A novel theranostic activity-based probe targeting kallikrein 7 for the diagnosis and treatment of skin diseases

Article abstract of DOI:10.1039/d1cc01673c

We applied a newin silicoapproach for using protease-substrate motifs to design a kallikrein 7 (KLK7)-specific phosphonate activity-based probe (ABP) to quantify the active KLK7in situ. Epidermal application of the ABP-inhibitor onSpink5^?/?Klk5

Full text of DOI:10.1039/d1cc01673c

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A novel theranostic activity-based probe targeting
kallikrein 7 for the diagnosis and treatment of skin
diseases†
Cite this: Chem. Commun., 2021,
57, 6507
Received 29th March 2021,
Accepted 26th May 2021
Evangelos Bisyris, Eleni Zingkou, Golfo G. Kordopati,
Minos Matsoukas,
Plato A. Magriotis, Georgios Pampalakis *‡ and Georgia Sotiropoulou
*
DOI: 10.1039/d1cc01673c
rsc.li/chemcomm
We applied a new in silico approach for using protease-substrate motifs and KLK7 represent potential drug compounds for NS and
to design a kallikrein 7 (KLK7)-specific phosphonate activity-based AD. On the other hand, the expression of KLK7 is altered in
probe (ABP) to quantify the active KLK7 in situ. Epidermal application various types of cancer for which it represents a new biomarker
of the ABP-inhibitor on Spink5À/ÀKlk5À/À mice, a Netherton syndrome for molecular diagnosis/monitoring and a putative therapeutic
model, reversed disease hallmarks, providing preclinical proof-of- target.8,9
concept for using ABPs as theranostics.
It must be noted that the biological function of enzymes,
including KLK7, and their implication in disease phenotypes
KLK7 is a serine protease with well-established functions in depend on their activity, which is often not linearly propor-
pathological skin desquamation and inflammation. It is pro- tional to protein abundance, since enzymes also occur in
duced as a zymogen (proKLK7) that is assumingly activated by inactive forms, i.e. zymogen, in complex with inhibitors, trun-
KLK5.1 In vivo, the activity of KLK7 is further regulated by cated. However, the assays used in clinics and in research are
specific endogenous inhibitors such as the Lympho-Epithelial mainly designed to determine enzyme concentrations indepen-
Kazal-Type related Inhibitor (LEKTI). Increased KLK7 expres- dent of their activation status. So far, there has been no
sion has been found in the epidermis of Netherton syndrome available method to determine the levels of active KLK7 in
(NS) and atopic dermatitis (AD) patients.2 NS is caused by loss- biological and/or clinical specimens. This significantly reduces
of-function mutations in the SPINK5 gene encoding LEKTI. It is the predictive value of diagnostic assays used to measure KLK7.
a rare form of ichthyosis highlighted by abnormally elevated In general, an assay to detect the active KLK7 would have a
proteolysis, excessive desquamation and sustained inflamma- great impact for elucidation of its putative function(s) in
tion in the epidermis associated with a severe barrier defect various (patho)physiologies and assessment of its diagnostic
leading to dehydration that is often lethal.3 AD is a frequent and therapeutic potential in cancer and skin disorders.
disease for which predisposing gene polymorphisms in SPINK5
Currently, inhibitors or ABPs specific for KLK7 are not
have been identified.4 Recently, we showed that targeting the available. Previous attempts have been made to generate rever-
KLK5 protease can rescue neonatal lethality in Spink5À/À mice sible or suicide KLK7 inhibitors but their main drawback is
that recapitulate NS5 but KLK7 must be additionally inhibited their lack of specificity, while none of these compounds has
in Spink5À/ÀKlk5À/À epidermis to sustain low proteolysis, nor- ever been pharmacologically validated in preclinical
mal desquamation rates and suppressed inflammation.6 KLK7 models.10,11 Thus, we aimed to develop an ABP that (a) will
could regulate the inflammatory pathways by various mec- specifically and selectively bind to active KLK7, and (b) will
hanisms such as activation of pro-IL-1b or cathelicidin anti- inhibit its enzymatic activity so that it could ideally be used for
microbial peptide precursors.7 Thus, KLK7 emerged as a novel diagnosis and therapy combined, i.e. as a theranostic agent.
therapeutic target and synthetic inhibitors against KLK5
To design ABPs for KLK7, we first developed here a specific
KLK7 synthetic inhibitor. We describe a novel strategy that
entails MEROPS (http://www.ebi.ac.uk/merops) database
mining to identify specific substrates for KLK7 based on
mapped protease cleavage sites in various proteins, peptides
and synthetic substrates.12 It was revealed that KLK7 can cleave
after the Phe–Phe motif (Fig. S1, ESI†). Similarly, ABPs for
monoamine oxidases (MAOs) were designed by modification
Department of Pharmacy, School of Health Sciences, University of Patras,
Rion-Patras, 26504, Greece. E-mail: gpampalakis@pharm.auth.gr,
gdsotiro@upatras.gr
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1cc01673c
‡ Current address: Department of Pharmacognosy-Pharmacology, School of
Pharmacy, Aristotle University of Thessaloniki, Thessaloniki, 54124, Greece.
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of known MAO inhibitors.13 Reciprocal analysis showed that
the ability of KLK7 to cleave after Phe–Phe is rather specific,
