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Biol. Chem., Vol. 391, pp. 357–374, April 2010 • Copyright by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2010.037

Review

Natural and engineered inhibitors: an emerging pharmacopoeia

Joakim E. Swedberg, Simon J. de Veer and ulated activation cascades, suggesting an involvement in a Jonathan M. Harris* diverse range of physiological processes (Pampalakis and Sotiropoulou, 2007). Both liver-derived KLKB1 and tissue- Institute of Health and Biomedical Innovation, Queensland derived KLK1, as well as KLK2 and KLK12 in vitro (Giusti University of Technology, Brisbane, Queensland 4059, et al., 2005), participate in the progressive activation of Australia , a bioactive involved in blood pressure * Corresponding author homeostasis and inflammation initiation (Bhoola et al., e-mail: [email protected] 1992). Although this is the only demonstration of classical kininogenic activity that was the original hallmark of kallik- Abstract rein , the contribution of subsets of KLKs to vital physiological processes is well appreciated. Prostate- The and kallikrein-related peptidases are expressed KLK2, 3, 4, 5 and 14 are involved in seminogelin proteases that control a plethora of developmental and home- hydrolysis (Lilja, 1985; Deperthes et al., 1996; Takayama et ostatic phenomena, ranging from semen liquefaction to skin al., 2001b; Michael et al., 2006; Emami and Diamandis, desquamation and blood pressure. The diversity of roles 2008), KLK6 and 8 have reported functions in defining neu- played by kallikreins has stimulated considerable interest in ral plasticity (Shimizu et al., 1998; Scarisbrick et al., 2002; these from the perspective of diagnostics and drug Tamura et al., 2006; Ishikawa et al., 2008) and KLK5, 7, 8 design. Kallikreins already have well-established credentials and 14 assist in epidermal remodelling via controlled pro- as targets for therapeutic intervention and there is increasing teolysis of corneodesmosomal (Caubet et al., 2004; appreciation of their potential both as biomarkers and as tar- Brattsand et al., 2005; Stefansson et al., 2006; Kishibe et al., gets for inhibitor design. Here, we explore the current status 2007). These kallikrein-driven phenomena are critical pro- of naturally occurring kallikrein -inhibitor complex- cesses within the prostate, central nervous system and skin, es and illustrate how this knowledge can interface with strat- respectively. egies for rational re-engineering of bioscaffolds and design In parallel to identification of the kallikrein locus (and of small-molecule inhibitors. serine proteases generally), attempts have been made to pro- duce inhibitors to regulate these enzymes and thus maintain Keywords: bioscaffold; drug design; inhibitor; phage the delicate homeostatic balance between degradation display; protease positional scanning synthetic and synthesis in crucial biological pathways. It has been un- combinatorial library; sunflower inhibitor. equivocally shown that failure of inhibition in kallikrein- related pathways causes detrimental effects that in turn have Introduction led to a burgeoning interest in the design and synthesis of kallikrein-specific inhibitors. The kallikrein peptidase family Kallikrein structure The extended kallikrein peptidases are defined by homology to either of two ancestral serine proteases, Collectively, the KLKs exhibit a number of structural and or tissue kallikrein, which differ in their location, functional similarities that need to be considered and can be sequence and structure. Whereas plasma kallikrein (KLKB1, exploited in targeted inhibitor design. All tissue KLK located at 4q34-q35) has no related homologue, the tissue consist of five exons of almost identical size with intermit- kallikrein-related peptidases (KLKs) form a highly conserved tent introns displaying a fully conserved phase (Yousef and multi-gene locus encoding enzymes with either trypsin- or Diamandis, 2001). This has consequences in terms of trans- -like activity (Yousef and Diamandis, 2001; lated protease sequence; each KLK contains the conserved Clements et al., 2004). Fifteen KLKs have been characterised (His57, Asp102, Ser195) and to date, arranged as a tandemly clustered array on chromo- N-terminal regulatory pre- and pro- sequences, meaning that some 19q13.3-13.4 (Clements et al., 2004). This represents KLKs are expressed as inactive precursors (zymogens). the largest known continuous collection of proteases within Although the within the classical KLK the (Puente et al., 2003). cluster (KLK1–3) is markedly higher (62–72%) than simi- Typically, KLK proteins are produced according to strict larities with any of the more recently characterised KLKs temporal and spatial expression patterns and function in reg- (25–49%) (Harvey et al., 2000), emerging crystallographic

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358 J.E. Swedberg et al.

studies have revealed that KLK tertiary structure across the Rationale for kallikrein inhibition locus is highly conserved (Figure 1). Indeed, the kallikrein ‘molecular chassis’ conforms to the canonical trypsin fold, Consistent with the important role of kallikrein proteases in consisting of an almost entirely b-sheet arrangement (SCOP normal physiology, defective control of expression or activity trypsin-like serine proteases; SCOP ID 50493). Furthermore, is strongly linked to development of disease. Most prominent although the KLKB1 gene encodes four additional N-terminal is the potential significance of KLKs to certain cancers typ- domains, its level of sequence similarity across the catalytic ified by aberrant KLK expression including lung, pancreatic, domain is not dissimilar to that observed for the tissue KLK colon (Yousef et al., 2004) and hormone-dependent cancers, locus (Figure 1). particularly those of the prostate (Magklara et al., 2000;

Figure 1 Structural alignment of KLK1–15, KLKB1 and b-trypsin. (A) Ribbon plot of KLK1 (PDB accession no. 1SPJ) as a representative KLK structure. b-Sheets and a-helices are shown in yellow and blue, respectively, with important features highlighted in green and purple, with catalytic residues displayed as stick models. (B) Sequence similarity between KLK1–15 and KLKB1 mapped onto the surface of b-trypsin with conservation visualised as a gradient from blue (high), cyan, green and light green to yellow (low). The alignment was produced in Swiss-PdbViewer 4.0.1 using the KLK1, 3-7, KLKB1 and b- trypsin crystal structures (PDB accession nos. 1SPJ, 2ANY, 2ZCH, 2BDH, 2PSX, 1L2E and 2QXI, 1SFI) and KLK2 and 8–15 models created using Swiss Model (Guex et al., 2009). (C) Structural alignment of sequence conservation between KLK1–15, KLKB1 and b- trypsin. Location of b-sheets and a-helices are indicated by yellow arrows and blue cylinders, respectively, with catalytic residues shown in cyan. Important features are highlighted in green and purple and cysteine residues participating in disulfide bonds are shown in yellow. Completely conserved residues and conservative substitutions are shown in dark grey and light grey, respectively.

