(InCat vitro) B: 59 Bromme1993 [110] et al., 7 acidcarbamoylN-peptidyl-O- hydroxamates amino 2nd(M931800− 2nd1Order s−1) Order (MPapain:CatCat1 S:sB: 10 159184) Irreversible(In vitro) Bromme1993 [110] et al., Irreversible Bromme et al., 1993 7 acidcarbamoyl hydroxamates amino (M931800−1 s−1) Papain:Cat− S:− 10 184 Irreversible(In vitro) Bromme1993 [110] et al., 7 7 acidcarbamoyl hydroxamatesamino amino acid (M931800−1 s−1) Papain:Cat S: 10184 (InCat vitro) S: 10 1993 [110] acid hydroxamates 931800 931800Papain: 184 (In vitro) 1993 [110] (In vitro) [110] acid hydroxamateshydroxamates 931800 Papain: 184 Papain: 184

Cat S: 7 2nd Order Cat S: 7 N-peptidyl-O-acyl 2nd Order CatCat B: S: 101 7 IrreversibleCat S: 7 Bromme et al., N-peptidyl-O-acyl 2nd −Order1 −1 CatCat B: S: 101 7 Irreversible Bromme et al., 8 N-peptidyl-O-acyl 2nd(M Order−1 s−1) CatCat B: S: 101 7 Irreversible Bromme et al., 8 N-peptidyl-O-acylhydroxamatesN-peptidyl-O-acyl 2nd(M Order−2nd1 s−1) Order (MCatCat1 H:B:s 10146551) IrreversibleCat(In vitro) B: 101 Bromme1993 [109] et al., Irreversible Bromme et al., 1993 8 N-peptidyl-O-acylhydroxamates 2nd(M3538000− 1Order s−1) CatCat− H:B:− 1014655 Irreversible(In vitro) Bromme1993 [109] et al., 8 8 N-peptidyl-O-acylhydroxamates (M3538000−1 s−1) CatCat H: B:a 4655101 Irreversible(In vitro) Bromme1993 [109] et al., hydroxamateshydroxamates 3538000−1 −1 3538000CatSP H: a :4655 II Cat(In vitro) H: 4655 1993 [109] (In vitro) [109] 8 hydroxamates 3538000(M s ) CatSP H: a :4655 II (In vitro) 1993 [109] hydroxamates 3538000 CatSP H: a : 4655II (In vitro)a 1993 [109] 3538000 SP a:: IIII SP : II SP a: II

2nd Order Peptidyl 2nd Order Cat S: 7 Irreversible Krantz, 1994 Peptidyl 2nd −Order1 −1 Cat S: 7 Irreversible Krantz, 1994 9 Peptidyl 2nd(M Order−1 s−1) Cat1 S:1 7 Irreversible Krantz, 1994 9 (acyloxy)methanesPeptidylPeptidyl 2nd(M Order−2nd1 s−1) Order (MCat− sS:B:− 74) Irreversible(InCat vitro) S: 7 Krantz,[112] 1994 Irreversible 9 9 (acyloxy)methanesPeptidyl 2nd(M− 1Order s−1) Cat S:B: 74 Irreversible(In vitro) Krantz,[112] 1994 Krantz, 1994 [112] 9 (acyloxy)methanes(acyloxy)methanesPeptidyl 10700000(M−1 s−1) 10700000Cat B:S: 74 Irreversible(InCat vitro) B: 4 Krantz,[112] 1994 (In vitro) 9 (acyloxy)methanes 10700000(M−1 s−1) Cat B: 4 (In vitro) [112] (acyloxy)methanes 10700000 Cat B: 4 (In vitro) [112] 10700000

Peptidyl 2nd Order Peptidyl 2nd Order Irreversible Torkar et al., Peptidyl 2nd −Order1 −1 1 1 Irreversible Torkar et al., 10 acyloxymethylPeptidylPeptidyl 2nd(M Order−2nd1 s−1) Order (MCat− sB:− 11) Irreversible Torkar et al.,Irreversible Torkar et al., 2013 10 10 acyloxymethylPeptidyl 2nd(M Order−1 s−1) Cat B: 11 Irreversible(InCat vitro) B: 11 Torkar2013 [113] et al., 10 acyloxymethylPeptidyl 2nd(M− 1Order s−1 ) Cat B: 11 Irreversible(In vitro) Torkar2013 [113] et al., 10 acyloxymethylacyloxymethylketone ketone (M875000−1 s−1) 875000Cat B: 11 Irreversible(In vitro) Torkar2013 [113] et al., (In vitro) [113] 10 acyloxymethylketone (M875000−1 s −1) Cat B: 11 (In vitro) 2013 [113] 10 acyloxymethylketone (M875000 s ) Cat B: 11 (In vitro) 2013 [113] ketone 875000 (In vitro) 2013 [113] ketone 875000

2nd Order 2nd Order 1 1 2nd −2ndOrder1 −1 Order (M− s− ) 2nd(M Order−1 s−1) Cat B ~ 2(L) IrreversibleCat B ~ 2(L) MartichonokIrreversible et Martichonok et al., 11 Peptidyl aziridine 2nd(M Order−1 s−1) Cat B ~ 2(L) Irreversible Martichonok et 11 11 PeptidylPeptidyl aziridine aziridine 2nd(M− 1Order s−1) (L) ~ 20000Cat B ~ 2(L) Irreversible Martichonok et 11 Peptidyl aziridine (L)(L)(M ~~− 12000020000 s−1) Cat B ~ 2(L)2(D) CatIrreversible(In(In B vitro)vitro) ~ 2(D) Martichonokal., 1995 [114] et(In vitro) 1995 [114] 11 Peptidyl aziridine (L)(M ~− 120000 s−1)(D) ~ 110000Cat B ~ 2(D)2(L) Irreversible(In vitro) Martichonokal., 1995 [114] et 11 Peptidyl aziridine (D)(L) ~ 11000020000 Cat B ~ 2(D) (In vitro) al., 1995 [114] (D)(L) ~ 11000020000 Cat B ~ 2(D) (In vitro) al., 1995 [114] (D)(L) ~ ~ 110000 20000 Cat B ~ 2(D) (In vitro) al., 1995 [114] (D) ~ 110000 (D) ~ 110000 2nd Order 2nd−1 Order−1 N-acylated 2nd(M−1 Ordermin−1) Cat B: 13 Irreversible Schirmeister, 12 N-acylated 2nd(M−1 Ordermin−1) Cat B: 13 Irreversible Schirmeister, 12 N-acylated (M2nd−1 Ordermin−1 ) Cat B: 13 Irreversible Schirmeister, 12 aziridinesN-acylated (type I) (M(S,S+R,R):−1 min−1) Papain:Cat B: 13 22 Irreversible(In vitro) Schirmeister,1999 [115] 12 aziridinesN-acylated (type I) (M(S,S+R,R):−1 min −1) Papain:Cat B: 13 22 Irreversible(In vitro) Schirmeister,1999 [115] 12 aziridinesN-acylated (type I) (M(S,S+R,R): min ) Papain:Cat B: 1322 Irreversible(In vitro) Schirmeister,1999 [115] 12 aziridines (type I) (S,S+R,R):3227 Papain: 22 (In vitro) 1999 [115] aziridines (type I) (S,S+R,R):3227 Papain: 22 (In vitro) 1999 [115] 3227 3227

2nd Order 2nd−1 Order−1 N-unsubstituted 2nd(M−1 Ordermin−1) Cat B: 10(R,R) Irreversible Schirmeister, 13 N-unsubstituted 2nd(M−1 Ordermin−1) Cat B: 10(R,R) Irreversible Schirmeister, 13 N-unsubstituted (M2nd−1 Ordermin−1 ) Cat B: 10(R,R) Irreversible Schirmeister, 13 aziridinesN-unsubstituted (type II) (R,R):(M−1 min 16261−1) CatCat B: B: 10(R,R) 76(S,S) Irreversible(In vitro) Schirmeister,1999 [115] 13 aziridinesN-unsubstituted (type II) (R,R):(M −1 min 16261−1) CatCat B: B: 10(R,R) 76(S,S) Irreversible(In vitro) Schirmeister,1999 [115] 13 aziridinesN-unsubstituted (type II) (R,R):(M min16261) Cat B: 76(S,S)10(R,R) Irreversible(In vitro) Schirmeister,1999 [115] 13 aziridines (type II) (R,R):(S,S):(S,S): 16261 31303130 Cat B: 76(S,S) (In vitro) 1999 [115] aziridines (type II) (R,R):(S,S): 162613130 Cat B: 76(S,S) (In vitro) 1999 [115] (S,S): 3130 (S,S): 3130

2nd Order N-acylated 2nd Order N-acylated 2nd−1 Order−1 N-acylated 2nd(M−1 Ordermin−1) Cat B: 3(R,R) Irreversible Schirmeister, 14 N-acylatedbispeptidyl 2nd(M−1 Ordermin−1) Cat B: 3(R,R) Irreversible Schirmeister, 14 N-acylatedbispeptidyl (M2nd−1 minOrder−1) Cat B: 3(R,R) Irreversible Schirmeister, 14 bispeptidylN-acylated (M(R,R):(R,R):−1 min 58965896−1) CatCat B:B: 3(R,R)3(S,S) Irreversible(In(In vitro)vitro) Schirmeister,1999 [115] 14 aziridinesbispeptidyl (type III) (M(R,R):−1 min 5896−1) CatCat B:B: 3(R,R)3(S,S) Irreversible(In vitro) Schirmeister,1999 [115] 14 aziridinesbispeptidyl (type III) (R,R):(S,S): 58961210 Cat B: 3(S,S) (In vitro) 1999 [115] 14 aziridinesbispeptidyl (type III) (R,R):(S,S): 58961210 Cat B: 3(S,S) (In vitro) 1999 [115] aziridines (type III) (R,R):(S,S): 12105896 Cat B: 3(S,S) (In vitro) 1999 [115] aziridines (type III) (S,S): 1210 (S,S): 1210

Molecules 2020, 25, 698 9 of 40 Molecules 2020, 25, 698 9 of 40

2nd Order Peptidyldiazometha 2nd Order Cat B: 41 Irreversible Crawford et al., Peptidyldiazometha 2nd −Order1 −1 Cat B: 41 Irreversible Crawford et al., 4 Peptidyldiazometha (M−1 s−1) Cat B: 41 Irreversible Crawford et al., 4 ne (M−1 s−1) Calpain: NI (In vitro) 1988 [103] ne 1128000 Calpain: NI (In vitro) 1988 [103] 1128000

Peptidylchlorometha Peptidylchlorometha 2nd Order Peptidylchloromethane 2nd Order Cat B: 113 Irreversible Crawford et al., ne 2nd −Order1 −1 Cat B: 113 Irreversible Crawford et al., 5 ne (M−1 s−1) Cat B: 113 Irreversible Crawford et al., 5 (chloromethylketone (M−1 s−1) Calpain: <200 (In vitro) 1988 [103] 5 (chloromethylketone 21500000(M s ) Calpain: <200 (In vitro) 1988 [103] (chloromethylketone) 21500000 Calpain: <200 (In vitro) 1988 [103] ) 21500000 )

2nd Order Cat B: 436 N-peptidyl-O-acyl 2nd Order Cat B: 436 Irreversible Bromme et al., N-peptidyl-O-acyl 2nd −Order1 −1 Cat B: 436 Irreversible Bromme et al., 6 N-peptidyl-O-acyl (M−1 s−1) Cat S: 58 Irreversible Bromme et al., 6 hydroxylamines (M−1 s−1) Cat S: 58 (In vitro) 1989 [108] hydroxylamines 1222000 Cat H: ND (In vitro) 1989 [108] 1222000 Cat H: ND

N-peptidyl-O- 2nd Order Cat B: 59 N-peptidyl-O- 2nd Order Cat B: 59 Irreversible Bromme et al., N-peptidyl-O- 2nd −Order1 −1 Cat B: 59 Irreversible Bromme et al., 7 carbamoyl amino (M−1 s−1) Cat S: 10 Irreversible Bromme et al., 7 carbamoyl amino (M−1 s−1) Cat S: 10 (In vitro) 1993 [110] acid hydroxamates 931800 Papain: 184 (In vitro) 1993 [110] acid hydroxamates 931800 Papain: 184

Cat S: 7 2nd Order Cat S: 7 N-peptidyl-O-acyl 2nd Order CatCat B: S: 101 7 Irreversible Bromme et al., N-peptidyl-O-acyl 2nd −Order1 −1 Cat B: 101 Irreversible Bromme et al., 8 N-peptidyl-O-acyl (M−1 s−1) Cat B: 101 Irreversible Bromme et al., 8 hydroxamates (M−1 s−1) Cat H: 4655 (In vitro) 1993 [109] 8 hydroxamates 3538000(M s ) Cat H: 4655 (In vitro) 1993 [109] hydroxamates 3538000 CatSP H: a: 4655II (In vitro) 1993 [109] 3538000 SP a: II SP a: II

2nd Order Peptidyl 2nd Order Cat S: 7 Irreversible Krantz, 1994 Peptidyl 2nd −Order1 −1 Cat S: 7 Irreversible Krantz, 1994 9 Peptidyl (M−1 s−1) Cat S: 7 Irreversible Krantz, 1994 9 (acyloxy)methanes (M−1 s−1) Cat B: 4 (In vitro) [112] (acyloxy)methanes 10700000 Cat B: 4 (In vitro) [112] 10700000

Peptidyl 2nd Order Peptidyl 2nd Order Irreversible Torkar et al., Peptidyl 2nd −Order1 −1 Irreversible Torkar et al., 10 acyloxymethyl (M−1 s−1) Cat B: 11 Irreversible Torkar et al., 10 acyloxymethyl (M−1 s−1) Cat B: 11 (In vitro) 2013 [113] ketone 875000 (In vitro) 2013 [113] Molecules 2020, 25, 698 ketone 875000 10 of 41

2nd Order 2nd Order Table 2. Cont. 2nd −Order1 −1 (M−1 s−1) Cat B ~ 2(L) Irreversible Martichonok et 11 Peptidyl aziridine (M−1 s−1) Cat B ~ 2(L) Irreversible Martichonok et 11 Peptidyl aziridine (L)(M ~ 20000 s ) Cat B ~ 2(D)2(L) Irreversible(In vitro) Martichonokal., 1995 [114] et 11 Peptidyl aziridine (L) ~ 20000 Cat B ~ 2(D) (In vitro) al., 1995 [114]Mechanism of (D)(L) ~~ 11000020000Inhibitor ECatﬃcacy B ~ 2(D) (In vitro) al., 1995 [114] # Chemical Structure Inhibitor Class (D) ~ 110000 Selectivity Factor # Inhibition References (D) ~ 110000(Cathepsin L) (Demonstrated Utilities) 2nd Order 2nd Order 2nd−1 Order−1 N-acylated (M−1 min−1) Cat B:1 13 Irreversible Schirmeister, 12 N-acylated (M−1 min2nd−1) Order (MCat− B: 13 Irreversible Schirmeister, 12 aziridinesN-acylated (type aziridines I) (S,S+R,R): 1Papain: 22 (InCat vitro) B: 13 1999 [115] Irreversible Schirmeister, 1999 12 12 aziridines (type I) (S,S+R,R): min− Papain:) 22 (In vitro) 1999 [115] aziridines(type (type I) I) (S,S+R,R):3227 Papain: 22 Papain:(In vitro) 22 1999 [115] (In vitro) [115] 3227(S,S +R,R): 3227

2nd Order 2nd Order2nd Order (M 1 2nd−1 Order−1 − N-unsubstituted (M−1 min−1) Cat1 B: 10(R,R) Irreversible Schirmeister, 13 N-unsubstitutedN-unsubstituted (M−1 min−1) minCat) B: 10(R,R) CatIrreversible B: 10(R,R) Schirmeister,Irreversible Schirmeister, 1999 13 13 aziridinesN-unsubstituted (type II) (R,R):(M min 16261) −Cat B: 10(R,R)76(S,S) Irreversible(In vitro) Schirmeister,1999 [115] 13 aziridinesaziridines (type (type II) II) (R,R): 16261(R,R): 16261Cat B: 76(S,S) Cat(In B: vitro) 76(S,S) 1999 [115] (In vitro) [115] aziridines (type II) (R,R):(S,S): 162613130 Cat B: 76(S,S) (In vitro) 1999 [115] (S,S): 3130(S,S): 3130

2nd Order 1 N-acylated 2nd Order2nd Order (M− N-acylatedN-acylated 2nd−1 Order−1 1 N-acylated (M−1 min−1) min Cat) B: 3(R,R) CatIrreversible B: 3(R,R) Schirmeister,Irreversible Schirmeister, 1999 14 bispeptidyl (M−1 min−1) −Cat B: 3(R,R) Irreversible Schirmeister, 14 14 bispeptidylbispeptidyl aziridines (M(R,R): min 5896) Cat B: 3(R,R)3(S,S) Irreversible(In vitro) Schirmeister,1999 [115] 14 aziridinesbispeptidyl (type III) (R,R): 5896(R,R): 5896Cat B: 3(S,S) Cat(In B:vitro) 3(S,S) 1999 [115] (In vitro) [115] MoleculesMolecules 2020 2020, ,25 25, ,698 698 aziridines(type (type III) III) (R,R):(S,S): 12105896 Cat B: 3(S,S) (In vitro) 10101999 of of 40 40 [115] aziridines (type III) (S,S): 1210(S,S): 1210 (S,S): 1210

PeptidylPeptidylPeptidyl aryl aryl arylvinyl vinyl vinyl ICIC5050 (nM) (nM) IC (nM) IrreversibleIrreversible MendietaMendieta et et al., al.,Irreversible Mendieta et al., 2010 15 1515 50 CatCat B: B: 404 404 Cat B: 404 sulfonessulfonessulfones 2.62.6 2.6 (In(In vitro) vitro) 20102010 [116] [116] (In vitro) [116]

CatCat K: K: 100 100 Cat K: 100 CatCat B: B: 44000 44000 Cat B: 44000 CatCat S: S: 13 13 Cat S: 13 2nd2nd Order Order IrreversibleIrreversible PeptidylPeptidylPeptidyl 2nd Order (MCatCat1 H: sH: NI1 NI) Cat H: NI DanaDana et et al., al., Irreversible 16 1616 (M(M−−11 s s−−11)) − − (In(In vitro vitro Dana et al., 2014 [117] arylvinylsulfonatearylvinylsulfonatearylvinylsulfonate CatCat D: D: NI NI Cat D: NI 20142014 [117] [117](In vitro cellular) 43000004300000 4300000 cellular)cellular) CatCat G: G: NI NI Cat G: NI hPTP1B:hPTP1B: NI NI hPTP1B: NI Trypsin:Trypsin: NI NI Trypsin: NI