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Kallikrein inhibitors 359

Petraki et al., 2003; Veveris-Lowe et al., 2005), ovaries teases is commonly controlled by -based inhibitors (Dong et al., 2001; Yousef et al., 2003; Prezas et al., 2006) that adopt the form of a non-hydrolysable substrate. Whereas and breast (Yousef et al., 2002; Zhang et al., 2006; Pampa- almost all inhibitors bind to their cognate protease via an lakis et al., 2009). Contrasting effects of KLK overexpres- exposed motif, the mechanism by which they achieve inhi- sion on cancer physiology have been proposed. KLK bition varies from canonical inhibitors that simply block the overexpression can provide a pathway for tumour develop- protease to serpins that irreversibly disturb the ment and metastasis by degrading the restrictive localised delicate conformation of the protease (Laskowski and Kato, tissue architecture and shifting the cellular signalling axis to 1980). A number of canonical and serpin inhibitors of KLK drive increased proliferation. Alternatively, differential KLK proteases have been identified. These include physiological expression can have a protective function in certain cancers. inhibitors that provide an essential regulatory influence on A considerable amount of research has been devoted to KLK-mediated processes and activation cascades, as well as exploring the utility of KLK panels as hormone-dependent broad-range, exogenous inhibitors that function as valuable cancer biomarkers (Borgono and Diamandis, 2004; Clements research tools for determining the significance of KLK activ- et al., 2004). KLK3 (prostate-specific antigen, PSA) remains ity in experimental systems. Notable KLK inhibitors are dis- the gold-standard biomarker for prostate cancer diagnosis cussed in more detail below and a comprehensive inhibitory and prognosis (Stamey et al., 1987; Catalona et al., 1991), profile of natural and engineered inhibitors is provided in albeit with some degree of controversy. Figure 2A–D. In the context of the skin, a role for KLKs has been iden- tified in the rare but severe disorder Netherton syndrome Canonical inhibitors (OMIM: 256500) (Chavanas et al., 2000; Descargues et al., 2005; Briot et al., 2009), as well as the milder but much Standard-mechanism or canonical inhibitors currently form more frequent condition acne rosacea (Yamasaki et al., 2007; the largest class of protein-based inhibitors. Here, protease Stefansson et al., 2008). Furthermore, KLK1 and KLKB1 inhibition generally involves contact with the target protease have been implicated in a number of diseases associated with via the canonical loop, a characteristic exposed binding motif dysregulation of the kallikrein/kinin system, such as cardio- on the inhibitor surface that is complementary to the active- vascular, renal, inflammatory and gastrointestinal tract dis- site cleft. This interaction is often referred to as analogous ease (Devani et al., 2002; Stadnicki et al., 2003). Finally, to the formation of a protease-substrate complex, since both KLK6 and KLK8 have potential roles in neurodegenerative are non-covalent, tight-binding and reversible with contact conditions such as Alzheimer’s disease (Little et al., 1997; occurring across extended b-sheets (Tyndall et al., 2005). Shimizu-Okabe et al., 2001), multiple sclerosis (Terayama et However, in the protease-inhibitor complex, hydrolysis of the al., 2005; Scarisbrick et al., 2008), epilepsy (Momota et al., reactive-site P1-P19 peptide bond is rare, preventing subse- 1998), synucleinopathies (Iwata et al., 2003) and general quent dissociation of the complex and temporarily removing central nervous system inflammation (Blaber et al., 2004). the ability of the protease to bind substrate (Laskowski and The identification of dysfunctional KLK activity as a major Kato, 1980). The importance of this binding mechanism is pathological influence suggests that targeted inhibition of rel- highlighted by the fact that the conformation of the canonical evant KLKs is likely to be an effective therapeutic strategy. loop is very similar, even among diverse families of protease Transient inhibition of certain kallikrein-mediated pro- inhibitors (Figure 2E). cesses has already been explored and the use of aprotinin to reduce peri-operative bleeding in cardiac surgery by inhib- Kunitz-domain inhibitors iting proteases involved in haemostatic and inflammatory processes was widespread (Fritz and Wunderer, 1983; Terrell Canonical inhibitors of this class are characterised by the et al., 1996). Although initially thought to be successful, this Kunitz domain, a motif spanning 50–60 residues that con- therapy has recently been withdrawn from use following the tains the inhibitory binding loop within a central anti-parallel result of several larger clinical trials that were terminated b-sheet (Figure 2E). Considerable rigidity across the domain before completion because of a persistent trend of increased is imparted by a network of conserved disulfide bonds mortality (Murkin, 2009). Since this outcome is very recent, (Hynes et al., 1990; Perona et al., 1993). Most prominent is the probable physiological explanation is yet to be deter- bovine pancreatic (BPTI), also previously mined, although one might speculate that it involves inter- referred to as kallikrein inactivator, aprotinin or trasylol. Fol- ference with unrelated biological pathways owing to the lowing isolation and crystallisation from bovine pancreatic broad range activity of aprotinin, much like the first-gener- tissue extracts, biochemical characterisation revealed that ation matrix metalloprotease (MMP) inhibitors. BPTI was a potent inhibitor of not only bovine trypsin, but also KLKB1 (Kraut et al., 1930). Later studies that extended to the classical tissue kallikrein locus reported inhibitory activity against KLK1 (Hofmann and Geiger, 1983) and Naturally occurring kallikrein inhibitors KLK2 (Geiger et al., 1980). More recently, BPTI/aprotinin has been widely used as a non-specific reagent to block the Although proteases drive numerous vital physiological pro- activity of kallikrein proteases in vitro, including KLK7 cesses, activity must be tightly regulated to ensure correct (Lundstrom and Egelrud, 1988), KLK5 and 14 (Brattsand et spatial and temporal delivery. The function of mature pro- al., 2005), and KLK2 and 4 (Mize et al., 2008).

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360 J.E. Swedberg et al.

BPTI has no homologue expressed in humans and there- sor protein (APP) and its homologue APPH (Petersen et al., fore is not an endogenous modulator of kallikrein proteases, 1994). A further KLKB1 and KLK1 inhibitor, serine pepti- but several potent inhibitors of KLKB1 and KLK1 have been dase inhibitor Kunitz-type 2 (SPINT2) was later isolated isolated from human tissue, including tissue factor pathway from human placental tissue and termed bikunin because of inhibitor-2 (TFPI-2) (Petersen et al., 1996) and Kunitz-type its double-headed Kunitz domain structure (Delaria et al., protease inhibitor domains from Alzheimer amyloid precur- 1997). Plant material has also proven to be a rich source of

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Kallikrein inhibitors 361

. Figure 2 Comparison of the activity of naturally occurring and engineered kallikrein inhibitors and structural overlay of the conserved binding loop of protein kallikrein inhibitors. Representation of the relative kallikrein inhibitory activity for an extensive selection of reported naturally occurring (blue), engineered protein (orange) and synthetic small-molecule (green) inhibitors. Data were compiled using the reported Ki (canonical inhibitors, panel A and synthetic inhibitors, panel B), IC50 (metal ions, panel C) or ka (covalent inhibitors, panel D) value for each kallikrein-inhibitor complex. Importantly, the relative inhibition values encompass different modes of inhibition and, undoubtedly, different experimental conditions, and therefore solely provide a relative comparison and should not be taken as absolute. Abbreviations: APP(H) KPI, Alzheimer amyloid precursor protein (homologue) Kunitz protease inhibitor; BbTI, Bauhinia bauhinioides trypsin inhibitor; BfTI, Bauhinia forficata trypsin inhibitor; BuXI, Bauhinia ungulate factor Xa inhibitor; BvTIp, Bauhinia variegata trypsin inhibitor, purple variety; CeKI, Caesalpinia echinata kallikrein inhibitor; CMTI, Cucurbita maxima trypsin inhibitor; EcTI, Enterolobium contortisiliquum trypsin inhibitor; LlTI, Leucaena leucocephala trypsin inhibitor; PAI-1, inhibitor-1, STI, soybean trypsin inhibitor; SFTI, sunflower trypsin inhibitor; SLPI, secretory leukocyte protease inhibitor; SwTI, Swartzia pickellii trypsin inhibitor. (E) Overlay of the binding loops from various classes of naturally occurring kallikrein inhibitors (inhibitor/class/PDB accession number): LEKTI domain 6/Kazal/1HOZ; aprotinin/Kunitz/2FTL;

Barley Bowman-Birk inhibitor/Bowman-Birk inhibitor/1TX6; a1-antitrypsin/Serpin/1OPH; SFTI/Bowman-Birk-like/1SFI; E. coli trypsin inhibitor/Ecotin/1EZU; bound to the active site of b-trypsin. Trypsin is shown as grey ribbons, with catalytic residues as stick models. The inhibitors are shown as yellow b-sheets and green coils. Note that all inhibitors interact with the protease via an extended b-sheet (black arrow).