2nd2nd Order Order IrreversibleIrreversible GallinamideGallinamide A- A- BoudreauBoudreau et et al., al., 1717 (M(M−−11 s s−−11)) NDND (In(In vitro vitro analoganalog 20192019 [118] [118] 87300008730000 cellular)cellular)

CovalentCovalent and and ICIC5050 (nM) (nM) LynasLynas et et al., al., 1818 PeptidylPeptidyl aldehydes aldehydes CatCat B: B: 357 357 reversiblereversible 0.60.6 20002000 [119] [119] (In(In vitro) vitro)

CatCat K>20000 K>20000 CovalentCovalent and and Azepanone-basedAzepanone-based KKi,iapp,app (nM) (nM) MarquisMarquis et et al., al., 1919 CatCat S: S: 36 36 reversiblereversible InhibitorsInhibitors 0.430.43 20052005 [120] [120] CatCat B: B: 349 349 (In(In vitro) vitro)

CovalentCovalent and and NitrileNitrile group group ICIC5050 (nM) (nM) HardeggerHardegger et et 2020 NANA reversiblereversible containingcontaining inhibitor inhibitor 2222 al.,al., 2011 2011 [121] [121] (In(In vitro) vitro)

CovalentCovalent and and NitrileNitrile group group KiKi (nM) (nM) GiroudGiroud et et al., al., 2121 NDND reversiblereversible containingcontaining inhibitor inhibitor 44 20172017 [122] [122] (In(In vitro) vitro)

CovalentCovalent and and NitrileNitrile group group KKi i(nM) (nM) KuhnKuhn et et al., al., 2222 NDND reversiblereversible containingcontaining inhibitor inhibitor 1212 20172017 [123] [123] (In(In vitro) vitro)

ParkerParker et et al., al., 20172017 [124], [124], CovalentCovalent and and ParkerParker et et al., al., ICIC5050 (nM) (nM) reversiblereversible 20152015 [125] [125] 2323 ThiosemicarbazoneThiosemicarbazone 189189 (active (active CatCat B: B: >50 >50 (In(In vitro vitro KumarKumar et et al., al., inhibitor)inhibitor) b b cellular)cellular) 20102010 [126] [126] KumarKumar et et al., al., 20102010 [127] [127]

Molecules 2020, 25, 698 10 of 40 Molecules 2020, 25, 698 10 of 40 Molecules 2020, 25, 698 10 of 40 Molecules 2020,, 2525,, 698698 10 of 40

Peptidyl aryl vinyl IC50 (nM) Irreversible Mendieta et al., 15 Peptidyl aryl vinyl IC50 (nM) Cat B: 404 Irreversible Mendieta et al., Peptidyl aryl vinyl IC50 (nM) Irreversible Mendieta et al., 15 Peptidylsulfones aryl vinyl IC50 2.6(nM) Cat B: 404 Irreversible(In vitro) Mendieta2010 [116] et al., 15 Peptidylsulfones aryl vinyl IC50 2.6(nM) Cat B: 404 Irreversible(In vitro) Mendieta2010 [116] et al., 15 sulfones 2.6 Cat B: 404 (In vitro) 2010 [116] sulfones 2.6 (In(In vitro)vitro) 2010 [116]

Cat K: 100 Cat K: 100 CatCat B: K: 44000 100 Molecules 2020, 25, 698 CatCat B:K: 44000100 11 of 41 CatCat B: S:44000 13 2nd Order CatCat B: S:44000 13 Irreversible Peptidyl 2nd Order Cat H:S: 13NI Irreversible Dana et al., Peptidyl 2nd −Order1 −1 Cat S:H: 13 NI Irreversible Dana et al., 16 Peptidyl 2nd(M Order−1 s−1) Cat H: NI Irreversible(In vitro Dana et al., 16 arylvinylsulfonatePeptidyl 2nd(M −Order1 s−1 ) CatCat H: D: NI NI Irreversible(In vitro Dana2014 et[117] al., 1616 arylvinylsulfonatePeptidyl (M(M4300000−1 ss−1)) TableCatCat 2. ContH: D: NINI. (In(Incellular) vitrovitro Dana2014 et[117] al., 16 arylvinylsulfonatearylvinylsulfonate (M4300000−1 s−1) CatCat D:D:G: NINI (Incellular) vitro 20142014 [117][117] arylvinylsulfonate 43000004300000 CatCat D: G: NINI cellular)cellular) 2014 [117] 4300000 hPTP1B:Cat G: NI NI cellular) hPTP1B:Cat G: NI NI Mechanism of Inhibitor EhPTP1B:Trypsin:ﬃcacy NI # Chemical Structure Inhibitor Class hPTP1B:Trypsin: NI NI Selectivity Factor # Inhibition References (CathepsinTrypsin: L) NI Trypsin: NI (Demonstrated Utilities) 2nd Order Irreversible Gallinamide A- 2nd Order Irreversible Boudreau et al., Gallinamide A- 2nd −Order1 −1 Irreversible Boudreau et al., 17 2nd(M Order−1 s−1) ND Irreversible(In vitro 17 Gallinamide A- (M s ) ND (In vitro Boudreau et al., 17 Gallinamideanalog A- 2nd(M −Order1 s−1) 1ND 1 Irreversible(In vitro Boudreau2019 [118] et al., 17 Gallinamideanalog A- (M8730000−12nd s−1) Order (M−NDs− ) (Incellular) vitro Boudreau2019 [118] et al., Irreversible Boudreau et al., 2019 17 17 Gallinamideanaloganalog A-analog (M8730000−1 s−1) ND (Incellular) NDvitro 20192019 [118][118] analog 8730000 8730000 cellular) 2019 [118](In vitro cellular) [118] 8730000 cellular)

Covalent and IC50 (nM) Covalent and Lynas et al., 18 Peptidyl aldehydes IC50 (nM) Cat B: 357 Covalentreversible and Lynas et al., 18 Peptidyl aldehydes IC50 (nM) Cat B: 357 Covalentreversible and Lynas et al., IC50 0.6(nM) IC (nM) Covalent and Lynas2000Covalent et[119] al., and reversible 18 1818 PeptidylPeptidylPeptidyl aldehydesaldehydes aldehydes IC50 0.6(nM) 50 CatCat B:B: 357357 reversiblereversibleCat(In vitro) B: 357 Lynas2000 et[119] al., Lynas et al., 2000 [119] 18 Peptidyl aldehydes 0.60.6 Cat B: 357 reversible(In vitro) 20002000 [119][119] 0.6 0.6 (In vitro) 2000 [119] (In vitro) (In(In vitro)vitro)

Cat K>20000 Covalent and Azepanone-based Ki,app (nM) Cat K>20000 Covalent and Marquis et al., 19 Azepanone-based Ki,app (nM) CatCat K>20000 S: 36 CovalentCatreversible K>20000 and Marquis et al., Azepanone-based Ki,app (nM) Cat K>20000 Covalent and Marquis et al., 19 Azepanone-basedAzepanone-basedInhibitors Ki,app0.43 (nM) Ki,app (nM)CatCat K>20000 S: 36 Covalentreversible and Marquis2005Covalent [120] et al., and reversible Marquis et al., 2005 19 19 Azepanone-basedInhibitors Ki,app0.43 (nM) CatCat B:S: 34936 reversible(InCat vitro) S: 36 Marquis2005 [120] et al., 19 InhibitorsInhibitors 0.43 0.43 Cat S:B: 36349 reversible(In vitro) 2005 [120] (In vitro) [120] InhibitorsInhibitors 0.43 Cat B: 349 Cat(In vitro) B: 349 2005 [120] Cat B: 349 (In(In vitro)vitro)

Covalent and Nitrile group IC50 (nM) Covalent and Hardegger et 20 NitrileNitrile group group IC50 (nM) IC (nM) NA Covalentreversible and HardeggerCovalent et and reversible Hardegger et al., 2011 Nitrile group IC50 (nM) 50 Covalent and Hardegger et 20 20 containingNitrile group inhibitor IC50 22(nM) NA CovalentreversibleNA and Hardeggeral., 2011 [121] et 20 containingcontainingNitrile group inhibitor inhibitor IC50 22(nM) 22 NA reversible(In vitro) Hardeggeral., 2011 [121] et (In vitro) [121] 20 containing inhibitor 22 NA reversible(In vitro) al., 2011 [121] containing inhibitor 22 (In vitro) al., 2011 [121] (In(In vitro)vitro)

Covalent and NitrileNitrile group group Ki (nM) Ki (nM) Covalent and GiroudCovalent et al., and reversible Giroud et al., 2017 21 21 Nitrile group Ki (nM) ND CovalentCovalentreversibleND andand Giroud et al., 21 containingcontainingNitrileNitrile groupgroup inhibitor inhibitor KiKi (nM)(nM)4 4 ND Covalentreversible and GiroudGiroud2017 [122] etet al.,al., (In vitro) [122] 2121 containingNitrile group inhibitor Ki (nM)4 NDND reversiblereversible(In vitro) Giroud2017 [122] et al., 21 containingcontaining inhibitorinhibitor 44 ND reversible(In vitro) 20172017 [122][122] containing inhibitor 4 (In(In vitro)vitro) 2017 [122] (In vitro)

Covalent and Nitrile group Ki (nM) Covalent and Kuhn et al., 22 Nitrile group Ki (nM) ND Covalentreversible and Kuhn et al., Nitrile group Ki (nM) Covalent and Kuhn et al., 22 containingNitrile group inhibitor Ki (nM)12 ND Covalentreversible and Kuhn2017 et[123] al., 22 containingNitrile group inhibitor Ki (nM)12 ND reversible(In vitro) Kuhn2017 et[123] al., 22 containing inhibitor 12 ND reversible(In vitro) 2017 [123] 22 containing inhibitor 12 ND reversible 2017 [123] containing inhibitor 12 (In(In vitro)vitro) 2017 [123] (In vitro) Parker et al., Parker et al., Parker2017 [124], et al., Parker2017 [124],et al., Covalent and Parker20172017 [124],[124], et al., IC50 (nM) Covalent and Parker2017 [124], et al., IC50 (nM) Covalentreversible and Parker2015 [125] et al., IC50 (nM) Covalent and Parker et al., 23 Thiosemicarbazone IC18950 (active(nM) Cat B: >50 Covalentreversible and Parker2015 [125]et al., 23 Thiosemicarbazone 189IC50 (active(nM) Cat B: >50 reversible(In vitro Kumar2015 [125] et al., 23 Thiosemicarbazone 189 (activeb Cat B: >50 reversible(In vitro Kumar2015 [125] et al., 23 Thiosemicarbazone 189inhibitor) (active b Cat B: >50 (In vitro Kumar et al., 23 Thiosemicarbazone 189inhibitor) (active b Cat B: >50 (Incellular) vitro Kumar2010 [126]et al., inhibitor)inhibitor) b (Incellular) vitro Kumar2010 [126]et al., inhibitor) b cellular) Kumar2010 [126] et al., cellular) Kumar2010 [126] et al., Kumar2010 [127] et al., Kumar2010 [127]et al., Kumar2010 [127] et al., 2010 [127]

Molecules 2020,, 2525,, 698698 10 of 40

Peptidyl aryl vinyl IC5050 (nM)(nM) Irreversible Mendieta et al., 15 Cat B: 404 sulfones 2.6 (In vitro) 2010 [116]

Cat K: 100 Cat B: 44000 Cat S: 13 2nd Order Irreversible Peptidyl Cat H: NI Dana et al., 16 (M−−11 ss−−11) (In vitro arylvinylsulfonate Cat D: NI 2014 [117] 4300000 cellular) Cat G: NI hPTP1B: NI Trypsin: NI

2nd Order Irreversible Gallinamide A- Boudreau et al., 17 (M−−11 ss−−11) ND (In vitro analog 2019 [118] 8730000 cellular)

Covalent and IC5050 (nM)(nM) Lynas et al., 18 Peptidyl aldehydes Cat B: 357 reversible 0.6 2000 [119] (In vitro)

Cat K>20000 Covalent and Azepanone-based Kii,,appapp (nM)(nM) Marquis et al., 19 Cat S: 36 reversible Inhibitors 0.43 2005 [120] Cat B: 349 (In vitro)

Covalent and Nitrile group IC5050 (nM)(nM) Hardegger et 20 NA reversible containing inhibitor 22 al., 2011 [121] (In vitro)

Molecules 2020, 25, 698 12 of 41

Table 2. Cont. Covalent and Nitrile group Ki (nM) Giroud et al., 21 ND reversible containing inhibitor 4 2017 [122] (In vitro) Mechanism of Inhibitor Eﬃcacy # Chemical Structure Inhibitor Class Selectivity Factor # Inhibition References (Cathepsin L) (Demonstrated Utilities)

Covalent and NitrileNitrile group group Kii (nM)(nM) K (nM) KuhnCovalent et al., and reversible 22 22 i ND reversibleND Kuhn et al., 2017 [123] containingcontaining inhibitor inhibitor 12 12 2017 [123] (In vitro) (In vitro)

Parker et al., Parker et al., 2017 2017 [124], [124], Covalent and Parker et al., Parker et al., 2015 IC5050 (nM)(nM) reversible 2015 [125] 23 Thiosemicarbazone 189 (activeIC 50 (nM)Cat B: >50 Covalent and reversible [125] 23 Thiosemicarbazone b Cat(In vitro B: > 50 Kumar et al., inhibitor)inhibitor)189 (active b inhibitor) (In vitro cellular) Kumar et al., 2010 Molecules 2020, 25, 698 cellular) 112010 of 40 [126] [126] MoleculesMolecules 2020 2020, ,25 25, ,698 698 1111 of of 40 40 Molecules 2020, 25, 698 Kumar11 of 40 et al., Kumar et al., 2010 2010 [127] [127]

Chowdhury et ChowdhuryChowdhury et et Cat L propeptide IC50 (nM) Cat K: 310 Reversible Chowdhuryal., 2002 [95] et Chowdhury et al., 24 Cat L propeptide IC50 (nM) Cat K: 310 Reversible al., 2002 [95] 24 CatCat L L propeptide propeptide ICIC5050 (nM) (nM) CatCat K: K: 310 310 ReversibleReversible al.,al., 2002 2002 [95] [95] 2424 Catmimicmimic L propeptide 1919 IC50 (nM)CatCat B:B: 210210 (InCat(In vitro)vitro) K: 310 ShenoyShenoy etet al.,al.,Reversible 2002 [95] 24 mimicmimic 1919 CatCat B: B: 210 210 (In(In vitro) vitro) ShenoyShenoy et et al., al., mimic 19 Cat B: 210 20092009 [128][128] (In vitro) Shenoy et al., 2009 20092009 [128] [128] [128]

Cat V: 11 CatCat V: V: 11 11 Cat V: 11 IC50 (nM) * CatCat S:V: 14 11 Reversible Myers et al., 25 Thiocarbazate IC50 (nM) * Cat S: 14 Reversible Myers et al., 25 Thiocarbazate ICIC5050 (nM) (nM) * IC* 50 (nM)CatCat * S: S: 14 14 ReversibleReversibleCat S: 14 MyersMyers et et al., al., Reversible Myers et al., 2008 25 2525 ThiocarbazateThiocarbazateThiocarbazate 11 CatCat B:B: 5050 (In(In vitro)vitro) 20082008 [129–131][129–131] 11 1 CatCat B: B: 50 50 (In(InCat vitro) vitro) B: 50 20082008 [129–131] [129–131](In vitro) [129–131] CatCat K:K: 137137 CatCat K: K: 137 137 Cat K: 137

Myers et al., Reversible MyersMyers et et al., al., IC50 (nM) * Reversible Myers2008 [130] et al., Myers et al., 2008 IC50 (nM) * ReversibleReversible 2008 [130] 26 Oxocarbazate IC50 (nM) *IC 50 (nM)Cat * B: 714 (In vitro 2008 [130] Reversible 26 26 OxocarbazateOxocarbazate IC50 (nM) * Cat B: 714 Cat(In vitro B: 714 2008 [130] [130] 2626 OxocarbazateOxocarbazate 0.40.4 0.4 CatCat B: B: 714 714 (In(In vitro vitro ShahShah etet al.,al.,(In 20102010 vitro cellular) 0.40.4 cellular)cellular) ShahShah et et al., al., 2010 2010 Shah et al., 2010 [132] cellular)cellular) [132][132] [132][132]

IC50 (µM) IC (µM) Reversible Myers et al., Reversible Myers et al., 2008 27 Aza-peptide IC50 (µM) 50 ND Reversible Myers et al., 27 27 Aza-peptideAza-peptide ICIC5050 (µM) (µM) ND ReversibleReversibleND MyersMyers et et al., al., 2727 Aza-peptideAza-peptide 3.03.0 3.0 NDND (In(In vitro)vitro) 20082008 [130][130] (In vitro) [130] 3.03.0 (In(In vitro) vitro) 20082008 [130] [130]

# a Fractions were not# considered while calculating selectivity factor; Inhibitory eﬃcacy has been reported in whole numbers only; : Serine Proteases; II: Insigniﬁcant Inhibition; ND: Not # Fractions were not considered while calculating selectivity factor; Inhibitory efficacy has been b ## FractionsFractions werewere notnot consideredconsidered whilewhile calculatingcalculating selectivityselectivity factor;factor; InhibitoryInhibitory efficacyefficacy hashas beenbeen Determined; ActiveFractions form of inhibitor were not is devoid considered of thea phosphatewhile calculating group; * Preincubationselectivity factor; for 4 h. Inhibitory efficacy has been reported in whole numbers only; a: Serine Proteases; II: Insignificant Inhibition; ND: Not Determined; reportedreported inin wholewhole numbersnumbers only;only; a a:: Serine Serine Proteases; Proteases; II: II: Insignificant Insignificant Inhibition; Inhibition; ND: ND: NotNot Determined;Determined; breported in whole numbers only; : Serine Proteases; II: Insignificant Inhibition; ND: Not Determined; bActive form of inhibitor is devoid of the phosphate group; * Preincubation for 4 h. b Active form of inhibitor is devoid of the phosphate group; * Preincubation for 4 h. bActive Active form form of of inhibitor inhibitor is is devoid devoid of of the the phosphate phosphate group; group; * * Preincubation Preincubation for for 4 4 h. h. 3.4.3.4. PeptidylPeptidyl Acyloxymethanes/AcyloxymethylAcyloxymethanes/Acyloxymethyl KetonesKetones 3.4.3.4. PeptidylPeptidyl Acyloxymethanes/AcyloxymethylAcyloxymethanes/Acyloxymethyl KetonesKetones AnotherAnother classclass ofof peptidylpeptidyl inhibitorsinhibitors thatthat spansspans ththee activeactive sitesite andand utilizesutilizes thethe catalyticcatalytic machinerymachinery AnotherAnother class class of of peptidyl peptidyl inhibitors inhibitors that that spans spans th thee active active site site and and utilizes utilizes the the catalytic catalytic machinery machinery ofof cathepsincathepsin LL forfor effectiveeffective attenuationattenuation ofof enzymeenzyme acactivitytivity isis peptidylpeptidyl acyloxymethanes.acyloxymethanes. KrantzKrantz etet al.al. ofof cathepsincathepsin LL forfor effectiveeffective attenuationattenuation ofof enzymeenzyme acactivitytivity isis peptidylpeptidyl acyloxymethanes.acyloxymethanes. KrantzKrantz etet al.al. 2 developeddeveloped aa librarylibrary ofof inhibitoryinhibitory compoundscompounds withwith aa generalgeneral sequence,sequence, Z-Phe-X-CHZ-Phe-X-CH2OCO-R;OCO-R; herehere developeddeveloped aa librarylibrary ofof inhibitoryinhibitory compoundscompounds withwith aa generalgeneral sequence,sequence, Z-Phe-X-CHZ-Phe-X-CH22OCO-R;OCO-R; herehere theythey systematicallysystematically variedvaried thethe aminoamino acidacid residueresidue atat P1P1 (denoted(denoted asas X)X) andand P1P1′′ (denoted(denoted asas R)R) theythey systematicallysystematically variedvaried thethe aminoamino acidacid residueresidue atat P1P1 (denoted(denoted asas X)X) andand P1P1′ ′ (denoted(denoted asas R)R) 2 3 2 positionspositions [112].[112]. AmongAmong thethe synthesisynthesizedzed compounds,compounds, Z-Phe-Cys(SBn)-CHZ-Phe-Cys(SBn)-CH2OCO-2,6-(CFOCO-2,6-(CF3))2-Ph-Ph (Entry(Entry positionspositions [112].[112]. AmongAmong thethe synthesisynthesizedzed compounds,compounds, Z-Phe-Cys(SBn)-CHZ-Phe-Cys(SBn)-CH22OCO-2,6-(CFOCO-2,6-(CF33))22-Ph-Ph (Entry(Entry 9,9, TableTable 2)2) exhibitedexhibited almostalmost aa diffusion-controlleddiffusion-controlled inactivationinactivation kineticskinetics towardtoward cathepsincathepsin L,L, howeverhowever 9,9, TableTable 2)2) exhibitedexhibited almostalmost aa diffusion-controlleddiffusion-controlled inactivationinactivation kineticskinetics towardtoward cathepsincathepsin L,L, howeverhowever itit showedshowed onlyonly marginalmarginal selectivityselectivity overover cathepsincathepsin BB andand cathepsincathepsin S.S. ByBy analyzinganalyzing thethe structure-structure- itit showedshowed onlyonly marginalmarginal selectivityselectivity overover cathepsincathepsin BB andand cathepsincathepsin S.S. ByBy analyzinganalyzing thethe structure-structure- activityactivity relationshipsrelationships ofof thethe library,library, thethe authorauthor elucidatedelucidated thethe importanceimportance ofof S1S1′′ sitesite inin cathepsincathepsin LL activityactivity relationshipsrelationships ofof thethe library,library, thethe authorauthor elucidatedelucidated thethe importanceimportance ofof S1S1′ ′ sitesite inin cathepsincathepsin LL thatthat couldcould potentiallypotentially bebe usedused toto harnessharness selectivityselectivity amongamong otherother cysteinecysteine cathepsins.cathepsins. InIn aa separateseparate thatthat couldcould potentiallypotentially bebe usedused toto harnessharness selectivityselectivity amongamong otherother cysteinecysteine cathepsins.cathepsins. InIn aa separateseparate study,study, TorkarTorkar etet al.al. designeddesigned aa librarylibrary ofof peptidespeptides forfor cathepsincathepsin LL thatthat spannedspanned thethe activeactive sitesite andand study,study, TorkarTorkar etet al.al. designeddesigned aa librarylibrary ofof peptidespeptides forfor cathepsincathepsin LL thatthat spannedspanned thethe activeactive sitesite andand attenuatedattenuated thethe enzymeenzyme activityactivity viavia aa non-covalentnon-covalent interactioninteraction [113].[113]. TheyThey initiallyinitially evaluatedevaluated thethe attenuatedattenuated thethe enzymeenzyme activityactivity viavia aa non-covalentnon-covalent interactioninteraction [113].[113]. TheyThey initiallyinitially evaluatedevaluated thethe compounds’compounds’ activitiesactivities againstagainst cathepsincathepsin LL andand cathepsincathepsin B,B, andand comparedcompared theirtheir hitshits againstagainst cathepsincathepsin compounds’compounds’ activities activities against against cathepsin cathepsin L L and and cathepsin cathepsin B, B, and and compared compared their their hits hits against against cathepsin cathepsin KK andand S.S. TheThe authorsauthors discovereddiscovered fivefive mostmost selectiveselective non-covalent,non-covalent, peptidylpeptidyl inhibitorsinhibitors ofof cathepsincathepsin L,L, KK andand S.S. TheThe authorsauthors discovereddiscovered fivefive mostmost selectiveselective non-covalent,non-covalent, peptidylpeptidyl inhibitorsinhibitors ofof cathepsincathepsin L,L, andand transformedtransformed themthem intointo irreversibleirreversible inhibitorsinhibitors byby strategicallystrategically appendingappending electrophilicelectrophilic warheadwarhead andand transformedtransformed themthem intointo irreversibleirreversible inhibitorsinhibitors byby strategicallystrategically appendingappending electrophilicelectrophilic warheadwarhead (Figure(Figure 6)—acyloxymethyl6)—acyloxymethyl ketoneketone (AOMK)(AOMK) groupsgroups (Entry(Entry 10,10, TableTable 2).2). However,However, thethe attachmentattachment ofof (Figure(Figure 6)—acyloxymethyl6)—acyloxymethyl ketoneketone (AOMK)(AOMK) groupsgroups (Entry(Entry 10,10, TableTable 2).2). However,However, thethe attachmentattachment ofof thethe AOMKAOMK groupgroup drasticallydrastically impactedimpacted thethe selectivitselectivityy profilesprofiles ofof thesethese inhiinhibitors,bitors, suggestingsuggesting thethe thethe AOMKAOMK groupgroup drasticallydrastically impactedimpacted thethe selectivitselectivityy profilesprofiles ofof thesethese inhiinhibitors,bitors, suggestingsuggesting thethe importanceimportance ofof thethe adjuvantadjuvant effecteffect ofof primeprime sitesite targetingtargeting inin determiningdetermining thethe efficacyefficacy andand selectivityselectivity ofof importanceimportance of of the the adjuvant adjuvant effect effect of of prime prime site site targeting targeting in in determining determining the the efficacy efficacy and and selectivity selectivity of of thisthis classclass ofof compounds.compounds. ThisThis classclass ofof inhibitorsinhibitors foundfound wide-spreadwide-spread utilitiesutilities inin detectingdetecting proteaseprotease thisthis classclass ofof compounds.compounds. ThisThis classclass ofof inhibitorsinhibitors foundfound wide-spreadwide-spread utilitiesutilities inin detectingdetecting proteaseprotease activityactivity andand werewere utilizedutilized toto developdevelop activity-basedactivity-based probe;probe; thisthis willwill bebe discusseddiscussed inin thethe laterlater sections.sections. activityactivity and and were were utilized utilized to to develop develop activity-based activity-based probe;probe; this this will will be be discussed discussed in in the the later later sections. sections.

Molecules 2020, 25, 698 13 of 41 Molecules 2020, 25, 698 8 of 40

Figure 5. Postulated active-site interactions between pa papain,pain, a prototypical cysteine protease, and peptidyl hydroxamatehydroxamate inhibitor.inhibitor. Active-site Active-site cysteine cysteine forms forms a a covalent covalent bond bond with with the the carbonyl carbonyl group group of theof the inhibitor inhibitor and and the negativelythe negatively charged charged nitrogen nitrog engagesen engages in electrostatic in electrostatic interaction interaction with the positivelywith the chargedpositively histidine charged residue. histidine residue.

In a follow-up study, BrommeBromme etet al.al. synthesizedsynthesized and tested a series of newnew inhibitors,inhibitors, with the general formula of Z-Phe-Gly-NHO-CO-Aa (Aa: amino amino acid), acid), against against papain papain class class of of enzymes enzymes [110]. [110]. This class class of of inhibitors inhibitors covalently covalently modified modified active-site active-site Cys Cys residue residue via sulfenamidation, via sulfenamidation, like in like their in theirN-peptidyl-N-peptidyl-O-acylO hydroxamate-acyl hydroxamate counterparts, counterparts, as shown as shown by the bymass the spectrometric mass spectrometric analysis. analysis. These Theseinhibitors inhibitors further further provided provided the option the option of varying of varying the theleaving leaving group group that that targets targets S2 S2′ 0 site;site; more hydrophobic substituentssubstituents were we preferredre preferred at this at positionthis position as comparison as comparison of ki/Ki values of ki/K ofi thevalues inactivation of the exhibitedinactivation the exhibited following the trend following for Aa: trend Gly Cathepsin Z-Phe-Cys(SBn)-CH L) OCO-2,6-(CF ) -Ph (Entry 9, 2 Utilities)3 2 Table2) exhibited almost a di ffusion-controlled inactivation kinetics toward cathepsin L, however it showed only marginal selectivity over cathepsin B and cathepsin S. By analyzing the structure-activity relationships of the library, the author elucidated the2nd Order importance of S1 site in cathepsin L that could Cat B: 1.10 Irreversible Barrett et al., 1 Epoxy Succinyl (M−1 s−1) potentially be used to harness selectivity among other cysteine cathepsins.Cat H: 24 In a separate(In vitro)study, Torkar1982 [99] 96250 et al. designed a library of peptides for cathepsin L that spanned the active site and attenuated the enzyme activity via a non-covalent interaction [113]. They initially evaluated the compounds’ activities against cathepsin L and cathepsin B, and compared their hits against cathepsin K and S. The authors discovered five most selective non-covalent, peptidyl inhibitors of cathepsin L, and transformed them 2nd Order (103 into irreversible inhibitors by strategically appending electrophilic warhead (FigureIrreversible6)—acyloxymethyl Gour-Salin et 2 Epoxy Succinyl M−1 s−1) Cat S: 89 (In vitro) al., 1994 [111] ketone (AOMK) groups (Entry 10, Table2). However,73 the attachment of the AOMK group drastically impacted the selectivity profiles of these inhibitors, suggesting the importance of the adjuvant effect of

prime site targeting in determining the eﬃcacy and selectivity of this class of compounds. This class of inhibitors found wide-spread utilities in detecting protease activity and were utilized to develop Cat B: NI Irreversible activity-based probe; this will be discussed in the10 later−6 M (100% sections. Katunuma et 3 Epoxy Succinyl Cat S: 3 (In vitro inhibition) al.,1999 [101] Cat K: NI cellular)

Molecules 2020, 25, 698 14 of 41 Molecules 2020, 25, 698 12 of 40

Figure 6.6. A reversible cathepsin L inhibitor was transformedtransformed to an irreversible one by strategicallystrategically galvanizing an electrophilic warhead.warhead.

3.5. Peptidyl Aziridine A very interesting class of inhibitors, the concept of which was derived from E-64, is peptidylpeptidyl aziridines. Martichonok Martichonok et et al. developed developed a a series series of of aziridine derivatives of E-64 and tested themthem against papain and cathepsin L andand BB [[114].114]. Contrarily to E-64, in which (L)-diastereomer is more potent than (D)-isomer,(D)-isomer, aziridineaziridine analogsanalogs exhibited the opposite trend whilewhile stillstill inactivatinginactivating the enzymes. More More importantly, importantly, the the efficacy efficacy of ofthis this class class of inhibitors of inhibitors was was strongly strongly pH-dependent pH-dependent and andshowed showed maximal maximal inhibitory inhibitory potency potency at pH4; atthis pH4; is attributed this is attributedto the protonated to the form protonated of aziridine form ring of aziridinethat is more ring susceptible that is more to nucleophilic susceptible attack to nucleophilic by catalytic attack Cys residue. by catalytic Among Cys the residue. developed Among library, the developedHO-(D)-Az-Leu-NH-iAm library, HO-(D)-Az-Leu-NH-iAm (Entry 11, Table 2) (Entryanalog 11, exhibited Table2) analogmaximal exhibited inhibitory maximal potency inhibitory towards potencycathepsin towards L with cathepsinmoderate Lselectivity with moderate over cathepsin selectivity B; overthe corresponding cathepsin B; the L isomer, corresponding HO-(L)-Az-Leu- L isomer, HO-(NH-iAmL)-Az-Leu-NH-iAm (Entry 11) was (Entryalmost 11)10-fold was almostless activity 10-fold while less activity maintaining while maintainingthe similar trend the similar in general. trend inSince general. only protonated Since only protonated form of aziridine form of ring aziridine undergoes ring undergoes nucleophilic nucleophilic attack, presumably attack, presumably it does not it doesinvolve not involvewater molecule-mediated water molecule-mediated ring-opening ring-opening like likeits itsepoxide epoxide counterpart counterpart E-64. E-64. Although promising, aa fullfull potentialpotential ofof thisthis classclass of of compounds compounds could could presumably presumably be be achieved achieved only only below below pH pH 4 when4 when aziridine aziridine ring ring nitrogen nitrogen gets gets completely completely protonated. protonated. This This class class ofof inhibitorsinhibitors thusthus lacks practical utility asas thethe catalyticcatalytic cysteine cysteine of of cathepsin cathepsin L L starts starts to to lose lose its its activity activity below below pH pH 4 and 4 and the the majority majority of the of cell-basedthe cell-based assays assays are performedare performed mostly mostly at a pHat a higherpH higher than than 4.0. In4.0. a notableIn a notable study, study, Schirmeister Schirmeister et al. developedet al. developed three di threefferent different classes of classes inhibitors of inhibitors with aziridine-2,3-dicarboxylic with aziridine-2,3-dicarboxylic acid (Azi), anacid electrophilic (Azi), an warhead,electrophilic installed warhead, at di installedfferent positions at different of thepositi peptideons of chain the peptide (Figure 7chain)[ 115 (Figure]. They 7) performed [115]. They a thoroughperformed SAR a thorough analysis SAR of this analysis type of of inhibitors, this type of all inhibitors, off which all exhibited off which time-dependent exhibited time-dependent irreversible inactivationirreversible inactivation of cathepsin of L withcathepsin no-recovery L with ofno-r enzymeecovery activity, of enzyme even afteractivity, extensive even after dialysis extensive of the enzyme-inhibitordialysis of the enzyme-inhibitor complex. Among type-Icomplex. inhibitors, Among N-acylated type-I inhibitors, aziridines N-acylated with aziridine aziridines as C-terminal with aminoaziridine acid, as a C-terminal mixture of diastereomericamino acid, a peptides mixture with of diastereomeric procathepsin B sequencepeptides Leu-Gly-Glywith procathepsin (Entry 12,B Tablesequence2), exhibited Leu-Gly-Gly enhanced (Entry inhibition 12, Table toward 2), exhibited cathepsin enhanced L. This inhibition was attributed toward to ancathepsin overall uniqueL. This foldingwas attributed of cathepsin to an Loverall with a unique shallowness folding of of the cath S2 pocketepsin L due with to a the shallowness presence ofof anthe additional S2 pocket Met161 due to residue.the presence Type of II an class additional of inhibitors Met161 (Figure residue.7) resembleType II class classic of inhibitors aziridine sca(Figureffold, 7)N-unsubstituted resemble classic aziridinesaziridine scaffold, with aziridine N-unsubstituted as N-terminal aziridines amino with acid, aziridine analogously as N-terminal to E-64 where amino nitrogen acid, analogously of aziridine remainedto E-64 where unsubstituted. nitrogen of aziridine Among remained the type II unsu inhibitorsbstituted. tested, Among EtO-( theR type,R)-Azi-Leu-OBzl II inhibitors tested, (Entry EtO- 13, Table(R,R)-Azi-Leu-OBzl2) inactivated cathepsin (Entry 13, L Table with 2) higher inactivated second-order cathepsin rate L constantwith higher than second-order EtO-( S,S)-Azi-Leu-OBzl, rate constant althoughthan EtO-( theS,S latter)-Azi-Leu-OBzl, showed better although selectivity the latter over showed cathepsin better B. selectivity over cathepsin B. The type III inhibitor class (Figure7) is comprised of N-acylated bispeptidyl derivatives of aziridine, where aziridine ring rests in the middle of the peptide. BOC-Phe-(R,R)-(EtO)-Azi-Leu-Pro-OBzl (Entry 14, Table2) of this series was 5-fold more potent than the ( S,S) analog; however both exhibited only a marginal selectivity over cathepsin B with diminished eudysmic ratio. The authors then superimposed and analyzed the structures of certain epoxide and aziridines and postulated that these inhibitors can assume different orientations in the active site while still binding within the enzyme pockets.