Kunitz-type compounds that inhibit KLKs; KLKB1 (Oliva been identified. Purified from the periplasm of Escherichia et al., 1999), KLK7 (Lundstrom and Egelrud, 1988), KLK5 coli, ecotin is a 32-kDa dimeric serine protease inhibitor with and 14 (Brattsand et al., 2005) are inhibited by soybean tryp- broad-range activity. Characterisation revealed that it is a sin inhibitor (SBTI). Inhibition of KLKB1 has also been very potent inhibitor of proteases of the contact activation demonstrated using novel compounds from Enterolobium, system, including KLKB1 (Ulmer et al., 1995). Hirustasin, Leucaena, Swartzia and several Bauhinia species (Sampaio a serine protease inhibitor isolated from the medical leech et al., 1996), as well as Caesalpinia echinata kallikrein inhib- Hirudo medicinalis, contains a single antistasin-like domain itor (CeKI) (Cruz-Silva et al., 2004). and is a potent inhibitor of KLK1, but not KLKB1 (Sollner et al., 1994). A detailed investigation of KLK inhibitors iso- Kazal-domain inhibitors lated and purified from stratum corneum extracts identified the elafin-like protease inhibitor antileukoprotease as a skin- Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is expressed KLK7 inhibitor and the potato type-1 inhibitor produced as a multi-domain serine protease inhibitor encod- eglin C as an exogenous inhibitor (Franzke et al., 1996). ed by the SPINK5 gene ( 5q31-32) (Magert et More recently, several KLK inhibitors have been isolated al., 1999). Within the skin and thymus, the LEKTI pro-pro- from plant material; Cucurbita maxima trypsin inhibitor tein is processed into up to 15 individual subunits. Structur- (CMTI) -I and -II exhibit inhibitory activity against serine ally, the resulting fragments are typified by a Kazal-like proteases of the clotting cascade, including KLKB1 domain arrangement; an anti-parallel b-sheet bordered by (Grzesiak et al., 2000a), and sunflower trypsin inhibitor two short a-helices is held together by conserved disulfide (SFTI) is a non-specific Bowman-Birk-like inhibitor of links. Most LEKTI domains carry basic residues (Arg or KLK4 (Swedberg et al., 2009). Lys) at their putative P1 sites, suggesting that trypsin-like proteases are likely targets for LEKTI-derived (Magert et al., 1999). A range of LEKTI fragments can mod- Serpins ulate KLK activity, albeit with differing efficiency; KLK5, Abundant in circulating human plasma, serpins are large 7 and 14 are inhibited by LEKTI D8–11 and LEKTI D5 protein-based inhibitors that irreversibly attenuate the activ- (Deraison et al., 2007), and KLK5 and 7 are inhibited by ity of their target protease. Like canonical inhibitors, contact LEKTI D6–99 (Schechter et al., 2005) and LEKTI D6 (Egel- with the protease is made via an extended b-sheet (Figure rud et al., 2005). This activity, in synergy with physiological 2E). However, the serpin mechanism of inhibition is unique factors such as pH, provides an essential regulatory influence in that it is characterised by catalysis of the P1-P19 peptide on the cascade of kallikrein proteases associated with bond, which causes both a change in the serpin conformation desquamation (Borgono et al., 2007b; Deraison et al., 2007). and formation of a protease-inhibitor covalent bond (Loe- An additional SPINK-related product, LEKTI2 encoded by bermann et al., 1984). Recent crystallographic studies have SPINK9, has recently been identified within the skin and shows KLK5-specific inhibition restricted to the palmoplan- further clarified this phenomenon. Since the protease is tight- tar epidermis (Brattsand et al., 2009; Meyer-Hoffert et al., ly linked to the inhibitor, the serpin structural change due to 2009). cleavage at the reactive centre induces a shift in conforma- tion of the bound protease (Huntington et al., 2000). The Other canonical inhibitors resulting loss of protease structure has terminal effects on catalytic ability, namely distortion of the catalytic triad via Aside from the classical Kunitz- and Kazal-type inhibitors, displacement of Ser195 and collapse of vital interactions several other prominent canonical inhibitors of KLKs have formed during zymogen activation (Huntington et al., 2000).

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362 J.E. Swedberg et al.

Several endogenous serpin KLK inhibitors have been Inhibitor engineering identified to date. Best characterised are those found in the circulating plasma, such as a2-macroglobulin, antithrombin Design challenges: potency and specificity