Molecules 2020, 25, 698 13 of 40

Molecules 2020, 25, 698 15 of 41

This scaﬀold was further explored by Vicik et al. who extended the previous work and developed a series of compounds in which Boc-(S)-Leu-(S)-Azy-(S,S)-Azi(OBn)2 (Figure8), Type I analog, spanned from S2 to S20 pocket and inactivated cathepsin L with more than 700-fold selectivity over cathepsin B[133]. This motif was also used for aﬃnity labeling of cathepsin L, which will be discussed in the later sections. These classes of compounds, especially N-unsubstituted aziridinyl peptides and in Figure 7. Types of N-substituted and unsubstituted aziridines (color-coded for visual clarity) that special cases N-acylated ones, exhibited a high selectivity and potency and provided the premise for bind to the active site of cathepsin L and assume different orientations depending on the favorable the further development of chemical biology tools much needed for functional studies. Moleculesinteractions 2020, 25, 698 incurred by the R-groups. 13 of 40

The type III inhibitor class (Figure 7) is comprised of N-acylated bispeptidyl derivatives of aziridine, where aziridine ring rests in the middle of the peptide. BOC-Phe-(R,R)-(EtO)-Azi-Leu-Pro- OBzl (Entry 14, Table 2) of this series was 5-fold more potent than the (S,S) analog; however both exhibited only a marginal selectivity over cathepsin B with diminished eudysmic ratio. The authors then superimposed and analyzed the structures of certain epoxide and aziridines and postulated that these inhibitors can assume different orientations in the active site while still binding within the enzyme pockets. This scaffold was further explored by Vicik et al. who extended the previous work and developed a series of compounds in which Boc-(S)-Leu-(S)-Azy-(S,S)-Azi(OBn)2 (Figure 8), Type I analog, spanned from S2 to S2′ pocket and inactivated cathepsin L with more than 700-fold selectivity over cathepsin B [133]. This motif was also used for affinity labeling of cathepsin L, which will be discussed in the later sections. These classes of compounds, especially N-unsubstituted Figure 7. Types of N-substituted and unsubstitutedunsubstituted aziridines (color-coded for visual clarity) that aziridinylbind to peptides the active and site in of special cathepsin cases L andN-acylated assume didifferentones,fferent exhibited orientationsorientations a high depending selectivity on theand favorable potency and providedinteractions the premise incurred for byby thethethe R-groups.R-groups. further development of chemical biology tools much needed for functional studies. The type III inhibitor class (Figure 7) is comprised of N-acylated bispeptidyl derivatives of aziridine, where aziridine ring rests in the middle of the peptide. BOC-Phe-(R,R)-(EtO)-Azi-Leu-Pro- OBzl (Entry 14, Table 2) of this series was 5-fold more potent than the (S,S) analog; however both exhibited only a marginal selectivity over cathepsin B with diminished eudysmic ratio. The authors then superimposed and analyzed the structures of certain epoxide and aziridines and postulated that these inhibitors can assume different orientations in the active site while still binding within the enzyme pockets. This scaffold was further explored by Vicik et al. who extended the previous work and developed a series of compounds in which Boc-(S)-Leu-(S)-Azy-(S,S)-Azi(OBn)2 (Figure 8), Type I analog, spanned from S2 to S2′ pocket and inactivated cathepsin L with more than 700-fold selectivity over cathepsin B [133]. This motif was also used for affinity labeling of cathepsin L, which will be discussed in the later sections. These classes of compounds, especially N-unsubstituted aziridinylFigure peptides 8. BindingBinding and mode in specialof Boc-( Scases)-Leu-( N-acylatedS)-Azy-(S,,S )-Azi(OBn)ones,)-Azi(OBn) exhibited22 in cathepsin cathepsin a high selectivityL active site, and as predicted potency and providedby docking the premise studies (Vicik for the etet al.,al., further ChemMedChemChemMedChem developmen 20062006,, 11t, , 1021–1028).of1021–1028). chemical biology tools much needed for functional studies. 3.6. Peptidyl Aryl Vinylsulfones Another promising scascaffoldffold that acts as a MichaelMichael acceptor and hijacks the catalytic residue of cysteine cathepsins is is peptidyl peptidyl aryl aryl vinylsulfones. vinylsulfones. This This scaffold scaffold was was first first introduced introduced by byPalmer Palmer et al. et al.as an as anirreversible irreversible inhibitor inhibitor of cysteine of cysteine cathepsins cathepsins [134]. [134 Subsequently,]. Subsequently, they they extended extended their their study study by byperforming performing an SAR an SAR analysis analysis of this of thisclass class of inhi ofbitors inhibitors which which showed showed pan-cathepsin pan-cathepsin inhibition inhibition with withoccasional occasional selectivity selectivity towards towards cathep cathepsinsin S for certain S for certainscaffolds sca [135].ffolds However, [135]. However, in a separate in a study separate by studyMendieta by Mendieta et al., the et al.,authors the authors developed developed a structurally a structurally novel novel library library of oftwenty twenty peptidyl peptidyl 3-aryl vinylsulfones (Figure(Figure9) in9) which in which they introducedthey introduced extensive extensive diversity atdiversity the R 1 position. at the Subsequently,R1 position. theySubsequently, also varied they the also R2 position varied whilethe R2 keepingposition either while morpholine keeping either or N-methyl morpholine piperazine or N-methyl group intactpiperazine [116]. group Docking intact studies [116]. with Docking most activestudies and with selective most active inhibitor and (Entry selective 15, Tableinhibitor2) of (Entry this class 15, revealed that the inhibitor extends from S2 to S20 sites of cathepsin L and the β-vinylsulfone moiety residesFigure in a 8. close Binding proximity mode of of Boc-( Cys-25S)-Leu-( residueS)-Azy-( therebyS,S)-Azi(OBn) favoring2 in the cathepsin formation L active of Michael site, as predicted adduct. The authorsby docking postulated studies that (Vicik considering et al., ChemMedChem the efficacy 2006 of, peptidyl1, 1021–1028). aryl vinyl sulfones, strong anti-cancer candidates could be harnessed by cultivating this scaffold. 3.6. Peptidyl Aryl Vinylsulfones Another promising scaffold that acts as a Michael acceptor and hijacks the catalytic residue of cysteine cathepsins is peptidyl aryl vinylsulfones. This scaffold was first introduced by Palmer et al. as an irreversible inhibitor of cysteine cathepsins [134]. Subsequently, they extended their study by performing an SAR analysis of this class of inhibitors which showed pan-cathepsin inhibition with occasional selectivity towards cathepsin S for certain scaffolds [135]. However, in a separate study by Mendieta et al., the authors developed a structurally novel library of twenty peptidyl 3-aryl vinylsulfones (Figure 9) in which they introduced extensive diversity at the R1 position. Subsequently, they also varied the R2 position while keeping either morpholine or N-methyl piperazine group intact [116]. Docking studies with most active and selective inhibitor (Entry 15,

MoleculesMolecules 20202020,, 2525,, 698698 1414 ofof 4040

TableTable 2)2) ofof thisthis classclass revealedrevealed thatthat thethe inhibitorinhibitor extendsextends fromfrom S2S2 toto S2S2′′ sitessites ofof cathepsincathepsin LL andand thethe ββ-- vinylsulfonevinylsulfone moietymoiety residesresides inin aa closeclose proximityproximity ofof Cys-25Cys-25 residueresidue therebythereby favoringfavoring thethe formationformation ofof Michael adduct. The authors postulated that considering the efficacy of peptidyl aryl vinyl sulfones, MichaelMolecules 2020 adduct., 25, 698 The authors postulated that considering the efficacy of peptidyl aryl vinyl sulfones,16 of 41 strongstrong anti-canceranti-cancer candidatescandidates couldcould bebe harnessedharnessed byby cultivatingcultivating thisthis scaffold.scaffold.

XX OO RR1 HH 1 NN NN NN SS O HH O O OO RR22 O X=O/N-methylX=O/N-methyl R = Phenyl / 1-Napthyl / 2-Napthyl / Mesityl / 4'-Biphenyl R11 = Phenyl / 1-Napthyl / 2-Napthyl / Mesityl / 4'-Biphenyl R = Benzyl / Iso-butyl R22 = Benzyl / Iso-butyl [Michael-acceptor vinylsulfone is shown in red] [Michael-acceptor vinylsulfone is shown in red]

FigureFigure 9.9. TheThe generalgeneralgeneral structure structurestructure of ofof the thethe library librarylibrary of ofof peptidyl peptidpeptid arylylyl arylaryl vinylsulfones vinylsulfonesvinylsulfones where wherewhere substituents substituentssubstituents (R1 and (R(R11 andRand2) haveRR22)) havehave been beenbeen varied variedvaried to assess toto assessassess the favorablethethe favorablefavorable interactions ininteractionsteractions within withinwithin the cathepsin thethe cathepsincathepsin L active LL activeactive site. site.site. 3.7. Peptidyl Aryl Vinylsulfonate 3.7.3.7. PeptidylPeptidyl ArylAryl VinylsulfonateVinylsulfonate OneOne other other very very potent potent class class of of inhibitors inhibitors that that al alsoalsoso includes includes a a Michael Michael acce acceptoracceptorptor is is peptidyl peptidyl aryl aryl vinylsulfonatevinylsulfonate esters,esters, aa superiorsuperior MichaelMichael acceptoracceptor thanthan vinylvinyl sulfone.sulfone. They They serv servedserveded as as potentpotent inhibitorsinhibitors T. cruzi ofof cruzain—acruzain—a parasitic parasiticparasitic cysteine cysteinecysteine protease proteaseprotease from fromfrom T.T. cruzicruzithat thatthat is homologous isis homologoushomologous to cathepsin toto cathepsincathepsin L [136 LL , 137[136,137].[136,137].]. This ThisscaThisff old scaffoldscaffold was explored waswas exploredexplored by Dana byby DanaDana et al., etet who al.,al., who determinedwho detedeterminedrmined the superiority thethe superioritysuperiority of aryl ofof vinylsulfonatearylaryl vinylsulfonatevinylsulfonate ester esterester over overarylover vinylsulfone arylaryl vinylsulfonevinylsulfone and aryl andand vinylsulfonamide arylaryl vinylsulfonamidevinylsulfonamide counterparts counterpartscounterparts towards towardstowards cathepsin cathepsincathepsin L inhibition LL inhibitioninhibition [117]. [117].[117]. Thus, Thus,theyThus, synthesized theythey synthesizedsynthesized and screened andand screenedscreened the effi thecacythe efficacyefficacy of a library ofof aa librarylibrary of aryl of vinylsulfonateof arylaryl vinylsvinylsulfonateulfonate ester compounds esterester compoundscompounds against againstcathepsinagainst cathepsincathepsin L; 4-bromo L;L; 4-bromo4-bromo phenyl phenyl vinylsulfonatephenyl vinylsulfonatevinylsulfonate was found waswas to foundfound be the toto champion bebe thethe championchampion ligand ligand presumablyligand presumablypresumably due to duefavorabledue toto favorablefavorable interactions interactionsinteractions between betweenbetween the 4-bromo thethe 4-bromo4-bromo phenyl moiety phenylphenyl with moietymoiety the with primewith thethe site primeprime residues sitesite of residuesresidues cathepsin ofof cathepsinL.cathepsin They further L.L. TheyThey designed furtherfurther adesigneddesigned hybrid inhibitor, aa hybridhybrid KD-1, inhibitor,inhibitor, (Entry KD-1,KD-1, 16, Table(Entry(Entry2) 16,16, by Table strategicallyTable 2)2) byby strategicallystrategically appending appendingtheappending 4-bromophenyl thethe 4-bromophenyl4-bromophenyl vinylsulfonate vinylsvinyls moietyulfonateulfonate as electrophilic moietymoiety asas electrophilicelectrophilic warhead to warheadwarhead a modestly toto aa potent modestlymodestly reversible potentpotent reversiblecathepsinreversible L cathepsincathepsin inhibitor [LL113 inhibitorinhibitor]; this design [113];[113]; was thisthis based designdesign on waswas the hypothesisbasedbased onon thethe that hypothesishypothesis the developed thatthat compoundthethe developeddeveloped will compoundtargetcompound both thewillwill prime targettarget site bothboth and thethe the primeprime non-prime sitesite andand site thethe residues non-non-primeprime for interaction. sitesite residuesresidues KD-1 forfor interaction.interaction. indeed exhibited KD-1KD-1 indeedindeed almost exhibitedaexhibited diffusion-controlled almostalmost aa diffusion-controlleddiffusion-controlled inactivation kinetics whileinactivationinactivation maintaining kineticskinetics an excellentwhilewhile maintainingmaintaining selectivity profile anan excellentexcellent toward selectivitycathepsinselectivity L profileprofile (Figure towardtoward10). Furthermore, cathepsincathepsin KD-1LL (Figure(Figure was cell-permeable 10).10). Furthermore,Furthermore, and inhibited KD-1KD-1 waswas the cell-permeable intracellularcell-permeable activity andand inhibitedofinhibited cathepsin thethe Lintracellularintracellular in human MDA-MB-231 activityactivity ofof cathepsincathepsin breast LL cancer inin humanhuman cell lines.MDA-MB-231MDA-MB-231 KD-1 also breastbreast enhanced cancercancer the cellcell integrity lines.lines. KD-KD- of 1cell–cell1 alsoalso enhancedenhanced junctions thethe by integrityintegrity effectively ofof cell–cell attenuatingcell–cell junctionsjunctions the migratory byby effectivelyeffectively potential attenuatingattenuating of the cells, thethe asmigratorymigratory demonstrated potentialpotential by oftheof thethe scratch cells,cells, as assay.as demonstrateddemonstrated The authors byby anticipatedthethe scratchscratch assay.assay. that this TheThe class authorsauthors of inhibitors anticipatedanticipated may thatthat find thisthis extensive classclass ofof inhibitorsinhibitors usage in maydecipheringmay findfind extensiveextensive context-specific usageusage inin deciphering cathepsindeciphering L biology. context-specificcontext-specific cathepsincathepsin LL biology.biology.

FigureFigure 10. 10. 4-bromophenylvinylsulfonate4-bromophenylvinylsulfonate moiety moiety waswas tethered tethered to to a a known known reversible reversible inhibitor inhibitor of of only only modestmodest potency potency to to achieve achieve a a highly highly potentpotent andand selectiveselective irreversibleirreversible peptidylpeptidyl cathepsincathepsin L-selectiveL-selective inhibitor,inhibitor,inhibitor, KD-1;KD-1; thisthis inhibitoriinhibitornhibitor likelylikely bindsbinds toto bothboth prime-prime- andand non-primenon-primenon-prime sitessitessites ofofof thethethe enzyme.enzyme.enzyme. 3.8. Gallinamide A Analogs Recently, another interesting discovery that provided a wealth of information on cathepsin L-inhibitor interactions came from marine cyanobacterial extracts. Miller et al. ﬁrst reported gallinamide A as potent irreversible inhibitor of cathepsin L with an IC50 value of 5 nM and a 28- Molecules 2020, 25, 698 15 of 40

3.8. Gallinamide A Analogs

MoleculesRecently,2020, 25 ,another 698 interesting discovery that provided a wealth of information on cathepsin17 of L- 41 inhibitor interactions came from marine cyanobacterial extracts. Miller et al. first reported gallinamide A as potent irreversible inhibitor of cathepsin L with an IC50 value of 5 nM and a 28- to 320-foldto 320-fold greater greater selectivity selectivity over over cathepsin cathepsin V Vand and B, B, respectively respectively [138]. [138]. They They further performed molecular dockingdocking andand molecularmolecular dynamics dynamics simulations simulations and and learned learned that that the the peptidyl peptidyl backbone backbone of the of theinhibitor inhibitor spans spans the activethe active site whereas site whereas the side the chains side engagechains engage in favorable in favorable interactions interactions with diff erentwith differentactive site active pockets, site placing pockets, the placing Michael the acceptor Michael enamide acceptor in enamide close proximity in close to proximity the catalytic to the Cys catalytic residue. CysIn a follow-upresidue. In study,a follow-up Boudreau study, et al.Boudreau performed et al. molecular performed docking molecular studies docking to predict studies the to potential predict themodifications potential ofmodifications a gallinamide of A a sca gallinamideffold that would A scaffold harness that favorable would enzyme–inhibitorharness favorable interactions enzyme– inhibitorand enable interactions the development and enable of compounds the development with improved of compounds inhibitory with effi improvcacy [118ed]. inhibitory They synthesized efficacy [118].a panel They of compounds synthesized by a retainingpanel of gallinamidecompounds Aby and retaining only varying gallinamide the amino A and acids only at P1,varying P10, andthe aminoP20 positions. acids at (FigureP1, P1′ ,11 and). ThisP2′ positions. led to the (Figure discovery 11). of This the mostled to potent the discovery analog ofof this the seriesmost potent (Entry 17,analog Table of2 )this with series sub-nanomolar (Entry 17, ICTable50 value 2) with (94 pM)sub-nanomolar and fast time IC50 dependent value (94 inactivation pM) and fast kinetics, time dependentsuggesting inactivation an improved kinetics, binding suggesting and reactivity an improv of theed inhibitor binding and with reactivity the enzyme of the active inhibitor site. with The theauthors enzyme found active that site. this The class authors of compounds found that e thisffectively class of inactivated compounds cruzain, effectively a homologous inactivated cysteinecruzain, aprotease homologous from T. cysteine cruzi, using protease cell-based from T. assay. cruzi Gallinamide, using cell-based A and assay. its analogs Gallinamide thus provide A and a remarkableits analogs thusinhibitory provide sca ffaold remarkable that could inhibitory potentially scaffold be harnessed that could to build potentially selective be enzyme harnessed inhibitors to build for selective a variety enzymeof therapeutic inhibitors applications. for a variety of therapeutic applications.

Figure 11. GallinamideGallinamide was was reconstructed reconstructed with with altered amino acid sequence at P1, P1P10′, and P2P20′ positions. The acrylic group, the Mi Michaelchael acceptor, is shown in red.

3.9. Peptidyl Peptidyl Aldehydes Aldehydes One classical inhibitor that has been used over a lo longng period period of of time time to to dissect dissect cysteine cysteine proteinase proteinase activity is Leupeptin, a microbial product. Leupepti Leupeptinn is a peptidyl aldehyde that occupies the active site cleft of of cysteine cysteine cathepsins cathepsins and and forms forms a a thiohemi thiohemiacetalacetal intermediate intermediate by trapping trapping catalytic catalytic cysteine residue; this complex hydrolyzes over time, thus showing the covalent and reversible nature of the inhibitor (Figure(Figure 1212a).a). LeupeptinLeupeptin unfortunately unfortunately su suffersffers from from non-specific non-specific inhibition inhibition ofboth of both serine serine and andcysteine cysteine proteinases, proteinases, thus making thus making it unfavorable it unfavorable for clinical for usage clinical and usage chemical and biology chemical applications. biology applications.This issue was, This however, issue was, addressed however, by Woo addressed et al. who by designedWoo et al. and who synthesized designed six and peptidyl synthesized aldehyde six analogs that were more potent than Leupeptin (IC = 70.3 nM) and exhibited improved selectivity peptidyl aldehyde analogs that were more potent50 than Leupeptin (IC50 = 70.3 nM) and exhibited improvedtowards cathepsin selectivity L overtowards cathepsin cathepsin B and L calpainover ca IIthepsin [139]. B The and most calpain potent II cathepsin[139]. The L most inhibitor potent of this series was Z-Phe-Phe-H (IC = 0.74 nM) (Figure 12b) that showed more than 90-fold selectivity cathepsin L inhibitor of this series50 was Z-Phe-Phe-H (IC50 = 0.74 nM) (Figure 12b) that showed more thanover cathepsin90-fold selectivity B. Interestingly; over cathepsin their data B. demonstrated Interestingly; the their importance data demonstrated of aromatic the amino importance acids, such of aromaticas phenylalanine amino acids, and tyrosine, such as atphenylalanine the P1 position and in determiningtyrosine, at the the P1 potency position and in selectivity determining towards the potencycathepsin and L; O-alkylationselectivity towards of tyrosine cathepsin group L; diminishes O-alkylation the of inhibitory tyrosine egroupfficiency diminishes as in Z-Phe-Tyr(Bu)-H the inhibitory (IC : 6.96 nM). In a follow-up publication, they further tested the efficacy of Z-Phe-Tyr-H (IC : efficiency50 as in Z-Phe-Tyr(Bu)-H (IC50: 6.96 nM). In a follow-up publication, they further tested the50 0.85 nM, 100-fold selective over cathepsin B) (Figure 12b) in vitro and in vivo [140]. This compound efficacy of Z-Phe-Tyr-H (IC50: 0.85 nM, 100-fold selective over cathepsin B) (Figure 12b) in vitro and ineff vivoectively [140]. inhibited This compound parathyroid effectively hormone-stimulated inhibited parathyroid osteoclastic hormone-stimulated bone resorption osteoclastic in pit formation bone resorptionassays, and in suppressed pit formation bone assays, weight and loss suppressed of ovariectomized bone weight mouse loss in a of dose-dependent ovariectomized manner mouse when in a dose-dependentadministered intraperitoneally. manner when administered intraperitoneally.

Molecules 2020, 25, 698 16 of 40

Molecules 2020, 25, 698 18 of 41 MoleculesFigure 2020 , 12.25, 698(a) Proposed mode of cathepsin L inactivation by aldehyde inhibitors. (b) Chemical16 of 40 structure of inhibitors, as discussed in text, are shown here.

In another interesting study, Yasuma et al. developed a library of compounds by varying the amino acid substituents at P1, P2, P3 position of the inhibitor and carried out a thorough SAR study [21]. Their study revealed that the configuration of the stereogenic center (S-configuration is favored over R-configuration) at the P1 position, and not the steric factor, was key to the inhibitory efficacy. Apparently, the substituent at P1 position does not interact with S1 position residues; rather, proper stereogenicity allows the placement of the inhibitor in vicinity of the catalytic cysteine residue for interaction. S2 subsite of cathepsin L, on the other hand, preferred a hydrophobic and moderate-size group;FigureFigure α-branched 12. ((aa)) ProposedProposed alkyl chains mode mode ofbut cathepsinof not cathepsin the Lbulkier inactivation L inac groupstivation by aldehyde like by phenylalaninealdehyde inhibitors. inhibitors. (b was) Chemical favorable. (b) Chemical structure Further, the structureS3of inhibitors,subsite of inhibitors,showed as discussed asa discussedpreference in text, are in shown text,for arehydrop here. shownhobic here. and bulky moieties such as 1- and 2- naphthalenylsulfonyl substituents. Among synthesized compound, N-(1-naphthalenylsulfonyl-L- isoleucyl-InIn another anotherL-tryptophanal interesting interesting (IC study, study,50 = 1.9 Yasuma Yasuma nM, 789-fold et al. selectivedeveloped developed over a a library librarycathepsin of of compounds compoundsB; Figure 13) by byattenuated varying varying the the aminoreleaseamino acid acidof Ca substituents substituents2+ and hydroxyproline at atP1, P1, P2, P2, P3 from P3position positionbone inof anthe of in inhibitor the vitr inhibitoro bone and culture carried and carriedsystem out a outandthorough afurther thorough SAR restricted study SAR [21].bonestudy Their loss [21 ]. instudy Theirovariectomized revealed study revealed that mice the dosedconfiguration that the orally. configuration of the stereogenic of the stereogenic center (S-configuration center (S-configuration is favored overis favored R-configuration)A further over modification R-configuration) at the ofP1 this position, scaffold at the and P1was not position, report the stericed andby factor, Lynas not was theet al. sterickey [119] to factor,Here,the inhibitory authors was key designedefficacy. to the Apparently,andinhibitory developed e ffithecacy. di-substituent and Apparently, tri-peptidyl at P1 position the α-keto- substituent doesβ-aldehydes, not at interact P1 position based with on S1 does substrate position not interact residues;and inhibitor with rather, S1 specificity position proper stereogenicityprofilesresidues; of rather,cathepsin allows proper L.the The placement stereogenicity compound of theZ-Phe-Tyr(OBut)-COCHO allows inhibitor the in placement vicinity of of the(Entry the catalytic inhibitor 18, Table cysteine in 2) vicinity turned residue ofout for the as interaction.highlycatalytic potent cysteine S2 andsubsite selective residue of cathepsin forinhibitor interaction. L, of on cathepsin the S2other subsite L hand, with of Kpreferred cathepsini value of a 0.6 hydrophobic L, nM. on theThis other molecule and hand, moderate-size was preferred further group;adapteda hydrophobic α-branched by Shenoy and alkyl et moderate-size al. chains to assess but thenot group; structuralthe bulkierα-branched ba groupssis for alkyl likecathepsin chainsphenylalanine L but inhibiti not was theon bulkier[96].favorable. In their groups Further, study, like thephenylalaninethe authorsS3 subsite crystallized wasshowed favorable. thea preference glyoxal Further, inhibitor for the hydrop S3 with subsite hobiccathepsin showed and L;bulky the a preference β -aldehydemoieties for suchforms hydrophobic as a tetrahedral1- and and2- naphthalenylsulfonylthiohemiacetalbulky moieties and such α as -ketosubstituents. 1- and oxygen 2-naphthalenylsulfonyl atom Among is stabilized synthesized by substituents. the compound, oxyanion Among hole. N-(1-naphthalenylsulfonyl- The synthesized Tyr(OBut) compound,group wasL- l l isoleucyl-foundN-(1-naphthalenylsulfonyl- toL -tryptophanaloccupy S1 site (IC while50-isoleucyl- = 1.9 phenylnM, -tryptophanal789-fold and carbselectiveoxybenzyl (IC50 over= 1.9 cathepsin nM,groups 789-fold B;occupied Figure selective 13)S2 attenuated overand cathepsinS3 sites, the 2+ releaserespectively.B; Figure of Ca13)2+ attenuated Thisand hydroxyprolineclass theof inhibitors release from of Cahas bone successfuand in an hydroxyproline inlly vitr beeno bone deployed culture from bone systemin inthe an andfunctionalin vitrofurtherbone biology restricted culture of bonecathepsinsystem loss and in L. furtherovariectomized restricted mice bone dosed loss inorally. ovariectomized mice dosed orally. A further modification of this scaffold was reported by Lynas et al. [119] Here, authors designed and developed di- and tri-peptidyl α-keto-β-aldehydes, based on substrate and inhibitor specificity profiles of cathepsin L. The compound Z-Phe-Tyr(OBut)-COCHO (Entry 18, Table 2) turned out as highly potent and selective inhibitor of cathepsin L with Ki value of 0.6 nM. This molecule was further adapted by Shenoy et al. to assess the structural basis for cathepsin L inhibition [96]. In their study, the authors crystallized the glyoxal inhibitor with cathepsin L; the β-aldehyde forms a tetrahedral thiohemiacetal and α-keto oxygen atom is stabilized by the oxyanion hole. The Tyr(OBut) group was found to occupy S1 site while phenyl and carboxybenzyl groups occupied S2 and S3 sites, respectively. This class of inhibitors has successfully been deployed in the functional biology of cathepsin L. Figure 13. N-(1-naphthalenylsulfonyl-Ll-isoleucyl--isoleucyl-Ll-tryptophanal-tryptophanal orients orients itself itself into into the the active site of cathepsin L and findsfinds favorable interactions withinwithin thethe S1,S1, S2,S2, andand S3S3 pocketspockets ofof thethe enzyme.enzyme.

3.10. AAzepanone-based further modification Inhibitors of this scaffold was reported by Lynas et al. [119] Here, authors designed and developed di- and tri-peptidyl α-keto-β-aldehydes, based on substrate and inhibitor specificity profilesAzepanone-based of cathepsin L. compounds The compound were Z-Phe-Tyr(OBut)-COCHO first reported as orally bioavailable (Entry 18, Tableand extremely2) turned outpotent as inhibitors of cathepsin K, as shown by the pharmacokinetic studies in the rat [141]. Marquis et al. highly potent and selective inhibitor of cathepsin L with Ki value of 0.6 nM. This molecule was further subsequentlyadapted by Shenoy adopted et al.the to template assess the and structural extended basis their for cathepsinwork to acquire L inhibition a selective [96]. In inhibitor their study, of cathepsinthe authors L with crystallized similar potency the glyoxal [120]. inhibitor This class with of inhibitors cathepsin are L; thearmoredβ-aldehyde with keto forms functional a tetrahedral group

thiohemiacetal and α-keto oxygen atom is stabilized by the oxyanion hole. The Tyr(OBut) group was found to occupy S1 site while phenyl and carboxybenzyl groups occupied S2 and S3 sites, respectively. Figure 13. N-(1-naphthalenylsulfonyl-L-isoleucyl-L-tryptophanal orients itself into the active site of This class of inhibitors has successfully been deployed in the functional biology of cathepsin L. cathepsin L and finds favorable interactions within the S1, S2, and S3 pockets of the enzyme. 3.10. Azepanone-based Inhibitors 3.10. Azepanone-based Inhibitors Azepanone-based compounds were ﬁrst reported as orally bioavailable and extremely potent Azepanone-based compounds were first reported as orally bioavailable and extremely potent inhibitors of cathepsin K, as shown by the pharmacokinetic studies in the rat [141]. Marquis et al. inhibitors of cathepsin K, as shown by the pharmacokinetic studies in the rat [141]. Marquis et al. subsequently adopted the template and extended their work to acquire a selective inhibitor of cathepsin subsequently adopted the template and extended their work to acquire a selective inhibitor of cathepsin L with similar potency [120]. This class of inhibitors are armored with keto functional group

Molecules 2020, 25, 698 19 of 41 Molecules 2020, 25, 698 17 of 40

Lthat with act similaras an electrophilic potency [120 warhead]. This and class traps of inhibitors cysteine proteases are armored by forming with keto a transient functional covalent group bond that actwith as the an active-site electrophilic Cys warhead residue, andrendering traps cysteine inactivated proteases enzyme. by The forming authors a transient initiated covalenttheir work bond by withscrupulously the active-site studying Cys the residue, cathepsin rendering K-inhibitor inactivated complex enzyme. that revealed The authors the influence initiated theirof P2 work and byP3 scrupulouslysubstituents of studying the inhibitor the cathepsin in determining K-inhibitor the complexefficacy and that selectivity revealed theprofile influence of the of compound. P2 and P3 substituentsBased on these of the observations, inhibitor in determiningthey designed the and effi cacysynthesized and selectivity a series profile of compound of the compound. and secured Based a onhighly these potent observations, cathepsin they L inhibitor designed (K andi,app synthesized: 0.43 nM; Entry a series 19, of Table compound 2) that and exerted secured remarkable a highly potentselectivity cathepsin over cathepsin L inhibitor K and (Ki,app fairly: 0.43 modest nM; Entryselectivity 19, Table over2 both) that cathepsin exerted remarkable B and S. Interestingly, selectivity overSAR cathepsinshowed that K and replacement fairly modest of P2 selectivity leucine and over P3 both benzofuran cathepsin of Bcathepsin and S. Interestingly, K inhibitor SARwith showedbulkier thathydrophobic replacement aromatic of P2 leucinegroups andyielded P3 benzofuranan improved of potency cathepsin and K inhibitorthe selectivity with bulkiertowards hydrophobic cathepsin L aromatic(Figure 14). groups Molecular yielded docking an improved studies potencyfurther supp andorted the selectivity this observation towards as cathepsin cathepsin LK (Figure was found 14). Molecularto have a shallower docking studies S2 pocket further than supported cathepsin thisL, thus observation incorporation as cathepsin of bulkier K wasnapthyl found group to have at P2 a shallowerposition favored S2 pocket cathepsin than cathepsin L inhibition L, thusbut not incorporation cathepsin K. of On bulkier the other napthyl hand, group inclusion at P2 of positionanother favorednapthyl cathepsingroup at P3 L inhibitionposition promoted but not cathepsin a steric cl K.ash On rather the otherthan furthering hand, inclusion the desired of another hydrophobic napthyl groupinteractions at P3 position within the promoted S3 pocket a steric of cathepsin clash rather K, than thus furthering incurring the a better desired selectivity hydrophobic profile interactions towards withincathepsin the S3L over pocket cathepsin of cathepsin K. This K, thustemplate incurring has apr betteroven selectivityto be an important profile towards tool to cathepsin study cysteine L over cathepsincathepsins K. as This it has template been further has proven extended to be anto importantachieve potent tool tocathepsin study cysteine S-selective cathepsins inhibitor as itwith has beencellular further activity extended [142]. to achieve potent cathepsin S-selective inhibitor with cellular activity [142].

Figure 14.14. (a) PictorialPictorial representation of mechanismmechanism of azepinone-mediatedazepinone-mediated ca cathepsinthepsin L inhibition. ((bb)) GenericGeneric structurestructure ofof azepinoneazepinone inhibitors.inhibitors. The inclusion of bulky aromatic groups at P2 and P3P3 positionspositions leadsleads toto cathepsincathepsin LL inhibitorsinhibitors withwith improvedimproved potencypotency andand selectivity.selectivity.

3.11. Nitrile-Containing Inhibitors Nitrile group containingcontaining inhibitors have been widelywidely recognizedrecognized as covalentcovalent andand reversiblereversible inhibitorsinhibitors ofof aa certaincertain classclass ofof enzymesenzymes thatthat dependdepend onon cysteine-mediatedcysteine-mediated nucleophilicnucleophilic attack for catalysis; thethe nitrile residue trapstraps thethe sulfur and formsforms a thioimidate bond (Figure 1515)) that hydrolyzes over thethe timetime renderingrendering freefree enzyme.enzyme. Odanacatib Odanacatib is is one one of the prime examples of this class ofof compounds that has been evaluated as a clinical agent, although with limited success [143,144]. [143,144]. Because of nitrile’s tunable target engagement nature, this scascaffoldffold has been adapted to target other relevantrelevant enzymes,enzymes, includingincluding cathepsin.cathepsin. Hardegger et al. utilized nitrile warhead and examinedexamined thethe eeffectffect ofof halogenhalogen bondingbonding inin protein–ligand interactionsinteractions [[121].121]. TheyThey developed aa series of compounds and performed a thoroughthorough SAR analysisanalysis in which the nitrilenitrile electrophileelectrophile faced towards S1 sitesite andand trappedtrapped thethe catalyticcatalytic cysteine.cysteine. In the developed analogs,analogs, the substituents that occupied the S3 site werewere systematicallysystematically variedvaried byby strategicallystrategically alteringaltering thethe substituentssubstituents atat the para-position ofof the phenyl group (Figure 16).16). The The auth authorsors observed observed an an improvement improvement in in inhibi inhibitiontion profile profile with with the the placement placement of ofhalogen halogen at the at the para-position para-position of phenyl of phenyl ring ring which which followed followed a trend a trend Cl < ClBr< < BrI (Entry< I (Entry 20, Table 20, Table 2), with2), withthe F the substituent F substituent being being an anoutlier. outlier. Further Further analysis analysis of of the the enzyme-inhibitor enzyme-inhibitor co-crystal co-crystal structures revealedrevealed that halogen at at the the para para position position of of the the phenyl phenyl ring ring suitably suitably interacted interacted with with Gly61 Gly61 at the at the S3 S3site; site; fluorine fluorine analog analog pointed pointed away away to avoid to avoid the the electronic electronic repulsion repulsion from from the the oxygen oxygen lone lone pairs pairs of Gly61. The authors have also performed computational analysis which taken together with the crystal

Molecules 2020, 25, 698 20 of 41

Molecules 2020,, 2525,, 698698 18 of 40 of Gly61. The authors have also performed computational analysis which taken together with the datacrystal suggests data suggests O· X-C O angleX-C and angle the and distance the distance between between the interacting the interacting atoms atoms primarily primarily influenced inﬂuenced the · protein-ligandthe protein-ligand interaction. interaction. This This work work provides provides an an important important roadmap roadmap for for developing developing improved chemical biology tools where a halogen-protein interactioninteraction hashas successfullysuccessfully beenbeen utilizedutilized [[97].97].

FigureFigure 15. Nitrile 15. Nitrile group-containing group-containing inhibito inhibitorsrs forms forms hydrolytically hydrolytically an an unstable unstable thioimidate bondbond withwith the catalyticthe cysteine catalytic residue cysteine of residue the enzyme. of the enzyme.

FigureFigure 16. Substituted 16. Substituted aryl groups aryl groups were sc werereened screened and evaluated and evaluated to harness to harness favorable favorable halogen-protein halogen-protein interaction by targetinginteraction S3 pocket by targeting of cathepsin S3 pocket L enzyme. of cathepsin L enzyme. To examine what effect amide heteroarene π-stacking interactions may have on chalcogen To examine what effect amide···heteroarene··· π-stacking interactions may have on chalcogen bonding in the S3 pocket of cathepsin L, Giroud et al. utilized triazine-nitrile scaffold [145]. The authors bonding in the S3 pocket of cathepsin L, Giroud et al. utilized triazine-nitrile scaffold [145]. The synthesized a diverse set of triazine-nitrile compounds with a diversified heteroarenes targeting S3 authors synthesized a diverse set of triazine-nitrile compounds with a diversified heteroarenes pocket; the S1 and S2 substituents were kept constant. Among the developed compound library, targeting S3 pocket; the S1 and S2 substituents were kept constant. Among the developed compound 2-benzothienyl analog (Entry 21, Table2) exhibited maximum inhibitory potential; 2-benzofuranyl, library, 2-benzothienyl analog (Entry 21, Table 2) exhibited maximum inhibitory potential; 2- 2-benzothiazolyl, and 2-imidazopyridinyl, which are of similar geometry, also followed a similar benzofuranyl, 2-benzothiazolyl, and 2-imidazopyridinyl, which are of similar geometry, also inhibitory pattern (Figure 17). Molecular modelling based on co-crystal structures showed favorable followed a similar inhibitory pattern (Figure 17). Molecular modelling based on co-crystal structures chalcogen interaction to the backbone carbonyl of Asn66 (d(S O = CAsn66) = 3.5 Å and the angle showed favorable chalcogen interaction to the backbone carbonyl··· of Asn66 (d(S···O = CAsn66) = 3.5 α(OAsn66 S-C) = 158 ) at the S3 pocket; this was further supported by a conformational strain Å and the··· angle α(OAsn66···S-C)◦ = 158°) at the S3 pocket; this was further supported by a analysis, as chalcogen–enzyme interactions compensated for higher torsional strain in the S-containing conformational strain analysis, as chalcogen–enzyme interactions compensated for higher torsional ligands when compared to the benzofuranyl and imidazopyridinyl ligands. Their study demonstrated strain in the S-containing ligands when compared to the benzofuranyl and imidazopyridinyl ligands. the importance of both intermolecular interactions and conformational strain in assessing the effect Their study demonstrated the importance of both intermolecular interactions and conformational of heterobicyclic ligands at the S3 pocket that could be potentially be utilized to develop cathepsin L strain in assessing the effect of heterobicyclic ligands at the S3 pocket that could be potentially be selective inhibitors. utilized to develop cathepsin L selective inhibitors. In a subsequent study, Kuhn et al. systematically compared the effectiveness of four different approaches: (a) selection by a medicinal chemist (b) manual modeling (c) docking followed by manual filtering, and (d) free energy calculations (FEP). This systematic protocol enabled them to prioritize building blocks for effective targeting of cathepsin L enzyme [123]. The authors developed a series of 36 analogs by varying only S2 substituents and keeping S1 and S3 fixed (Figure 18). After analyzing the affinity by enzyme kinetics, they found that the FEP method was superior over other well-established methodologies; this method not only predicted the most relevant ligands but also identified the topological requirements of the substituents for a more effective engagement in the S2 pocket. Among the developed compounds, cyclopentylmethyl substituent in the S2 pocket (Entry 22, Table2) incurred the most favorable interaction as it optimally filled the front part of the pocket. This strategy certainly provided an edge over other conventional methodologies in predicting the optimal ligands for the S2 Figure 17. Triazine nitrile scaffold was utilized with varying substituents that explored the S3 pocket on the enzyme for cathepsin L inhibition.