III, a1-antitrypsin, a2-antiplasmin and inhibitor, which inhibit KLKB1 (Gallimore et al., 1979; Schapira et al., Protease inhibitors require high affinity if they are to be suc- 1982), KLK2 (particularly protein C inhibitor) (Deperthes cessful as therapeutic agents. The evolution of HIV-1 pro- et al., 1995; Frenette et al., 1997) and KLK3 (Christensson tease inhibitors revealed important design strategies when et al., 1990). The search for additional serum-based inhibi- analysed from a thermodynamic viewpoint (Freire, 2006). The affinity of the inhibitor for the protease is determined tors for KLK1 other than a1-antitrypsin (Geiger et al., 1981) led to the isolation and characterisation of kallistatin, a novel by the Gibbs free energy of binding, with enthalpy and entro- py changes corresponding to different types of atomic inter- 58-kDa serpin that complexes with KLK1 in vitro more read- actions (Freire, 2006). The change in enthalpy is proportional ily than other circulating inhibitors (Zhou et al., 1992). Fur- to the strength of binding between the inhibitor and the pro- thermore, the recently identified interaction between KLK8 tease, such as hydrogen bonds, van der Waals interactions and serpin B6 (Scott et al., 2007) is of particular interest and salt bridges. However, in aqueous medium the entropy since inhibition of KLK8 by common skin-based inhibitors increases on binding because water molecules are freed from (such as LEKTI fragments) is yet to be successfully solvation layers around hydrophobic areas and thus become achieved. Serpin inhibition of KLK5, 7, 8 and 11–14 has less ordered. Therefore, to achieve maximum affinity, hydro- also been examined in vitro using a -antitrypsin, a -antichy- 1 1 phobic repulsion from the aqueous medium must be com- motrypsin, kallistatin, antithrombin-III, protein C inhibitor, bined with strong inhibitor-protease interactions achieving plasminogen activator inhibitor-1, a -antiplasmin and C1 2 favourable entropy and enthalpy. The first-generation HIV-1 inhibitor (Luo and Jiang, 2006). protease inhibitors (indinavir, nelfinavir and saquinavir) were characterised by favourable entropy but unfavourable enthal- Divalent ions py (Velazquez-Campoy et al., 2001), whereas second-gen- eration inhibitors exhibited both favourable enthalpy and Although not strictly defined as protease inhibitors, several entropy with resulting higher affinity (Ohtaka and Freire, divalent ion species also show an ability to modulate KLK 2005). It thus appears that finding a balance between enthal- protease activity. Initially, studies were confined to the clas- pic and entropic changes on inhibitor-protease binding is sical KLK cluster and it was demonstrated that prostate- vital for high-affinity inhibitors. expressed KLK2 and KLK3 are inhibited by zinc (Lovgren The importance of inhibitor selectivity is underlined by et al., 1999; Malm et al., 2000). KLK3 is also inhibited by the high profile failure in clinical trials of a series of MMP copper, mercury, cobalt and cadmium ions in vitro, although inhibitors. These were designed to block MMP-mediated the extent of inhibition is considerably weaker (Malm et al., remodelling of the cellular microenvironment and thus halt 2000). More recently, zinc inhibition of KLK4 (Debela et al., tumour growth and metastasis (Zucker et al., 2000). Unfor- 2006a), KLK5 (Michael et al., 2006; Debela et al., 2007a), tunately, the majority have been terminated following Phase KLK7 (Debela et al., 2007b), KLK8 (Kishi et al., 2006) and III trials because of a lack of or even negative survival ben- KLK14 (Borgono et al., 2007c) was demonstrated and cor- efits. Critical evaluations of MMPI performance concluded responding crystallographic approaches suggest a potential that this was due, at least in part, to their broad-spectrum molecular mechanism for inhibition, at least for certain activity (Zucker et al., 2000; Coussens et al., 2002; Overall KLKs. For both KLK5 and KLK7, metal ion binding is coor- and Lopez-Otin, 2002; Turk, 2006). This resulted in blockade of important off-target cellular processes that were in fact dinated by exposed His residues (His96 or His99 for KLK5 anti-tumourigenic (Vazquez et al., 1999), as well as activa- and His99 for KLK7) (Debela et al., 2007a,b). The bound tion of pro-tumorigenic signalling pathways (Maquoi et al., divalent ion subsequently interacts with the catalytic His57 2002) or interference with unrelated functions with detri- residue, displacing it from the active site. KLK4 is subtly mental side effects, such as tendonitis-like musculoskeletal different in that the metal ion is produced by pain (Giavazzi et al., 1998). Selective inhibitors can be His25 and Gln77 (Debela et al., 2006a). designed by exploiting unique structural features within the Naturally occurring inhibitors have evolved over time to active site. These can be identified by screening high-affinity function in a temporal and spatial manner that elegantly off- ligands against a panel of the most closely related proteases sets their broad-range activity. However, since disease path- to establish selectivity, which, in the case of a KLK, should ogenesis is highly complex and can involve partial or even include all related tryptic or chymotryptic members of the multiple protease cascades, and since treatments are often family. administered systemically, therapeutic inhibition must be highly specific with low levels of off-target inhibition. This Design challenges: mapping the KLK active site explains why natural inhibitors, although effective at regulating key physiological processes, very rarely have The twin goals of specificity and potency described above appreciable value as therapeutics. Therefore, the focus has are attainable provided the engineered inhibitor has a com- shifted to engineered inhibitors that can deliver the require pletely complementary fit with the active site of its target selectivity. protease. Accordingly, irrespective of the inhibitor being

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Kallikrein inhibitors 363

designed, the first step is to probe the active site of the target . The most common strategies revolve around map- ping the active site of the target protease through bacterio- phage display, positional scanning and sparse matrix substrate libraries. Bacteriophage display (or biopanning) exploits the phys- ical link between peptides on the exterior of the bacterio- phage particle and the genetic material that carries the code for it. In its simplest form, phage display parallels natural evolution, with subpopulations of genetically diverse bacte- riophage libraries iteratively selected and amplified by bind- ing to an immobilised target; see Smith and Petrenko (1997) for a review. This technique has been spectacularly success- ful as a tool for sampling the chemical landscape of short peptides and monoclonal antibodies, resulting in a series of Figure 3 Phage-display cycle. drugs currently available in the clinic, including Avastin, Genetic diversity is generated by ligating degenerate oligonucleo- Rituxan and Lucentis (Dimitrov and Marks, 2009). An tides into the genome of filamentous bacteriophage (typically M13) ingenious twist in the application of phage display has refo- to produce fusion proteins with pIII, pV or pVII bacteriophage coat cused this technique as a tool for discovering protease cleav- proteins. The resultant bacteriophage library is then allowed to inter- age specificities. This strategy turns the affinity maturation act with an immobilised bait molecule (BINDING) after which concept on its head so that non-selected sequences are immo- weakly bound bacteriophages are removed by washes of increasing bilised on a solid support and sequences are selected follow- stringency (WASH) prior to harvesting of more specifically bound ing cleavage from the solid support. This technique, which bacteriophage particles by cycles of high- and low-pH washing (ELUTE). The resulting eluate is then reamplified in an E. coli was pioneered by Matthews and Wells (1993) revolves bacteriophage propagation strain (AMPLIFY) and iteratively around expression of the bacteriophage pIII protein as a screened and amplified through a further three or four cycles to fusion protein with an affinity tag, with the pIII portion of produce a final library through pseudo-evolutionary selection. Indi- the fusion protein being separated from the affinity tag by a vidual clones from this library are then sequenced and validated randomised sequence. The resulting bacteriophage library is through interaction or inhibition assays. then immobilised on an affinity matrix specific for the fusion partner on the modified pIII protein. Bacteriophages that fact that assays were based on peptide hydrolysis whereas have linker sequences that are substrates for a given protease selection was carried out on the basis of protein cleavage. will be cleaved off the solid support and can be harvested Selection bias inherent in biological libraries is circum- by filtration and subject to affinity maturation through iter- vented by the positional scanning synthetic combinatorial ative cycles of amplification, immobilisation and cleavage library (PS-SCL) approach, which uses solid-phase chemis- (Figure 3). To date, this technology has been applied to try to generate diversity. It consists of sub-libraries of pep- KLKB1 (Dennis et al., 1995), KLK2 (Cloutier et al., 2002), tides for each binding subsite of the protease; thus, if the KLK3 (Coombs et al., 1998), KLK6 (Sharma et al., 2008) P1–P4 sites are to be investigated, four sub-libraries are and KLK14 (Felber et al., 2006). These studies in turn guid- needed. These contain 20 pools of peptides, each with a dif- ed the design of the engineered bioscaffolds discussed below. ferent fixed amino acid for that protease subsite (P1, P2, P3 The strength of phage display lies in the magnitude and or P4), whereas the other positions have a mixture of all diversity of the libraries that can be screened. However, the amino acids (Dooley and Houghten, 1993). By screening the screening technique itself is not without inherent bias and rate of proteolysis against all 80 pools, the individual con- blind spots. In particular, pIII fusion peptides have the poten- tribution of each amino acid at each subsite to substrate rec- tial to modulate bacteriophage infectivity. In turn, this can ognition or inhibitor binding is revealed. Positional scanning lead to under- or over-representation of particular sequences has been used to probe the active site of a number of KLKs; during screening through their ability to propagate during the outcomes are summarised in Table 1. reamplification rather than by virtue of their affinity for an Although this information is invaluable, it is important immobilised ligand (Wilson and Finlay, 1998). Peptide when interpreting PS-SCL data to be conscious of the sequences can also be strongly selected by the solid support assumptions on which the method is based. First, it is used for immobilisation of the target protease. Immobilised assumed that the contributions of each position to biological metal affinity chromatography (IMAC) is notorious for its activity are independent of each other. One argument against ability to select for pIII fusion proteins with multiple histi- this is what can be termed P-site overlap: although the results dine residues. These issues are highlighted by studies by from the screen indicate that the same residue is favoured in Cloutier et al. (2002) that identified peptide substrates for adjacent positions, when assayed as an individual peptide, KLK2 using phage display. Although a number of substrates this is not the case. Two PS-SCL studies of KLK4 ranked for the enzyme were identified, none of these achieved the Gln favourably for the P2 and P3 positions (Matsumura et catalytic efficiency of the control substrate (TFRSA), nor al., 2005; Debela et al., 2006b), whereas assays of individ- was this sequence selected. However, this may reflect the ually synthesised peptides revealed that Gln is only favoured

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364 J.E. Swedberg et al.

at these subsites if one but not both Gln are present (Swed- thesised and verified peptides (typically 50–200 members) berg et al., 2009). This suggests that the residue interacts selected from the PS-SCL (Swedberg et al., 2009). This strat- with an area of the protease accessible from both subsites. egy is complementary to the positional scanning approach, Another misconception is that PS-SCL results can be uni- but circumvents the problems of subsite and versally applied to protein substrates. A PS-SCL study of incomplete and under-represented sequences. In addition, KLK3 revealed a strong preference for Met at the P1 site since each peptide is individually assayed, there is often far (Table 1), although comparison with known cleavage sites greater selectivity apparent for each position in the peptide. for protein substrates indicates that Tyr and Gln are preferred (Merops database, http://merops.sanger.ac.uk/). An inspec- Design challenges: bioavailability and stability tion of the S1 pocket of the KLK3 structure suggests that it is too narrow to fit Tyr or Gln, but not Met. Therefore, it For protease inhibitors to be viable therapeutic agents, they may well be that binding of a larger protein substrate, which must be not only potent and specific towards their target occurs over a considerably larger surface area, causes an protease, but also readily bioavailable (Fear et al., 2007). The induced fit and enables access to larger residues such as Tyr bioavailability of a drug in a particular tissue is governed by and Gln. its ability to cross biological barriers to reach the site versus A final inherent problem of the PS-SCL method is the its rate of degradation and excretion. Orally bioavailable assumption that all amino acid couplings occur with the same drugs in humans commonly fulfil Lipinski’s rule of five, efficiency. Considering the popularity of the PS-SCL meth- which states that the compound should contain not more than od, it is surprising that very few examinations of differential five hydrogen bond donors or ten acceptors, an octanol-water reaction rates in mixtures of amino acids have been made. partition coefficient of less than five and a molecular mass Early solid-phase experiments noted differential reaction below 500 Da (Lipinski et al., 2001). Conversely, the poten- rates (Bayer and Hagenmaier, 1968; Ragnarsson et al., 1971, cy of inhibition depends on the number and nature of contact 1974; Sandberg and Ragnarsson, 1974) and compensation points between the protease and inhibitor, whereas specificity methods were later used to solve this problem (Kramer et relies on their uniqueness. Therefore, the design of potent, al., 1993; Ostresh et al., 1994; Ivanetich and Santi, 1996). specific and bioavailable KLK inhibitors represents quite a This issue has been highlighted by Boutin et al. (1997), who challenge, especially considering the high level of conser- found ten-fold variations in concentration between different vation among the KLKs described earlier (Figure 1). sequences and an absence of 35% of all theoretical sequen- Methods that both reduce enzymatic degradation and renal ces. These findings may explain why two PS-SCL (Matsu- clearance of peptide and protein drugs include polysialyla- mura et al., 2005; Debela et al., 2006b) screens did not detect tion and PEGylation, in which polysialic acid or poly the strong preference of KLK4 for Phe at P4 (Swedberg et (ethylene glycol) is attached to one or both termini of the al., 2009). peptide/protein. Polysialylation is a natural strategy to To overcome these problems, positional preferences sug- improve peptide and protein half-life (Jain et al., 2004) and gested by PS-SCL can be verified by screening against a was recently used to increase the in vivo availability of an sparse matrix library, a small sub-library of individually syn- antitumour monoclonal antibody (H17E2 Fab fragment) by

Table 1 Extended substrate specificity of KLK1–15 and KLKB1.

P1 P2 P3 P4 Method Reference KLKB1 R n/a n/a n/a PDL Dennis et al., 1995 KLK1 F/K/Y L/F n/a n/a PDL Li et al., 2008 KLK2 R H/L/R n/a n/a Peptides Janssen et al., 2004 KLK3 M)n)A L)A/M/Q)n Y)V)M/n/I V)M/A/Q)T PS-SCL Debela et al., 2006b KLK4 R)K Q)T/L)V)P V)Q)T)S/A F)I)V)Y/W SML Swedberg et al., 2009 KLK5 RS)A/N/T)P M)Y)K/F/n G)Y)V/P/W PS-SCL Debela et al., 2006b KLK6 R)A/n/M R)K A)K/S/Y/M V)Y/I/M/G PS-SCL Debela et al., 2006b KLK7 Y)A/M/n Y)L)T/F/n T/M)H/A/V K/Q/M/V PS-SCL Debela et al., 2006b KLK8 R/K n/a n/a n/a Peptides Rajapakse et al., 2005 KLK9 n/a n/a n/a n/a n/a n/a KLK10 R)K/n/M D)E/R)K/M E/D)S/Y/M M)D/E/V/n PS-SCL Debela et al., 2006b KLK11 M)n)K/R)A R)K/D)E)S D/E/M)Y)S M)D/E/V/M PS-SCL Debela et al., 2006b KLK12 K)R n/a n/a n/a Peptides Memari et al., 2007 KLK13 R)N N)L)F)Y/A RV)Y)I/P/G PS-SCL Borgono et al., 2007a KLK14 R N/A)P)H/S K/A)R/S/n Y)W)F/V/I PS-SCL Borgono et al., 2007a KLK15 R n/a n/a n/a Peptide Takayama et al., 2001a Amino acids are shown in one-letter code, with ‘n’ representing norleucine. Bold font indicates the most preferred residue at each subsite. When several sources of data were available, selections were made according to the following priority order: Sparse Matrix Library (SML), Positional Scanning Synthetic Combinatorial Library (PS-SCL), Phage Display Library (PDL) and peptides. n/a, not applicable.