Molecules 2020, 25, 698 18 of 40 data suggests O· X-C angle and the distance between the interacting atoms primarily influenced the protein-ligand interaction. This work provides an important roadmap for developing improved chemical biology tools where a halogen-protein interaction has successfully been utilized [97].

Figure 15. Nitrile group-containing inhibitors forms hydrolytically an unstable thioimidate bond with the catalytic cysteine residue of the enzyme.

Figure 16. Substituted aryl groups were screened and evaluated to harness favorable halogen-protein interaction by targeting S3 pocket of cathepsin L enzyme.

To examine what effect amide···heteroarene π-stacking interactions may have on chalcogen bonding in the S3 pocket of cathepsin L, Giroud et al. utilized triazine-nitrile scaffold [145]. The authors synthesized a diverse set of triazine-nitrile compounds with a diversified heteroarenes targeting S3 pocket; the S1 and S2 substituents were kept constant. Among the developed compound library, 2-benzothienyl analog (Entry 21, Table 2) exhibited maximum inhibitory potential; 2- benzofuranyl, 2-benzothiazolyl, and 2-imidazopyridinyl, which are of similar geometry, also followed a similar inhibitory pattern (Figure 17). Molecular modelling based on co-crystal structures showed favorable chalcogen interaction to the backbone carbonyl of Asn66 (d(S···O = CAsn66) = 3.5 Å and the angle α(OAsn66···S-C) = 158°) at the S3 pocket; this was further supported by a conformationalMolecules 2020, 25, 698strain analysis, as chalcogen–enzyme interactions compensated for higher torsional21 of 41 strain in the S-containing ligands when compared to the benzofuranyl and imidazopyridinyl ligands.

TheirMolecules study 2020, 25demonstrated, 698 the importance of both intermolecular interactions and conformational19 of 40 strainpocket in targeting. assessing These the effect findings of heterobicyclic could benefit ligands the ongoing at the e ffS3ort pocket of achieving that could a suitable be potentially therapeutic be utilizedcandidate toIn fordevelopa subsequent cathepsin cathepsin Lstudy, enzyme. L Kuhnselective et al. inhibitors. systematical ly compared the effectiveness of four different approaches: (a) selection by a medicinal chemist (b) manual modeling (c) docking followed by manual filtering, and (d) free energy calculations (FEP). This systematic protocol enabled them to prioritize building blocks for effective targeting of cathepsin L enzyme [123]. The authors developed a series of 36 analogs by varying only S2 substituents and keeping S1 and S3 fixed (Figure 18). After analyzing the affinity by enzyme kinetics, they found that the FEP method was superior over other well-established methodologies; this method not only predicted the most relevant ligands but also identified the topological requirements of the substituents for a more effective engagement in the S2 pocket. Among the developed compounds, cyclopentylmethyl substituent in the S2 pocket (Entry 22, Table 2) incurred the most favorable interaction as it optimally filled the front part of the pocket. This

strategy certainly provided an edge over other conventional methodologies in predicting the optimal FigureligandsFigure 17. for Triazine 17.theTriazine S2 nitrilepocket nitrile scaffold targeting. scaﬀ wasold wasutilizedThese utilized findingswith with varying varyingcould substituents benefit substituents the that ongoing that explored explored effort the the S3 of S3pocket achieving pocket on the a enzymeon for the cathepsin enzyme for L inhibition. cathepsin L inhibition. suitable therapeutic candidate for cathepsin L enzyme.

Figure 18. Diverse set of pyrimidine-based nitrile Inhibitors, with with ligands targeting the S2 pocket of cathepsin L for inhibition.

The readers are encouraged to refer toto studiesstudies byby FalgueyretFalgueyret etet al.al. andand Asaad et al.al. where, where, although not selective, nitrile-containing inhibitors targeting cathepsin L have also successfully been reported [146,147]. [146,147].

3.12. Thiosemicarbazone Thiosemicarbazone The thiosemicarbazonethiosemicarbazone moiety moiety was was ﬁrst first recognized recogniz ased a relevantas a relevant covalent covalent and reversible and reversible warhead warheadof cathepsin of cathepsin L homologous L homologous enzyme cruzain, enzyme a proteasecruzain, froma proteaseT. Cruzi from. The T. mechanism Cruzi. The of mechanism inactivaction of inactivactioninvolves the formationinvolves the of aformation transient covalentof a transient bond withcovalent the catalyticbond with Cys the residue catalytic (Figure Cys 19 residue)[148]. (FigureIn an interesting 19) [148]. In study, an interesting Kishore Kumar study, et Kishore al. ﬁrst Kumar utilized et this al. ideafirst utilized and synthesized this idea aand small synthesized library of acompounds small library in of which compounds the most in active which class the ofmost inhibitors active class were of comprised inhibitors of were one meta-bromocomprised of substituted one meta- bromoaryl ring substituted along with aryl another ring along one with with optimally another substitutedone with optimally functionalities substituted [127]. functionalities The inhibitor places[127]. Theitself inhibitor in the active places site itself cleft in of cathepsinthe active Lsite where cleft meta-bromo of cathepsin substituted L where meta-bromo aryl ring occupies substituted the S2 aryl site ringand thiosemicarbazoneoccupies the S2 sitemotif and thiosemicarbazone lies near the active motif site cysteine. lies near the active site cysteine.

Figure 19. CatalyticCatalytic cysteine cysteine residue residue of of cathepsin cathepsin L L forms forms a a covalent and reversible bond with the thiosemicarbazonethiosemicarbazone group rendering the inhibited enzyme.enzyme. However, when the motif was extended to capture S1 site interaction by placing the aryl/alkyl However, when the motif was extended to capture S10′ site interaction by placing the aryl/alkyl group at the terminal nitrogen of thiosemicarbazone, the inhibitory potency was completely diminished. group at the terminal nitrogen of thiosemicarbazone, the inhibitory potency was completely Overall, this class of inhibitors showed a good selectivity over cathepsin B and exhibited low cytotoxicity diminished. Overall, this class of inhibitors showed a good selectivity over cathepsin B and exhibited low cytotoxicity when tested on human cancer cell lines. In follow up studies, Kishore et al. and Parker et al. further expanded the scope of thiosemicarbazone scaffold and developed diversely

Molecules 2020, 25, 698 22 of 41 when tested on human cancer cell lines. In follow up studies, Kishore et al. and Parker et al. further expanded the scope of thiosemicarbazone scaffold and developed diversely functionalized analogs that exhibited an enhanced inhibitory potency and promising cellular activities while still retaining the selectivity over cathepsin B [125,126]. In their latest study, Parker et al. strategically transformed an active inhibitor with limited aqueous solubility into a water-soluble prodrug (Entry 23, Table2), by phosphorylation of phenolic hydroxy group; this group was readily hydrolyzable by alkaline phosphatases, rendering the active pharmacophore [124]. The phosphate prodrug exhibited a remarkable 600-fold increase in solubility over the parent drug and did not disintegrate in aqueous solution, even after prolonged exposure at the physiological temperature. Furthermore, this compound did not show any significant cytotoxicity on normal primary HUVECs cells in comparison to other FDA-approved cytotoxic drugs, Doxorubicin and Paclitaxel. This prodrug thus far has shown promise to be a desirable clinical candidate and the authors have proposed to evaluate its in vivo efficacy in a preclinical setup.

3.13. Propeptide Mimics As noted earlier, cathepsin L, like other cysteine cathepsins, contains an inhibitory propeptide domain that spans the active site of the enzyme in the inverse direction to the regular substrate binding mode. Chowdhury et al., in their seminal study, exploited this concept by examining the eﬀect of a series of synthesized tripeptidyl compounds that mimicked cathepsin L inhibitory propeptide [95]. Importantly, the developed tripeptidyl motifs also exhibited nanomolar potency; however, a moderate truncation of the full-length propeptide drastically lost all activities [47]. Notably, while the full-length propeptide showed only 2-fold selectivity over cathepsin K, the most potent analog of this series (Entry 24, Table2) demonstrated a far-improved selectivity (310-fold). The authors further investigated the binding mode of this class of inhibitors by means of co-crystal structure and molecular modeling. This revealed that (a) arginine residue of the inhibitor occupied the S1 pocket, (b) phenyl alanine residue found favorable hydrophobic interactions within the S2 pocket, (c) 2-phenylethyl group pointed toward S3 pocket, (d) the methionine residue showed optimal interaction within S10 pocket, and (e) the biphenyl acetyl group extended to the S30 pocket for favorable interactions [95,128]. This class of inhibitors has shown resistance to enzyme-dependent hydrolysis and demonstrates the reversible mode of enzyme inactivation. Overall, this inhibitor class provides a wealth of information on inhibitor binding to cathepsin L and provides a general template for the development of therapeutic candidates for other relevant enzymes as well.

3.14. Thiocarbazate, Oxocarbazate and Azapeptides In an effort to discover small molecule inhibitors of cathepsin L, Myers et al. performed high throughput screening (HTS) of the NIH Molecular Libraries Small Molecule Repository (MLSMR); they identified 2,5-disubstituted oxadiazoles (Figure 20a) as potent hit compounds [129]. Surprisingly, upon synthesis and purification of the putative inhibitory lead compounds, a complete loss of activity was observed. The authors then investigated the compounds’ integrity from NIH MLSMR library by LC-MS; this showed the presence of additional impurities. To trace back the active impurity, the authors hypothesized the presence of impurities resulting from an acid-catalyzed ring-opening reaction of thiocarbazate. The resulting azapeptides was likely the active pharmacophore that inhibited enzyme via acylation of active site Cys; this was validated by synthesis of azapeptides and performing the enzyme assay [149]. The (S)-stereoisomer of newly synthesized compound (Entry 25, Table2; Figure 20b) indeed attenuated the activity of cathepsin L with an IC50 value of 56 nM. In follow up studies, the authors further developed a series of compounds with structural diversity and performed a computational analysis to recognize the basis of potent enzyme inhibition [130,150] One of the thiocarbazate analogs developed this way (Figure 20c) showed improved potency over the parent compound. Molecular modeling studies performed with parent compound (Entry 25, Table2) in complex with papain indicated that indole motif preferably bound to S2 subsite, -NHBoc group engaged Molecules 2020, 25, 698 23 of 41

inMolecules favorable 2020, hydrophobic25, 698 interactions within the S3 subsite and 2-ethylphenyl anilide extended21 to of S1 400 pocket. To further probe the importance of thiocarbazate moiety, the authors synthesized compound containing2) motifs. The oxocarbazate oxocarbazate (Entry showed 26, Table a fairly2) and improved azapeptide IC50 (Entry value 27,(7 nM) Table towards2) motifs. cathepsin The oxocarbazate L, whereas showedthe azapeptide a fairly was improved at best IConly50 valuea modest (7 nM) inhibitor towards (IC50 cathepsin = 3 µM). L,Consistently, whereas the the azapeptide binding mode was atof bestoxocarbazate only a modest exerted inhibitor similarity (IC 50to= that3 µ M).of thio Consistently,carbazate when the binding investigated mode ofby oxocarbazate molecular modeling exerted similaritystudies [150]. to that of thiocarbazate when investigated by molecular modeling studies [150].

Figure 20.20. (a) Structure ofof HTSHTS hithit thatthat waswas aa falsefalse positive.positive. (b) Structure ofof thethe impurityimpurity thatthat turnedturned out toto be be an an active active pharmacophore. pharmacophore. (c) The(c) The most most potent potent analog analog of this of series, this achievedseries, achieved by SAR analysis.by SAR analysis. In a separate study, Shah et al. carried out a thorough enzymatic analysis of the champion thiocarbazateIn a separate compound study, (EntryShah et 25, al. Tablecarried2) thatout showeda thorough a time-dependent enzymatic analysis improvement of the champion in the inhibitionthiocarbazate profile; compound the IC50 value(Entry went 25, downTable to2) 1that nM showed when preincubated a time-dependent with cathepsin improvement L for 4 hin [131 the]. LC-MSinhibition and pr kineticofile; the analysis IC50 value of enzyme-inhibitor went down to 1 complexnM when (inhibition preincubated rate with constants: cathepsin kon L= for24,000 4 h 1 1 5 1 M[131].− s− LC-MSand k oandff = kinetic2.2 10 analysis− s− , and of enzyme-inhibitor binding constant: complex Ki = 0.89 (inhibition nM) demonstrated rate constants: a slow-binding kon = 24,000 × kineticsM−1s−1 and and koff reversibility = 2.2 × 10−5 s of−1, inhibition.and binding The constant: selectivity Ki = over0.89 nM) other demonstrated members of thea slow-binding enzyme family kinetics was modest.and reversibility Interestingly, of inhibition. the compound The selectivity inhibited over propagation other members of malaria of the parasite enzymePlasmodium family was falciparum modest. [ICInterestingly,50 = 15.4 µ M],the andcompoundLeishmania inhibited major propagation[IC50 = 12.5 µofM], malaria and did parasite not exhibit Plasmodium any significant falciparum toxicity [IC50 = against15.4 µM], human and Leishmania aortic endothelial major [IC cells50 = and 12.5 zebrafish. µM], and Although did not exhibit thiocarbazate any significant motif showed toxicity promise against as anhuman inhibitory aortic sca endothelialffold, the lackcells of and reasonable zebrafish. stability Although (it decomposes thiocarbazate even motif in DMSO) showed and promise only modest as an inhibitoryinhibitory activityscaffold, in the cell-based lack of reasonable assays probably stability ceased (it decomposes any further developmenteven in DMSO) of theand sca onlyffold modest [129]. Theinhibitory authors activity also extended in cell-based their studiesassays probably to evaluate ceased the potential any further of oxocarbazate development inhibitor of the scaffold that showed [129]. anThe improved authors also IC50 extendedvalue of 0.4their nM studies upon 4to h evalua preincubationte the potential with the of enzyme. oxocarbazate Like asinhibitor in the casethat ofshowed thiocarbazate, an improved they IC performed50 value of an 0.4 enzyme nM upon kinetic 4 h analysispreincubat ofion the with enzyme-inhibitor the enzyme. Like complex as inand the 1 1 obtainedcase of thiocarbazate, the following they parameters: performed inhibition an enzyme rate kinetic constants: analysis kon =of153,000 the enzyme-inhibitor M− s− and ko ffcomplex= 4.4 5 1 × 10and− obtaineds− , and bindingthe following constant: parameters: Ki = 0.29 inhibition nM [132]. rate The constants: inhibitor k blockedon = 153,000 SARS-CoV M−1s−1 and (IC 50koff= =273 4.4 × ± 4910− nM)5 s−1, andand Ebolabinding virus constant: (IC50 =Ki193 = 0.2939 nM nM) [132]. entry The into inhibitor the human blockedembryonic SARS-CoVkidney (IC50 (HEK) = 273 293T ± 49 ± cells,nM) and a process Ebola thatvirus utilizes (IC50 cathepsin= 193 ± 39 L-mediatednM) entry in proteolysisto the human for hostembryonic cell infection. kidney The(HEK) oxocarbazate, 293T cells, a process that utilizes cathepsin L-mediated proteolysis for host cell infection. The oxocarbazate, when treated with HEK 293T lysate in the presence of DCG-04, an activity-based cysteine cathepsin probe, showed reduced cathepsin L labeling when assessed by a Western-blot analysis; this further

Molecules 2020, 25, 698 24 of 41 when treated with HEK 293T lysate in the presence of DCG-04, an activity-based cysteine cathepsin probe, showed reduced cathepsin L labeling when assessed by a Western-blot analysis; this further corroborated the results obtained from the virus pseudotype infection assay. Overall, oxocarbazate inhibitor not only provided a promising template for further exploitation but also rendered a new direction for intervening SARS and Ebola virus infections.

Molecules 2020, 25, 698 22 of 39 4. Molecular Probes 4. MolecularUbiquitous Probes expression of human cathepsin L in most human tissues possesses a significant challengeUbiquitous in targeting expression this enzyme of human for therapeutic cathepsin development.L in most human This tissues problem possess is furtheres a significant exacerbated withchal recentlenge in findings targeting that this alternative enzyme for spliced therapeutic isoforms development. could exist This at problem distinct is cellular further locationsexacerbated (e.g., nucleus,with recent cytosol, findings and ECM that space)alternative [37,38 spliced,151]. Whileisoforms several could unique exist at functional distinct cellular roles of locations cathepsin (e.g. L, are known,nucleus, it has cytosol, also beenand ECM reported space) that [37,38, some15 of1]. its While function several can unique also be accomplishedfunctional roles by of other cathepsin members L ofare the cathepsinknown, it familyhas also (i.e., been functional reported redundancy);that some of its for function example, can both also cathepsin be accomplished L and B can by mediateother a mutuallymembers compensatoryof the cathepsin role family in the (i.e. inflammatory, functional redundancy); response signaling for example pathways, both [cathepsin152]. In this L and regard, B accuratecan me functiondiate a mutually of cathepsin compensatory L must be role first in determinedthe inflammatory in di ffresponseerent cell signaling types individually pathways [15 before2]. consideringIn this regard, significant accurate investment function of in cathepsindrug development. L must be Since first determined the function in of different an enzyme, cell types such as cathepsinindividually L, depends before primarilyconsidering on significant its activity investment profile and in given drug thatdevel theopment. activity Since profiles the function of differentially of an processedenzyme, cathepsinsuch as cathepsin L isoforms L, depends may be veryprimarily different, on its probes activity capable profile ofand reporting given that accurate the activity activity statusprofiles in di offf erentdifferentially cell types processed (and cellular cathepsin location) L isoforms are anticipated may be very to advancedifferent, our probes understanding capable of if cathepsinreporting L accuratebiology. activi Overty the status years, in the different concept cell of types Activity-Based (and cellular Probes location) (ABPs) are anticipatedhas emerged to as a valuableadvance chemicalour understanding biology tool if cathepsin for monitoring L biology. the enzymeOver the activity years, the (not concept just the of expression Activity-Based profile Probes (ABPs) has emerged as a valuable chemical biology tool for monitoring the enzyme activity alone) in cells at the proteome levels [153–156]. (not just the expression profile alone) in cells at the proteome levels [153–156]. In most cases, existing covalent inhibitors containing a recognition motif are adopted and In most cases, existing covalent inhibitors containing a recognition motif are adopted and transformed to ABPs by conjugating optimal detection modalities; these include fluorescent probes, transformed to ABPs by conjugating optimal detection modalities; these include fluorescent probes, affinity labels, radiotracer, and many others (Figure 21). Indeed, the use of ABPs has rather established affinity labels, radiotracer, and many others (Figure 21). Indeed, the use of ABPs has rather cathepsinsestablished as keycathepsins diagnostic as key marker diagnostic for various marker disease for various conditions, disease and conditions, have even and enabled have evenoptical surgicalenabled navigation, optical surgical leading navigation to an improved, leading surgical to an precision improved [ 157 surgical,158]. Inprecision the following [157,158 sections,]. In the we discussedfollowing cathepsin sections, L-selective we discuss moleculared cathepsin probes L-selective that have molecular been developed probes that and have utilized been for developed monitoring itsand activity. utilized A thorough for monitoring overview its activity. of cathepsin A thorough probe development overview of forcathepsin imaging probe purpose development could be foundfor elsewhereimaging [purpose10,159–161 could]. be found elsewhere [10,159–161].