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Kallikrein inhibitors 365

a factor of five (Constantinou et al., 2008). PEGylation is synthesis of multi N-methylated peptides (Biron et al., 2006) approved by the FDA as a vehicle for pharmaceuticals have yielded a somatostatin analogue with four-fold higher (Harris and Chess, 2003) and is used in a number of protein- half-life after oral administration (Biron et al., 2008). A num- based drugs currently on the market (Veronese and Harris, ber of less commonly used amide bond surrogates also exist,

2008) with an annual market value of over $US4 billion including retro bonds (NH-CO), carba bonds (CH2CH2), aza

(Krishan, 2007). bonds (CO-NH-NR-CO), reduced amide (CH2NH) and urea Peptide-based inhibitors particularly suffer from short bonds (HN-CO-NH). These have previously been reviewed half-lives since they are often hydrophilic and thus show elsewhere (Adessi and Soto, 2002; Lozano et al., 2006). poor oral absorption and are rapidly cleared by the kidneys Curiously, certain naturally occurring small protein inhib- (Werle and Bernkop-Schnurch, 2006). In addition, peptide itors that deviate from Lipinski’s rule of five are readily bio- drugs are often quickly degraded by the numerous exo- and available. For example, orally administrated Bowman-Birk endo-proteases present in most tissues (Werle and Bernkop- inhibitors (;8 kDa) are readily bioavailable (Kennedy, Schnurch, 2006). Even if intravenously administered, most 1998) and the same is true for cyclotides (;3 kDa), at least peptides are cleared from the bloodstream within minutes. in insects (Whetstone and Hammock, 2007). There is a grow- To overcome these particular challenges, peptide lead com- ing realisation that many of the issues above can be solved pounds often need to be modified in various ways, depending at a stroke by using naturally occurring inhibitors of serine on the route of administration and the target tissue. N- and proteases. For example, the inhibitors aprotinin (Terrell et al., C-terminal modifications such as N-acetylation and C-ami- 1996) and hirudin (Zeymer, 1998) have seen clinical use as dation can reduce degradation by exoproteases (McGregor, modulators of bleeding after thoracic surgery and thrombo- 2008). For example, the natural peptide thymopoietin lysis, respectively. The obvious next step for these naturally involved in T-cell maturation has a half-life of 1 min in plas- occurring inhibitors is to produce variants with redirected ma, whereas a double end-capped version shows no detect- inhibitory specificity and enhanced potency. Use of these able degradation (Heavner et al., 1986). Similarly, capping bioscaffolds is becoming increasingly common as their with fatty acids can improve both the half-life and lipophi- advantages in terms of stability, specificity and potency are licity of a peptide, a technique used to improve serum recognised. stability of an anti-proliferative somatostatin analogue (Das- gupta et al., 2002). Backbone- and/or disulfide bond-circularised peptides are Engineered inhibitors: bioscaffolding intrinsically stable to degradation, a common feature in nat- urally occurring peptides. A diverse range of plants produce Bioscaffolding revolves around the use of natural template cyclotides, circular miniproteins with a cysteine knot of three structures (bioscaffolds) in contrast to more conventional intertwined disulfides, believed to be involved in host small-molecule approaches. Extensive use is made of pre- defence (Craik et al., 2002; Gruber et al., 2008). Many bac- existing natural protease inhibitors, which are re-engineered teria also produce circular peptides (bacteriocins) to target using recombinant DNA techniques or solid-phase peptide competing species (Mercedes et al., 2008), as do many synthesis. The chief features of successful bioscaffolds are a marine microorganisms (Hamada and Shioiri, 2005). This structure that is both robust and flexible enough to tolerate approach was recently used by Clark et al. (2005) to produce multiple amino acid substitutions, and a template structure a backbone-cyclised snail peptide (a-conotoxin) that has intrinsic protease inhibitor activity. Thus, the engi- analogue with improved stability in serum. neering goal is simply to redirect and enhance existing A more significant challenge is to improve peptide resis- inhibitory activity. Further advantages come from facile pro- tance to endoprotease activity, since this involves peptide duction of inhibitor molecules in living systems, thus facil- backbone and/or side chain modifications that are likely to itating biopanning (phage display) and cheap and efficient have major effects on inhibitor potency and specificity. One large-scale bioproduction in microbial or plant systems. strategy involves the substitution of certain amino acids that Currently there are four classes of bioscaffold with rele- are known to be targets of proteolysis with non-natural D- vance to kallikreins: serpins, Kunitz-domain inhibitors, eco- amino acids, as has been used to produce a variety of pro- tin and SFTI derivatives. All four classes of inhibitor interact tease-resistant reagents including small peptides (Silvia et al., with their target proteases through the canonical loop 2008) and antibodies (Webb et al., 2005). Alternatively, sub- described above (Tyndall et al., 2005; Figure 2E), forming stitution of peptide backbone atoms, in particular participants an extension of the protease b-sheet structure. However, the of the amide bond, can be used to produce what are com- scaffolding presenting the reactive loop shows considerable monly referred to as pseudopeptides. For example, the amide structural diversity and plays varying roles in stabilising the nitrogens of scissile bonds can be methylated (N-methyla- protease-inhibitor complex. In addition to these four estab- tion) to prevent hydrolysis, as for the fungal peptide immuno- lished bioscaffolds, there is also a move towards production suppressant cyclosporine A. This peptide has seven N-meth- of specific monoclonal antibodies that can inhibit kallikreins, ylations, as well as one D-amino acid, and is highly bioa- with Dyax developing a specific KLK1 inhibitor for treat- vailable since it is chiefly degraded by the hepatic ment of asthma (Sexton et al., 2009). This antibody was cytochrome P450 3A enzyme system rather than by prote- selected from a so-called Fab-on-phage library (Steukers et ases (Dunn et al., 2001). Recent advances in methods for al., 2006), which contains genes encoding the antibody

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366 J.E. Swedberg et al.

heavy- and light-chain variable regions that are then Kunitz domain displayed on the phage surface as Fabs and selected using surface plasmon resonance screening. Other non-clinical Originally identified in BPTI, the Kunitz-domain inhibitory examples include antibody-mediated inhibition of KLK13 in motif is remarkably compact and shows considerable thermal a cell-based model of extracellular matrix degradation stability (Moses and Hinz, 1983; Makhatadze et al., 1993). (Kapadia et al., 2004) and use in biochemical characterisa- Both BPTI and the Alzheimer’s amyloid b-protein precursor tion of KLK12 (Memari et al., 2007). Both the bioscaffold (APPI), which has a similar structure to BPTI, have been and monoclonal antibody approaches make extensive use of exploited as bioscaffolds. APPI was the first bioscaffold sub- phage display as a primary design tool. However, bioscaffold jected to phage display. Screening against KLKB1 achieved design is also driven by information from positional scanning a Ki value of 15 pM (Dennis et al., 1995). A later study and sparse matrix libraries, as outlined below. searched for potent inhibitors of six distinct serine proteases, including KLKB1. This study focused on the BPTI canonical Serpins loop, substituting positions P1, P3 and P4 with amino-acid side chains gauged to explore the diversity of charge, shape To date, three serpins have been exploited as bioscaffolds; and hydrophobicity. The best inhibitor against KLKB1

C1-inhibitor protein, which inhibits the showed a ki value of 5.4 nM, a modest 2.2-fold improvement (Kase and Pospisil, 1983); human a1-antitrypsin (AAT), over the wild-type inhibitor; considerable thermal destabilis- which protects lung tissue from the effects of human neutro- ation was observed when P4 mutations were undertaken phil (Sun and Yang, 2004); and human a1-antichy- (Grzesiak et al., 2000b). motrypsin (ACT), which is associated with the development The Kunitz-domain approach has also had some success of Alzheimer’s disease (Potter et al., 2001). All three inhib- in the world of commercial drug design and development, itors are high-molecular-weight ()50 kDa) plasma proteins with a Dyax product, ecallantide (DX-88), reaching Phase that contact their target proteases via a canonical loop, which III clinical trials for treatment of hereditary angioedema. is termed a reactive loop since it is cut by the target protease. Ecallantide was derived from a phage display screen of the Although the reactive loop does in theory control the major- Kunitz domain of human lipoprotein-associated ity of the specificity of a given serpin, there is also a signif- inhibitor (LACI) using immobilised KLKB1 as bait. It has a icant contribution from serpin exosites such that swapping reported Ki value of 44 pM and is highly specific for KLKB1 reactive loops alone between serpin subtypes is not sufficient (Williams and Baird, 2003). At the time of writing, FDA to completely redirect their inhibitory activity (Djie et al., approval for use of ecallantide was awaiting completion of 1997). Nonetheless, the reactive loop is very tolerant of sub- a risk evaluation and mitigation strategy. stitution and this has led to the development of a series of recombinant kallikrein inhibitors. Ecotin Initially, an inhibitor for KLKB1 was developed using a variant of C1-inhibitor (Sulikowski et al., 2002). C1-inhibitor Ecotin (E. coli trypsin inhibitor) was isolated from the peri- effectively blocks KLKB1 activity and the aim of the re- plasmic space of E. coli and is a dimeric, bidentate protease engineering was to increase the inhibition specificity and inhibitor (Chung et al., 1983). It is thought to provide pro- prevent the blockade of other complement proteases. Accord- tection against the effects of (Eggers et ingly, a reactive loop variant was designed on the basis of al., 2004), which it inhibits with a Ki value of 12 pM. Inter- the plasma KLK substrate specificity, which was previously action between the protease and ecotin is driven by four assessed using a relatively small pool of methyl loops (two from each ecotin monomer) and results in the esters (Levison and Tomalin, 1982). The resulting variant formation of a heterotetramer with a very extensive buried showed the specificity required and maintained potency, with surface area of 2850 A˚ 2. Interestingly, binding by the contact -1 -1 ka of 382 180 M s . Following this success, a KLK2 inhib- loops is not restricted to the trypsin active site, but also itor was designed using ACT as bioscaffold (Cloutier et al., includes residues that are highly divergent within the trypsin 2004). The ACT reactive loop was substituted with a KLK2- superfamily. Accordingly, these secondary contacts have the specific sequence found through phage display (Cloutier potential to provide greater selectivity than that of inhibitors et al., 2002), yielding an inhibitor with a ka value of 6261 that bind at the active site alone. This prompted Craik and M-1 s-1. co-workers (Stoop and Craik, 2003) to re-engineer the ecotin More recently, Felber et al. (2006) re-engineered the reac- bioscaffold to produce a series of variant molecules that can tive loop of both AAT and ACT serpins using pentapeptides inhibit KLKB1, , MT-SP1 and FXIIa. Two distinct derived from a phage display scan against immobilised design strategies were used, a simple progressive substitution KLK14 (Felber et al., 2005). Currently, KLK2 ACT-derived of the ecotin apparent P1 residue and biopanning using phage inhibitors are being developed by Med Discovery, with the display. Ecotin, like the C1-inhbitor bioscaffold, is a potent lead compound, MDKP67b, close to entering clinical trials inhibitor of KLKB1 but lacks selectivity against other con- as a targeted treatment for prostate cancer. The success of tact activation proteases. Given the role of the contact loops, this approach owes much to the adaptability of the serpin as well as the canonical loop, in protease complex formation, reactive loop. In addition, using a human serpin as a bio- a library of ecotin variants was produced by gene shuffling scaffold, the problem of patient immune response to a protein to target all five motifs. This library was screened against therapeutic is neatly avoided. immobilised KLKB1 as described above and, in a novel

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Kallikrein inhibitors 367

deviation from the normal phage display procedure, the library was pre-blocked with soluble proteases from classes against which inhibitory activity was to be selected. This led to the isolation of an ecotin variant with a Ki value for KLKB1 of 11 pM but with activity against factor Xa, factor XIa, -type plasminogen activator thrombin, and membrane-type serine protease 1 that is four to seven orders of magnitude lower (Stoop and Craik, 2003).

Sunflower trypsin inhibitor

SFTI belongs to the Bowman-Birk serine protease inhibitor family and is a potent inhibitor of trypsin, G and suppressor of tumourigenesis (14 ST14//MT-SP1). It was originally discovered in sunflower (Helianthus annuus) seeds and characterised by determination of its three-dimensional structure in complex with bovine b-tryp- sin (Luckett et al., 1999). Consisting of just 14 cyclised ami- no acids bisected by a disulfide bond, SFTI is intermediate between the macromolecular scaffolds represented by the bioscaffolds above and the small-molecule inhibitors Figure 4 Tetrahedral transition-state analogues of serine proteases. described below. Its smaller size allows for facile chemical (A) Structure of the P1-P19 residues and adjoining peptide bond on synthesis (Zablotna et al., 2002; Korsinczky et al., 2005) and a typical amide substrate (left) and putative tetrahedral intermediate rapid generation of variants. In addition, a very recent pub- formed coordinately with the Ser195 (catalytic) side chain prior to lication described expression of a library of fully cyclic SFTI cleavage of the peptide bond (right). (B) Representation of a boronic variants in E. coli (Austin et al., 2009), opening up the pos- acid inhibitor (e.g., P8720, Figure 2B) (left) and subsequent com- sibility of rapid in vivo screening. plex formation via covalent bond formation with the Ser195 (cata- Similar to the other bioscaffolds described above, SFTI lytic) hydroxyl group (right). (C) Structure of a peptide aldehyde interacts with its target proteases via a canonical-type loop. inhibitor (e.g., hK2p01, Figure 2B) (left) and covalent bond for- mation with the Ser195 (catalytic) hydroxyl group (right). (D) Rep- However, unlike the larger molecules, SFTI has essentially resentation of a chloromethyl ketone inhibitor (e.g., CH-2856, no molecular scaffolding behind it, relying on its fully cyclic Figure 2B) (left) and mechanism of inhibition via dual linkage to backbone and a disulfide bond for stability. Interestingly, the the target protease at Ser195 (catalytic) as well as the adjacent His57 structure of SFTI is maintained even when the disulfide is (catalytic) by the exposed CH2-Cl group (right). removed (by substituting glycine or aminobutyric acid), being preserved by an internal hydrogen bond network (Korsinczky et al., 2005). However, this variant is not stable pharmacokinetics are currently unavailable for either wild- to cleavage by proteases. Surprisingly, there have been few type or engineered versions of this scaffold. In addition, the attempts to use the SFTI molecule as a bioscaffold and the other bioscaffold-based inhibitors show considerable cross- small number of variants produced have not been tested in reactivity between the kallikreins. Clearly there is consider- cell culture or animal systems. To date, only KLK4 has been able scope for further investigation focused on these actively targeted; Swedberg et al. (2009) used a sparse matrix remarkable reagents, which have the potential to become as library approach to probe the KLK4 active site, and then successful as the small-molecule approaches conventionally substituted the optimal sequence obtained into the SFTI bio- applied to protease inhibitor design. scaffold. This strategy improved both the potency and spec- ificity of KLK4 inhibition by SFTI, increasing its potency by nearly two orders of magnitude to give a Ki value of Engineered inhibitors: small-molecule inhibitors 3.6 nM. Moreover, the specificity of inhibition improved to give 500-fold selectivity against the closely related KLK14. The most commonly used small-molecule kallikrein inhibi- Thus, in contrast to the situation for serpins described above, tors are serine protease-specific tetrahedral transition-state re-engineering of just three residues is sufficient to complete- analogues. These include peptides in which the C-terminal ly redirect the inhibitory activity of SFTI. This reflects the carboxyl acid is substituted for boronic acid, an aldehyde or lack of interactions with regions outside the active-site cleft halogenated methyl ketones to form a tetrahedral adduct with in the SFTI/protease complex. Ser 195 (Figure 4). For example, Fareed et al. (1981) used Despite the inhibitory potency of these bioscaffolds, only KLK2 and KLKB1 cleavage sites to design pep- ecallantide has successfully made the transition to clinical tide aldehyde inhibitors with Ki values of 10–20 mM, use. This may reflect the fact that ecotin, Kunitz-domain and although these inhibitors showed little specificity against a serpin-based inhibitors are all macromolecular inhibitors number of serine proteases of the blood clotting cascade. A requiring intravenous administration. Although the much small- boronic acid inhibitor based on a similar peptide sequence er SFTI may yet prove to be readily bioavailable, detailed produced a potent (Ki 150 pM) and more specific KLKB1