FigureFigure 21. 21Schematic. Schematic representation representation ofof enzyme-labelingenzyme-labeling and and proteome proteome-wide-wide activity activity profiling proﬁling studies studies usingusing probes probes for for selective selective detection detection of of activeactive andand functional cathepsin cathepsin L L enzyme. enzyme.

Molecules 2020, 25, 698 25 of 41

4.1. Radio-Labelled Radio-labeled inhibitors have long been used as a primary mode for detecting active cysteine proteases both in vitro and in vivo. Docherty et al. first used a chloromethyl inhibitor containing 125 radioactive iodine, I-Tyr-Ala-Lys-ArgCH2Cl, and detected cathepsin B in crude granule fraction of islet cells [162]. In their follow up study, they also presumably identified cathepsin L in insulin Molecules 2020, 25, 698 24 of 40 secretory granuleMolecules 2020 using, 25, 698 the same radio-isotopically labeled inhibitor [163]. The24 of mechanism 40 of 4.1. Radio-Labelled detection involves4.1. Radio-Labelled covalent modification of the target protein that shows up as a distinct band upon performing autoradiography.Radio-labeled inhibitors This have chloromethyl long been used containing as a primary inhibitormode for detecting turned active out cysteine to be non-selective Radio-labeled inhibitors have long been used as a primary mode for detecting active cysteine proteases both in vitro and in vivo. Docherty et al. first used a chloromethyl inhibitor containing due to its reactivityproteases both towards in vitro and trypsin, in vivo.a Docherty serine et protease. al. first used This a chloromethyl was followed inhibitor by containing the discovery of a 125 2 radioactive iodine, 125I-Tyr-Ala-Lys-ArgCH2Cl, and detected cathepsin B in crude granule fraction of radioactive iodine, 125I-Tyr-Ala-Lys-ArgCH2Cl, and detected cathepsin B in crude granule fraction of radioactive-peptidyldiazomethaneislet cells [162]. In their follow compoundup study, they (Entryalso presumably P1, Table identified3), a selectivecathepsin L cysteinein insulin proteinase islet cells [162]. In their follow up study, they also presumably identified cathepsin L in insulin inhibitor [164secretory]. Mason granule et al. using adopted the same the radio-isotopically peptidyldiazomethane labeled inhibitor scaff old,[163]. which The mechanism potently of inhibited both secretory granule using the same radio-isotopically labeled inhibitor [163]. The mechanism of detection involves covalent modification of the target protein that shows up as a distinct band upon cathepsin Ldetection and B. Thisinvolves sca covalentffold showed modification improved of the target inhibition protein that profile shows uponup as a iodination,distinct band upon as demonstrated performing autoradiography. This chloromethyl containing inhibitor turned out to be non-selective performing autoradiography. This chloromethyl containing inhibitor turned out to be non-selective by Crawforddue etto al.its reactivity [103] This towards inhibitory trypsin, a agent serine protease. was then This transformed was followed by to the a radio-labeleddiscovery of a probe via due to its reactivity towards trypsin, a serine protease. This was followed by the discovery of a incorporationradioactive-peptidyldiazomethane of 125I[164]. The developed compound probe (Entry was P1, utilizedTable 3), a to selective detect cysteine both cathepsinproteinase L and B in radioactive-peptidyldiazomethane compound (Entry P1, Table 3), a selective cysteine proteinase inhibitor [164]. Mason et al. adopted the peptidyldiazomethane scaffold, which potently inhibited Kirsten-virus-transformedinhibitor [164]. Mason KNIH et al. 3T3adopted cells. the Thepeptidyldiazomethane incubation of cellularscaffold, which extracts potently with inhibited P1 followed by gel both cathepsin L and B. This scaffold showed improved inhibition profile upon iodination, as electrophoresisboth showedcathepsin theL and presence B. This scaffold of two showed protein im bandsproved atinhibition 30 and profile 23 kDa, upon showing iodination, two as active forms demonstrated by Crawford et al. [103] This inhibitory agent was then transformed to a radio-labeled demonstrated by Crawford125 et al. [103] This inhibitory agent was then transformed to a radio-labeled of cathepsinprobe L. Active via incorporation cathepsin of 125 BI [164]. was The also developed detected probe at was around utilized 33–35 to detect kDa. bothInterestingly, cathepsin L and pulse-chase probe via incorporation of 125I [164]. The developed probe was utilized to detect both cathepsin L and B in Kirsten-virus-transformed35 KNIH 3T3 cells. The incubation of cellular extracts with P1 followed experimentsB in with Kirsten-virus-transformed [ S]methionine-labeled KNIH 3T3 proteins cells. The incubation only detected of cellular two extracts separate with P1 bands followed at 36 kDa and by gel electrophoresis showed the presence of two protein bands at 30 and 23 kDa, showing two 39 kDa, whichby gel correspond electrophoresis to showed the intracellular the presence of inactive two protein precursors bands at 30 ofand cathepsin 23 kDa, showing L and two B respectively. active forms of cathepsin L. Active cathepsin B was also detected at around 33–35 kDa. Interestingly, active forms of cathepsin L. Active35 cathepsin B was also detected at around 33–35 kDa. Interestingly, This indicatedpulse-chase that the experiments inactive with precursor [35S]methionine-labeled proteins did proteins not only react detected with two P1, separate demonstrating bands at its unique pulse-chase experiments with [35S]methionine-labeled proteins only detected two separate bands at 36 kDa and 39 kDa, which correspond to the intracellular inactive precursors of cathepsin L and B ability to quantify36 kDa and only 39 kDa, active which protein. correspond Further, to the intracellular P1 wasutilized inactive precursors to probe of activecathepsin cathepsin L and B L and B in respectively. This indicated that the inactive precursor proteins did not react with P1, demonstrating respectively. This indicated that the inactive precursor proteins did not react with P1, demonstrating different humanits unique tissues ability as to wellquantify as only in lysosomes active protein. and Further, whole P1 was cells utilized [165, 166to probe]. In active a follow-up cathepsin L study, Xing et its unique ability to quantify only active protein. Further, P1 was utilized to probe active cathepsin L al. developedand Fmoc-[IB in different2]Tyr-Ala-CHN human tissues as2 well(Entry as in P2,lysosomes Table 3and) that whole selectively cells [165,166]. detected In a follow-up cathepsin L and and B in different human tissues as well as in lysosomes and whole cells [165,166]. In a follow-up 2 2 B over cathepsinstudy, Xing S, exhibiting et al. developed a faster Fmoc-[I rate2]Tyr-Ala-CHN of inactivation2 (Entry towardsP2, Table 3) cathepsin that selectively L [167 detected]. The developed study, Xing et al. developed Fmoc-[I2]Tyr-Ala-CHN2 (Entry P2, Table 3) that selectively detected cathepsin L and B over cathepsin S, exhibiting a faster rate of inactivation towards cathepsin L [167]. compoundcathepsin successfully L and B probedover cathepsin the amountS, exhibiting of a faster active rate cathepsin of inactivation L andtowards B incathepsin different L [167]. cell-lines; two The developed compound successfully probed the amount of active cathepsin L and B in different The developed compound successfully probed the amount of active cathepsin L and B in different unknown proteinscell-lines; two also unknown got labeled proteins in also certain got labeled cases. in certain Overall, cases. Overall, these probesthese probes enabled enabled the the detection of cell-lines; two unknown proteins also got labeled in certain cases. Overall, these probes enabled the detection of active cathepsin enzymes with their cellular location, thereby advancing the knowledge active cathepsindetection enzymes of active cathepsin with their enzymes cellular with location,their cellular thereby location, thereby advancing advancing the the knowledge knowledge of cathepsin of cathepsin L biology. L biology. of cathepsin L biology.

Table 3. Reported probes of cathepsin L, their detection module, efficacy, and utilities. Table 3. ReportedTable 3. Reported probes probes of cathepsinof cathepsin L, their their detection detection module, module, efficacy, eandﬃcacy, utilities. and utilities. Mechanism Mechanism of Probe Efficacy (Cathepsin of MechanismProbe of # Chemical Structure Probe Class Efficacy (Cathepsin Selectivity Factor Action References # Chemical Structure Probe Class EﬃL)cacy (CathepsinSelectivitySelectivity Factor ActionProbe ActionReferences # Chemical Structure Probe Class L) (Demonstrate References L) Factor (Demonstrate(Demonstrated d Utilities) d Utilities)Utilities) Covalent and Mason et al., 2nd order rate Covalent and Mason et al., Radio- 2nd order rate CovalentirreversibleCovalent and andMason1989 [165] etMason al., et al., Radio- 2nd order2nd rate− order1 −1 rate irreversible 1989 [165] P1 Radio- constant (M−1s−1) Cat B: 23 irreversible 1989 [165] P1 labelled constant (M−1s−1) 1 1 Cat B: 23 (In vitroirreversible Wilcox et1989 al., [165] P1P1 Radio-labelledlabelled constantconstant (M s (M) s ) Cat B:Cat 23 B: 23 (In vitro Wilcox et al., labelled 240000 − − (In vitro(In vitro Wilcox Wilcoxet al., et al., 240000 Cellular) 1992 [166] 240000 Cellular) 1992 [166] Cellular) 1992 [166]

Covalent and 2nd order rate CovalentCovalent and and Radio- 2nd order2nd rate order rate Cat B: 2.4 Covalentirreversible and Xing et al., 1998 P2 Radio- constant2nd order (M rate−1s−1 ) Cat B:Cat 2.4 B: 2.4 irreversibleirreversible Xing et al.,Xing 1998 et al., P2 Radio- constant (M−1s−1) 1 1 Cat B: 2.4# irreversible Xing et al., 1998 P2P2 Radio-labelledlabelled constantconstant (M−1s−1 (M) − s− ) Cat S: NI # # (In vitro [167] labelled 60900 Cat S:Cat NI S:# NI (In vitro(In vitro [167] 1998 [167] 60900 60900 Cellular) Cellular)Cellular)

Gelhaus et Gelhaus et al., InhibitorInhibitor CovalentCovalent and andGelhaus et al.,al., 2004 Affinity- Inhibitor Cat B:Cat 36 B: 36 Covalent and 2004 [168] P3P3 AAffinity-ﬃnity-labeled Constant(µM)Constant( µM) Cat B: 36 irreversibleirreversible 2004 [168] [168] P3 labeled Constant(µM) Papain:Papain: 4 4 irreversible Vicik et al., 2006 labeled (S,S) 1.4(S,S) 1.4 Papain: 4 (In vitro)(In vitro)Vicik et al.,Vicik 2006 et al., (S,S) 1.4 (In vitro) [169] [169] 2006 [169]

Molecules 2020, 25, 698 26 of 41

Table 3. Cont.

Mechanism of Eﬃcacy (Cathepsin Selectivity Probe Action #Molecules Chemical 2020, Structure25, 698 Probe Class 25 of 40 References Molecules 2020,, 2525,, 698698 L) Factor (Demonstrated25 of 40 Utilities)

Cat B:Cat 9 B: 9 Cat B: 9 Inhibitor Constant Cat K:Cat 3 K: 3 InhibitorInhibitor Constant Constant Cat K: 3 (µM) Cat S:Cat 0.3 S: 0.3 (µM) (µM) Cat S: 0.3 Covalent and Photoaffinity 3.6 Cat V:Cat 0.15 V: 0.15 CovalentCovalent and andTorkar et al., P4 Photoaffinity 3.6 3.6 Cat V: 0.15 irreversible Torkar etTorkar al., et al., P4P4 Photoa-basedﬃ nity-based(able to detect [P4 did[P4 show did show irreversibleirreversibleirreversible 2012 [170] -based (able to(able detect to detect [P4 did show (In vitro) 2012 [170]2012 [170] picomolar amount of remarkableremarkable (In vitro)(In vitro) picomolarpicomolar amount amount of ofremarkable protein) selectivityselectivity in vivo protein)protein) selectivity in vivo and notin in vivovitro] and and not in vitro] not in vitro]

Reversible Two-photon ReversibleReversible Na et al., 2012 5 Two-photonTwo-photon N.D N.D (In vitro Na et al., 2012Na et al., 5 5 FRET-based N.D N.DN.D N.D (In vitro(In vitro [171] FRET-basedFRET-based Cellular) [171] 2012 [171] Cellular)Cellular)

CathepsinCathepsin L specific L speciﬁc Cat V:Cat 847 V: 847 CovalentCovalent and and Cathepsin L specific Cat V: 847 Covalent and (some(some degree degreeof of Cat B:Cat 413 B: 413 irreversibleirreversible Poreba Porebaet al., et al., 6 6 FluorescentFluorescent (some degree of Cat B: 413 irreversibleirreversible Poreba et al., 6 Fluorescent labellinglabelling was seen was for seen forCat S:Cat 1431 S: 1431 (In vitro(In vitro 2018 [172]2018 [172] labellinglabelling waswas seenseen forfor Cat S: 1431 (In vitro 2018 [172] cat V andcat B) V and B) Cat K:Cat >1600 K: > 1600 Cellular)Cellular) cat V and B) Cat K: >1600 Cellular)

Cat B:9556Cat B:9556 Cat B:9556 Cat K:Cat 29 K: 29 Cat K: 29 CovalentCovalent and and Cat H: NI Covalent and −1 −1 1 1 Cat H:Cat NI H: NI Clickable and 2nd Order2nd Order (M−1 s− (M1) s ) Cat H: NI irreversibleirreversible Dana et al.,Dana 2019 et al., 7 7 Clickable 2nd Order (M−1 ss−1) − − Cat D:Cat NI D: NI irreversibleirreversible Dana et al., 2019 7 and taglesstagless 430000430000 Cat D: NI (In vitro(In vitro [173] 2019 [173] and tagless 430000 Cat G:Cat NI G: NI (In vitro [173] Cat G: NI Cellular)Cellular) hPTP1B:hPTP1B: NI NI Cellular) hPTP1B: NI Trypsin: NI Trypsin:Trypsin: NI NI

−1 −1 Selectivity > 2nd Order (M−1 s−1) Selectivity > 150- Covalent and 2nd Order (M−−11 s−−11) 1 Selectivity1 > 150- Covalent and 2nd229300 Order2nd (mix-Gd) Order (M s (M ) − sfold:−Selectivity) cat 150-fold:B, V, > and150- S cat CovalentirreversibleCovalent and andPoreba et al., 8 TOF-based 229300 (mix-Gd) fold: cat B, V, and S irreversible Poreba et al., 8 TOF-based 229300232500229300 (mix-Gd)(159-Tb) (mix-Gd) fold:N.I: cat cat B,B, K V, V,and and and S S irreversible(In vitroirreversible Poreba2019 [174] Porebaet al., et al., 8 8 TOF-basedTOF-based 232500 (159-Tb) N.I: cat K and (In vitro 2019 [174] 227000232500232500 (175-Lu)(159-Tb) (159-Tb) N.I:Legumain cat N.I:K and cat K Cellular)(In vitro(In vitro 2019 [174]2019 [174] 227000 (175-Lu) Legumain Cellular) 227000227000 (175-Lu) (175-Lu) Legumainand Cellular)Cellular)

Legumain The detecting agents—radionuclide (P1, P2), biotin (P3), fluorophore (P4, P5, P6), clickable acetylene The detecting agents—radionuclide (P1, P2), biotin (P3), fluorophore (P4, P5, P6), clickable acetylene The detecting(P7), agents—radionuclide and lanthanide containing (P1, DOTA P2), biotin (P8)—is (P3), red-color fluorophore coded and (P4, the P5, photoactivatable P6), clickable benzoyl acetylene (P7), and (P7), and lanthanide containing DOTA (P8)—is red-color coded and the photoactivatable benzoyl lanthanide containinggroup (P4) and DOTA quencher (P8)—is (P5) red-coloris coded in codedblue. #: andNo Inhibition. the photoactivatable benzoyl group (P4) and quencher group (P4) and quencher (P5) is coded in blue. #: No Inhibition. (P5) is codedgroup in blue. (P4)# and: No quencher Inhibition. (P5) is coded in blue. : No Inhibition. 4.2. Affinity-Based 4.2. Affinity-Based 4.2. A nity-BasedGelhaus et al. first developed biotinylated aziridine-2,3-dicarboxylate and demonstrated its anti- ffi Gelhaus et al. first developed biotinylated aziridine-2,3-dicarboxylateine-2,3-dicarboxylate andand demonstrateddemonstrated itsits anti-antiplasmodial activity using cell-based studies [168]. Since, the biotinylated compound inhibited plasmodial activity using cell-based studies [168]. Since, the biotinylated compound inhibited Gelhausplasmodial et al. protease first developed falcipain and biotinylated cathepsin L, authors aziridine-2,3-dicarboxylate suggested that this scaffold could and be utilized demonstrated its plasmodial protease falcipain and cathepsin L, authors suggested that this scaffold could be utilized for the development of cell-permeable, non-radioactive reagents that selectively labels enzymes anti-plasmodialfor theactivity development using of cell-permeable, cell-based studies non-radioa [168ctive]. reagents Since, thethat biotinylatedselectively labels compound enzymes inhibited involved in parasite pathogenicity. Later on, Vicik et al. adopted this motif (Entry P3, Table 3) and plasmodialinvolved proteaseinvolved inin falcipain parasiteparasite pathogenicity.pathogenicity. and cathepsin LaterLater on, L,on, authorsVicikVicik etet al.al. suggested adoptedadopted thisthis that motifmotif this (Entry(Entry sca P3,P3,ffold TableTable could 3)3) andand be utilized for developed an affinity label to probe cathepsin L activity [133]. The aziridine analog irreversibly developed an affinity label to probe cathepsin L activity [133]. The aziridine analog irreversibly the developmentinactivates of cell-permeable,the enzyme via covalent non-radioactive modification, as reagents discussed thatpreviously. selectively The conjugated labels enzymes biotin involved inactivatesinactivates thethe enzymeenzyme viavia covalentcovalent modification,modification, as discussed previously. The conjugated biotin moiety is utilized for affinity pull down and target identification. When (S,S) isomer of the in parasite pathogenicity.moiety is utilized Laterfor affinity on, Vicikpull down et al. and adopted target identification. this motif (EntryWhen (S P3,,,S) isomer Table 3of) andthe developed biotinylated probe was incubated with cathepsin L and subjected to gel electrophoresis, electro- an affinity labelbiotinylated to probe probe cathepsin was incubated L activity with cathepsin [133]. L Theand subjected aziridine to analoggelgel electrophoresis,electrophoresis, irreversibly electro-electro- inactivates the transferred to a membrane, and exposed to streptavidin-alkaline phosphatase conjugate, a strong transferred to a membrane, and exposed to streptavidin-alkaline phosphatase conjugate, a strong enzyme vialabeling covalent of the modification, enzyme-inhibitor as discussedcomplex was previously. observed. However, The conjugated when the enzyme biotin was moiety treatedis utilized for labelinglabeling ofof thethe enzyme-inhibitorenzyme-inhibitor complexcomplex waswas observed.observed. However,However, whenwhen thethe enzymeenzyme waswas treatedtreated with E-64, an active site-directed competitive and irreversible cathepsin inhibitor, prior to incubation affinity pullwith down E-64, and an active target site-directed identification. competitive When and i (rreversibleS,S) isomer cathepsin of the inhibitor, biotinylated prior to incubation probe was incubated with P3, the labeling was diminished, clearly demonstrating that P3 competes for the active site of with cathepsinwith LP3, and the labeling subjected was todiminished, gel electrophoresis, clearly demonstrating electro-transferred that P3 competes tofor athe membrane, active site of and exposed cathepsin L. In line with this observation, a desthiobiotinylated analog also exhibited the same trend cathepsin L. In line with this observation, a desthiobiotinylated analog also exhibited the same trend to streptavidin-alkalinebut with reduced phosphatase labeling due to conjugate,its weaker bind aing strong affinity labeling to streptavidin. of the Although enzyme-inhibitor P3 has a complex but with reduced labeling due to its weaker bindinging affinityaffinity toto streptavidin.streptavidin. AlthoughAlthough P3P3 hashas aa was observed. However, when the enzyme was treated with E-64, an active site-directed competitive and irreversible cathepsin inhibitor, prior to incubation with P3, the labeling was diminished, clearly demonstrating that P3 competes for the active site of cathepsin L. In line with this observation, a desthiobiotinylated analog also exhibited the same trend but with reduced labeling due to its weaker binding affinity to streptavidin. Although P3 has a modest binding affinity (Ki = 1.4 µM) to cathepsin L, it exerted a 36-fold selectivity over cathepsin B. Certainly, the affinity labeling technique not only Molecules 2020, 25, 698 27 of 41 served as an ABP for cathepsin L but also provides a premise for developing aziridine-based chemical tools for functional proteomics.