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368 J.E. Swedberg et al.

inhibitor (Dela Cadena et al., 1995). Concurrently, Evans et therapeutic intervention. With application of the strategies al. (1996a,b) reported conservative non-natural amino acid we have discussed here, the next decade could see a blos- substitutions of KLKB1 kininogen cleavage sites combined soming of research in this area and the production of a new with a fluoroalkyloxymethyl ketone functionality to develop generation of protease inhibitor drugs to rival the antiviral a series of specific KLK1 inhibitors with Ki values in the protease inhibitors. lower nM range. These inhibitors attenuated breast cancer cell invasion in a Matrigel-based model (Wolf et al., 2001). Acknowledgements More recently, the boronic acid approach was used to devel- Work in the corresponding author’s laboratory is supported by the op inhibitors to PSA; the best has a K value of 65 nM i Cancer Council Queensland (Grant 44323) and the Prostate Can- (LeBeau et al., 2008). However, there are limited specificity cer Foundation of Australia (Grant PR09). SJD receives funding data available for these compounds and their biological from the Smart Futures Fund (Queensland Government, Australia). impact is ambiguous. Using a different strategy, Wanaka et al. (1990) capped phenylalanine N-terminally with benzylamine to yield a References competitive KLKB1 inhibitor (Ki 810 nM). This strategy, pre- viously used to produce trypsin, and thrombin inhib- Adessi, C. and Soto, C. (2002). Converting a peptide into a drug: itors (Markwardt et al., 1968), capitalises on mimicking the strategies to improve stability and bioavailability. Curr. Med. P1 arginine by the benzylamine group; the ‘peptide bond’ is Chem. 9, 963–978. displaced, resulting in a non-hydrolysable compound. Vari- Adlington, R.M., Baldwin, J.E., Chen, B., Cooper, S.L., McCoull, ous derivatives of this compound were later screened to W., and Pritchard, G.J. (1997). Design and synthesis of novel produce a more potent KLKB1 inhibitor with a Ki value of monocyclic b-lactam inhibitors of prostate-specific antigen. 130 nM (Teno et al., 1993), although it suffers from the same Bioorg. Med. Chem. Lett. 7, 1689–1694. lack of specificity as previous benzylamine derivatives Adlington, R.M., Baldwin, J.E., Becker, G.W., Chen, B., Cheng, L., Cooper, S.L., Hermann, R.B., Howe, T.J., McCoull, W., (Markwardt et al., 1968). b-Lactam analogues have also been McNulty, A.M., et al. (2001). Design, synthesis and proposed used to produce a number of serine protease inhibitors active site binding analysis of monocyclic 2-azetidinone inhib- (Konaklieva, 2002), including a series of KLK3 inhibitors itors of prostate-specific antigen. J. Med. Chem. 44, 1491–1508. with inhibition in the nM range, although the specificity of Austin, J., Kimura, R.H., Woo, Y.H., and Camarero, J.A. (2009). In this inhibition was not evaluated (Adlington et al., 1997, vivo biosynthesis of an Ala-scan library based on the cyclic pep- 2001). tide SFTI-1. Amino Acids, in press. DOI: 10.1007/s00726-009- Recently, phage display has been used to identify a series 0338-4. of KLK2 peptide inhibitors with inhibition constants in the Bayer, E. and Hagenmaier, H. (1968). Solid phase synthesis of oxy- lower micromolar range and selectivity over a number of tocin. Tetrahedron Lett. 17, 2037–2039. Bhoola, K.D., Figueroa, C.D., and Worthy, K. (1992). Bioregulation tryptic serine proteases, including KLKB1 (Hekim et al., of kinins: kallikreins, , and kininases. Pharmacol. 2006). The stability of these peptides was later improved by Rev. 44, 1–80. head-to-tail cyclisation and/or internal disulfide bond for- Biron, E., Chatterjee, J., and Kessler, H. (2006). Optimized selective mation, without loss of potency (Pakkala et al., 2007). The N-methylation of peptides on solid support. J. Pept. Sci. 12, same group also produced three non-peptide KLK3 inhibitors 213–219. with inhibition in the nanomolar range by screening against Biron, E., Chatterjee, J., Ovadia, O., Langenegger, D., Brueggen, J., a library of small drug-like molecules (Koistinen et al., Hoyer, D., Schmid, H.A., Jelinek, R., Gilon, C., Hoffman, A., 2008b). The development of these inhibitory peptides has et al. (2008). Improving oral bioavailability of peptides by mul- recently been reviewed (Koistinen et al., 2008a). tiple N-methylation: somatostatin analogues. Angew. Chem. Int. Ed. 47, 2595–2599. Small-molecule kallikrein inhibitors have not had the same Blaber, S.I., Ciric, B., Christophi, G.P., Bernett, M.J., Blaber, M., clinical success as bioscaffold-based inhibitors. However, Rodriguez, M., and Scarisbrick, I.A. (2004). Targeting kallikrein transition-state analogues are currently being investigated for 6 proteolysis attenuates CNS inflammatory disease. FASEB J. use as activity-based probes, utilising the platform initially 18, 920–922. established for organophosphate peptide inhibitors (Liu et al., Borgono, C.A. and Diamandis, E.P. (2004). The emerging roles of 1999), which were developed to detect KLK6 (Oikonomo- human tissue kallikreins in cancer. Nat. Rev. Cancer 4, 876–890. poulou et al., 2008). Clearly, the bioscaffold approach Borgono, C.A., Gavigan, J.A., Alves, J., Bowles, B., Harris, J.L., and conventional small-molecule design techniques are Sotiropoulou, G., and Diamandis, E.P. (2007a). Defining the extended substrate specificity of kallikrein 1-related peptidases. complementary. Biol. Chem. 388, 1215–1225. Borgono, C.A., Michael, I.P., Komatsu, N., Jayakumar, A., Kapadia, R., Clayman, G.L., Sotiropoulou, G., and Diamandis, E.P. Conclusions (2007b). A potential role for multiple tissue kallikrein serine proteases in epidermal desquamation. J. Biol. Chem. 282, 3640– 3652. There is increasing appreciation of the potential roles played Borgono, C.A., Michael, I.P., Shaw, J.L., Luo, L.Y., Ghosh, M.C., by the kallikrein proteases as regulators of human physiol- Soosaipillai, A., Grass, L., Katsaros, D., and Diamandis, E.P. ogy, as biomarkers in disease diagnosis and as points for (2007c). Expression and functional characterization of the can-

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