4.3. Photoaffinity-Based Although covalent modifiers of proteins have been vastly exploited as chemical tools for target identification and functional proteomics, photoaffinity probes offer unique mode of action. They bind proteins non-covalently (affinity based solely on non-covalent interactions) first and form a non-selective covalent bond with the closest amino acid residue only upon irradiation. Torkar et al. took advantage of this technique and developed the first photoaffinity-based probe (Entry P3, Table3) to detect active cathepsin L selectively over other members of the family [170]. The photoaffinity-based probe was designed based on existing peptidyl acetyloxymethyl ketone (AOMK), a known covalent modifier of cysteine proteases. During the design, the AOMK group at the C-terminus was replaced by a short di(ethylene glycol) moiety that increased the aqueous solubility and altered the character of the inhibitor from irreversible to a reversible one. The probe was comprised of a lysine residue that was strategically placed to append fluorescent cyanine-3 (Cy3) group for detection. A photoactivatable benzoylphenylalanine amino acid was placed to accommodate the S2 pocket of cathepsin L. The developed probe detected recombinant cathepsin L upon incubation and subsequent irradiation for 40 min at 365 nm as demonstrated by SDS-PAGE. The protein band became invisible when enzyme was incubated with known cathepsin L inhibitors, E-64 and GB111-NH2, respectively, prior to the probe treatment [175,176]. The probe also showed preferential selectivity towards cathepsin L when compared to other two fluorescent-based probes. The probe P3 exhibited a remarkable selectivity for cathepsin L over all other cathepsins in light-mediated labeling experiments with recombinant proteins. The relative labeling percentages (cathepsin B and K: 4%; cathepsin S: 1%) were insignificant, except for cathepsin V (27%), the closest homolog of cathepsin L, relative to cathepsin L. Interestingly, U87-MG glioma cell extracts did not present any cathepsin L for detection with the P3 probe. However, when the same cell extracts were treated with recombinant cathepsin L added externally and subjected to a labeling experiment, the probe selectively detected the desired protein in complex proteome. The mechanism of protein labeling by P3 was attributed to putative bond formation between benzophenone and non-conserved Met161 at the S2 site of cathepsin L. Although the developed probe lacks cell-penetrability, the authors envisioned that the technique might have diagnostic and prognostic value where cathepsin L overexpression is high, such as in malignant tissues.

4.4. Fluorescence-Based

4.4.1. Two-Photon Based Although several fluorescent-based molecular probes have been reported for cysteine cathepsins, the superiority of two-photon-based over one-photon based imaging technique inspired Na et al. to develop probes with a better cellular imaging profile [171]. Notably, the two-photon fluorescence imaging technique provides increased tissue penetration depth with reduced photobleaching, and a lower tissue autofluorescence. The authors first fabricated a microarray with 105 different peptidyl aldehydes and screened against GFP-labeled cathepsin L enzyme; this led to the identification of two inhibitory hits that potently inactivated enzyme with IC50 values of 14.5 and 20.8 nM. The lead inhibitors were further structurally modified to include (a) a two-photon dye, DL-1 (a 4,6-bis(4-hydroxystyryl)pyrimidine derivative), at P1 position, and (b) Disperse Red 1 dye, a fluorescence quencher, in the place of aldehyde moiety. The resulting imaging probes (Entry P5 in Table3 is one of such examples) showed a time-dependent increase in fluorescence signals when treated with HepG2 cell lysates, a mammalian liver cancer cell-line known to overexpresses cysteine cathepsins. Enhanced fluorescence signal is due to the release of the quencher upon successful proteolytic cleavage. Furthermore, to assess the suitability of P5 as imaging agents for live cells, HepG2 cells were incubated with the probe and subjected to a live-cell imaging analysis. There were strong fluorescence signals Molecules 2020, 25, 698 28 of 41 from endolysomal compartments that disappeared completely when cells were pretreated with E-64, validating the target specificity of the probe. As the probe was developed using cathepsin L as a model enzyme, it likely will lack specificity and perhaps interact with other members of the family. Still, however, this motif could potentially be utilized to develop selective ABPs targeting respective cathepsins and assess their activities in a tissue environment, as proposed by the authors.

4.4.2. One-Photon Based Activity-based probes with a single photon fluorescent tag have been successfully utilized for the functional analysis of target proteins both in vitro and in vivo. Poreba et al., in their pursuit of developing cathepsin L selective imaging agent, took advantage of this technology [172]. They realized the importance of developing selective cathepsin L substrate that will bind to the active site of the enzyme over other homologous proteins. To do so, they employed Hybrid Combinatorial Substrate Library (HyCoSuL) technology that provides information on optimal chemical space inside the enzyme active sites by strategically scanning diverse peptide library containing both natural and unnatural amino acids. This led to the acquisition of a panel of compounds with desired properties. The potent hits discovered by HyCoSuL technology were further optimized to gain selectivity over other cathepsins while identifying the most efficient substrate based on Michaelis Menten parameters. Unfortunately, when the chosen peptide substrate transformed to an activity-based probe by appending an acyloxymethylketone warhead and a biotin tag, it showed cross-reactivity with cathepsin B. Despite biotinylated probe’s somewhat compromised selectivity, the authors still assessed its activity-based labeling profile in HEK293T cells; as expected, cathepsin L labeling was primarily observed with minor amounts of cathepsin B. Further optimization by swapping the biotin and Arg with cyanine-5 and Cys(Bzl) (Entry P6, Table3) groups was carried out next. The newly developed fluorescent ABP, P6, showed an enhanced selectivity (in comparison to pan-cathepsin probes) against a panel of other recombinant cathepsins (cathepsin V,B, S, and K), as well as cellular extracts derived from HEK293T and MDA-MB-231 cells. The developed probe served as an effective imaging agent for cellular cathepsin L activity in human MDA-MB-231 breast cancer cells when incubated for 8 h. The selectivity of the probe started to recede with longer incubation time. The importance of optimizing the probe concentration and time-course of the reaction was evident from these experiments. Interestingly, P6 only detected active cathepsin L and not procathepsin L; this certainly signifies the effectiveness of the developed probes as an activity-based probe. The authors further examined the colocalization of the probe with both cathepsin L and B in MDA-MB-231 cells by treating the cells with respective cathepsin antibodies and performing a quantitative pixel analysis from a set of fluorescence microscopy images. This supported the previous observation as the highest weighted colocalization coefficient was obtained for cathepsin L and not for cathepsin B. The developed compound certainly harbors the key traits of an effective activity-based probe, as the authors duly envisioned its significance in deciphering cathepsin L biology in the coming years.

4.5. Clickable and Tagless As noted above, fluorescence-based imaging probes have been successfully developed to gain access to unknown functionalities of cathepsin enzymes. However, the bulkiness and often multiple charges associated with the fluorophore and/or quencher structures on these probes likely render them poorly cell-permeable and reduce their target affinity. To address this issue, Dana et al. adopted a previously reported peptidyl vinylsulfonate inhibitor KD-1—a highly potent, selective, covalent and irreversible inhibitor of cathepsin L discussed in Section 3.7—and tactically appended a small alkynyl group at the para-position of the Cbz group [117]. This led to the development of a clickable and tagless activity-based probe (catABP) of cathepsin L that retained the key desirable traits of KD-1; i.e., cell permeability, charge profile, molecular weight, potency and selectivity profile [170]. This strategy eliminated the requirements of including bulky and charged fluorophore/quencher moiety to the probe. One of the inherent advantages of this approach is that the labeling can be performed in live Molecules 2020, 25, 698 29 of 41 cellular environment with high efficiency. After cell lysis, the labeled cathepsin L can be quantified by performing click chemistry protocol with a fluorescent azide containing dye, resolving the protein using gel-electrophoresis, and directly scanning the gel for fluorescence signal. As anticipated, the developed KDP-1 probe (Entry P7, Table3), exhibited rapid inactivation kinetics, retained selectivity for cathepsin L, and labeled recombinant active cathepsin L in an activity-dependent manner; the heat-denatured and E-64 treated cathepsin L showed no labeling when subjected to the identical labeling protocol. A mass-spectrometric analysis of the enzyme-probe complex concluded that the probe was active-site directed, and covalently modified the catalytic Cys residue for inactivation. Since the KDP-1 probe was developed with the intention of capturing active cathepsin L in vivo and in human live cell culture, the probe was tested for its cytotoxicity in MDA-MB-231 cells; no cytotoxicity was observed in these cells, even at a concentration as high as 10 µM. Incubation of MDA-MB-231 cells overexpressing cathepsin L with KDP-1 attenuated the intracellular cathepsin L activity in a dose-dependent manner; this was demonstrated by live-cell imaging and wound healing assay. Finally, KDP-1 also interfered with the hatching process of post-fertilized zebrafish embryos, further validating probe’s in vivo activity; notably, cathepsin L serves as the key regulator of the hatching process [177–179]. In conclusion, KDP-1 demonstrated many desirable attributes of a good probe and is anticipated to find extensive applications in probing cathepsin L function in cells from diverse origins.

4.6. Mass Cytometry-Compatible Activity-Based Probes Although fluorophores-containing molecules have been appreciated as useful imaging probes, commonly used fluorophores often suffer from spectral overlapping that limits the number of targets that can be analyzed concomitantly [180]. To address this issue, Poreba et al. recently developed protease-selective lanthanide-labeled probes compatible with mass cytometry which allows subsequent analysis by both mass and imaging mass cytometry (IMC) [171]. These metal-tagged, time of flight activity-based probes (TOF) allowed them to determine cellular activities and location of three lysosomal proteases. Thus, using cathepsin L, B, and legumain as the model systems, they elegantly crafted an activity-based probe by incorporating (a) a protease-selective peptide sequence for specific enzyme recognition, (b) the acyloxymethylketone as electrophilic warhead to trap the target enzyme, and (c) the dodecanetetraacetic acid (DOTA) for metal chelation that is tethered to the peptide sequence via a linker (Entry P8, Table3). They further incorporated three di fferent lanthanides, 159Tb, 175Lu, and Gd (a mixture of naturally occurring six isotopes), to validate their approach and to further evaluate the influence of isotopes on enzyme binding specificity. The newly developed probes exerted promising selectivity toward both recombinant proteases and proteases from cancer cell lines, HCT-116 and MDA-MB-231.The authors were able to simultaneous detect the activities of proteases in HCT-116 cells. Moreover, each of the protease-specific probes exerted a similar labelling efficiency in HCT-116 cells, regardless of their metal counterparts, which further reinforces the compatibility of this class of probes as cytometry-labelling agents. They extended their investigation to THP-1 cells, a non-adherent monocyte-like cell line, that expresses both cathepsin B and L but contains very low levels of legumain enzyme. Probe treated THP-1 showed a clear labeling of both cathepsin B and L with no detectable activity of legumain. This finding was consistent with their transcriptional data. Finally, the developed probes not only allowed to detect the activome of cathepsin L, B, and legumain in peripheral blood mononuclear cells (PBMC) but also enabled to categorize NK cells in two distinct populations based on protease activome levels. This strategy, unlike many existing technologies, thus allowed the simultaneous detection of target proteases, thereby providing a more holistic understanding of the activome. This certainly merits the future development of TOF-based probes for multiplexed enzyme activity detection.

5. Final Perspectives The inhibition of cathepsin L has continue to emerge at the forefront of drug development for several human diseases. Yet, no inhibitory agents targeting cathepsin L have advanced to clinical Molecules 2020, 25, 698 30 of 41 trials. While inhibitors of cathepsin B and S are currently being evaluated in clinical trials, recent failure of Odanacatib, a cathepsin K inhibitor for osteoporosis, in late stage clinical trial has made pharmaceutical industries wary of targeting cathepsins. The key challenge remains gaining inhibitor selectivity with respect to the other members of cathepsins and directing them to the targeted cell types for selective functional perturbation. With its ubiquitous expression profile, the function of cathepsin L in individual cell types must be precisely defined first; this is especially important since several isoforms of cathepsin L have been reported in the distinct cellular locations, and their activity and functional profile in individual cell types still remain poorly documented. While recently developed cathepsin inhibitors and probes have significantly advanced our understanding of cathepsin L function in both normal and disease cells, more efforts are needed for the development of isoform-selective reagents for further advancement; perhaps new allosteric modules on cathepsin L enzyme can be explored and exploited for precision targeting. Fortunately, several structural coordinates for cathepsin L enzyme forms, some with a diverse set of complexing ligands and some without, are now available (Table4). These could aid in the development of isoform-selective inhibitory probes that will enable researchers to assess the context-specific needs of targeting cathepsin L in different cellular states. In recent years, disease-associated protease activatable prodrugs have gained much recognition and garnered breakthrough therapies in the area of antibody-drug conjugation (ADC) [181]. The majority of the ADCs are constructed by tethering a cytotoxic drug with an antibody via a protease-sensitive module for targeted delivery. Cathepsin B-specific module, for example, has been successfully implemented for this role, which led to the development of FDA approved therapies; for example, ® Brentuximab vedotin (Adcetris ) for CD30-positive relapsed or refractory Hodgkin0s lymphoma. In addition, protease cleavable prodrug strategy has also inspired the development of cathepsin B selective probe and even enabled real-time monitoring of drug release [182,183]. Interestingly, Ueki et al. slightly maneuvered this approach to acquire a prodrug which gets serially activated by histone deacetylase (HDAC) and cathepsin L, and subsequently delivers the cytotoxic payload, puromycin, to cancer cells [184]. This strategy has thus enabled selective targeting of cancer cells, specifically with high HDAC and cathepsin L activities. Taken together, these developments surely lay the foundation for the development of future cathepsin L-based therapies. Much excitement remains as new cathepsin L functions continue to emerge from specific cell types in the coming years.

Table 4. Reported structural studies of cathepsin L enzyme.

PDB Entry Method Resolution (Å) Reference 1CJL X-ray 2.20 [42] 1CS8 X-ray 1.80 [42] 1ICF X-ray 2.00 [42] 1MHW X-ray 1.90 [95] 2NQD X-ray 1.75 [185] 2VHS X-ray 1.50 [186] 2XU1 X-ray 1.45 [121] 2XU3 X-ray 0.90 [121] 2XU4 X-ray 1.12 [121] 2XU5 X-ray 1.60 [121] 2YJ2 X-ray 1.15 [97] 2YJ8 X-ray 1.30 [97] 2YJ9 X-ray 1.35 [97] 2YJB X-ray 1.40 [97] 2YJC X-ray 1.14 [97] Molecules 2020, 25, 698 31 of 41

Table 4. Cont.

PDB Entry Method Resolution (Å) Reference 3BC3 X-ray 2.20 [187] 3H89 X-ray 2.50 [128] 3H8B X-ray 1.80 [128] 3H8C X-ray 2.50 [128] 3HHA X-ray 1.27 [147] 3HWN X-ray 2.33 [147] 3IV2 X-ray 2.20 [188] 3K24 X-ray 2.50 [188] 3KSE X-ray 1.71 [189] 3OF8 X-ray 2.20 [96] 3OF9 X-ray 1.76 [96] 4AXL X-ray 1.92 [190] 4AXM X-ray 2.80 [190] 5F02 X-ray 1.43 [191] 5I4H X-ray 1.42 [192] 5MAE X-ray 1.00 [145] 5MAJ X-ray 1.00 [145] 5MQY X-ray 1.13 [123] 6EZP X-ray 1.37 [98] 6EZX X-ray 2.34 [98] 6F06 X-ray 2.02 [98]

Funding: S.K.P. gratefully acknowledges the financial support from the National Science Foundation (NSF); Grant no. 1,709,711 for this work. Acknowledgments: Authors also wish to thank Senthil Perumal for providing critical feedback during the preparation of this manuscript. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

MA Methacrylate Phe Phenylalanine Gly Glycine Z/Cbz Carboxybenzyl His Histidine NBz Nitrobenzyl iAm isoamyl SAR Structure Activity Relationship AOMK Acyloxymethanes/Acyloxymethyl Ketones Leu Leucine Met Methionine Azi Aziridine FRET Fluorescence Resonance Energy Transfer catABP Clickable and Tagless Activity-based Probe Molecules 2020, 25, 698 32 of 41

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