thus, this motif was used to rationally design an inhibitor and
a theranostic ABP (3b and 5b, respectively). Both have
a-aminomethylphosphonate reactive groups and carry a mod-
ified Phe–Phe dipeptide as a recognition sequence. The ABP
was further biotinylated at the Na-terminus. Chemical synth-
eses of the compounds are shown in Schemes 1 and 2.
For initial optimization, the a-carbon length at the P1-like
position was varied in analogues 3a and 3c. Compound 3b (Boc-
FFP) along with 3a (Boc-FBP) and 3c (Boc-FCP) was tested by
casein gel zymography for specific inhibition of KLK7. Fig. S2
(ESI†) shows that only Boc-FFP inhibits the activity of KLK7, a
finding supported by docking studies. Compound 3b (both R,
S isomers at a-carbon) demonstrates the highest docking scores
with 3b-R performing better (Table S1, ESI†). 3b-R interacts
with KLK7 by inserting the benzyl group located between the
two amide bonds deep in the catalytic pocket and specifically in
the main S1 hydrophobic subpocket14 to interact with A190 and
V213. The two phenyl groups attached to the phosphonic
oxygens interact with the aromatic rings of H99 and W215
and the tert-butyl group of Boc interacts via sigma–pi position-
ing with the aromatic ring of H57 in subpocket S1’. One of the
phosphonic oxygens forms a hydrogen bond with the main
chain NH group of F218 and one of the amides forms hydrogen
bond with the side chain of N192. Finally, the benzyl group at
the a-carbon of 3b forms a pi–pi interaction with the aromatic
ring of F218, providing an explanation for its better perfor-
mance relative to 3a and 3c that do not allow for correct
positioning of the aromatic ring and thus, strong interactions
with F218 (Fig. 1).
Scheme 2 Synthesis of the KLK7-ABP biotin-FBP (5a) and biotin-FFP (5b).
Reagents and conditions: (a) TFA/CH2Cl2 for 2 hours; and (b) biotina-
midohexanoyl-6-aminohexanoic acid N-hydroxysuccinimide ester, HOBt,
DIPEA in DMSO for 16 hours.
Therefore, 3b was selected for validation in vitro and in vivo.
3b has an IC50 of 1.91 mM, and when tested against various
serine proteases as shown in Fig. S3 (ESI†) it was found to be
selective for KLK7. Then, we tested whether the Boc-group is
required for inhibition. Following removal of the Boc-protective
group we purified the free amino-terminated FFP (H2N-FFP,
compound 4b) and tested its inhibitory activity against KLK7,
as shown in Fig. S4A (ESI†). H2N-FFP inhibits KLK7 activity to
Fig. 1 Docking pose of 3b-R bound to the catalytic site of KLK7. The
protein in shown in teal color, compound 3b-R is depicted with brown
sticks, and hydrogen bonds are highlighted with yellow dashed lines.
the same extent as the Boc-FFP 3b indicating that modifications at the N-terminus do not affect recognition by the active KLK7
protease.
Subsequently, a biotin derivative was attached at the
N-terminus to yield KLK7-ABP 5b (biotin-FFP). Fig. S4B (ESI†)
shows that the KLK7-ABP (biotin-FFP) can detect active KLK7 by
Western blotting. We further generated the biotin-FBP (5a) that
also detected KLK7, albeit with significantly lower affinity than
the biotin-FFP (Fig. S4B, ESI†) enforcing that the length of the
a-carbon chain on the P1-like position is crucial for optimal
recognition by KLK7. Next, an ELISA-type reaction was set up to
investigate whether the new ABP can be used to determine the
levels of active KLK7 in biological samples as shown in Fig. S5
(ESI†).
Human KLK7 and mouse Klk7 show 76% identity and 86%
positivity, while all residues that determine the substrate
Scheme 1 Synthesis of the inhibitor and the ABP. Reagents and condi-
tions: (a) HOAc/reflux for 2 hours; (b) 1,4-cyclohexadiene, 10% Pd/C in
specificity are highly conserved (Fig. S6, ESI†). Therefore, it is
expected that the Boc-FFP and the biotin-FFP would likely react
ethanol for 2 hours or triethylsilane, 10% Pd/C in MeOH/CH2Cl2 for 1 hour;
and (c) Boc-L-Phe-OH, TBTU, HOBt, DIPEA in CH2Cl2 for 16 hours.
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Fig. 3 Tissue localization of active KLK7. Skin cryosections were sub-
jected to activography with the biotin-FFP. As expected, normal healthy
skin was stained only at the superficial stratum corneum. In contrast, skin
samples from NS patients were stained at the regions of stratum corneum
rupture (red asterisks, Patient 1) or throughout the damaged SC (Patient 2),
and staining was evident at the interface of SC with stratum granulosum
(separated by a green dashed line) consistent with the known spatial
localization of the KLK7 expression in the NS epidermis. The red dashed
line indicates the epidermal–dermal junction. Due to large acanthosis
(thickening of the living epidermal layer) the epidermal–dermal junction is
not shown in NS samples.
Fig. 2 Detection of the endogenous mouse Klk7 protein. (A) Detection of
Klk7 in Spink5À/ÀKlk5À/À mouse skin extracts by Western blotting. (B) The
biotin-FFP detects only the Klk7 in the same sample. The nonspecific band
of B75 kDa detected in the absence of ABP is likely due to endogenous
biotinylated proteins. (C) TPA application on mice skin induces the expres-
sion of KLK7 as shown by western blotting (upper). a-Tubulin was used as a
loading control (lower). (D) Quantification of western blots in C with
ImageJ. Data shown are median Æ s.e.m. (E) The TPA-induced Klk7 activity
is quantified spatially in the epidermis by activography using the biotin-FFP.
expected. Nevertheless, in NS epidermis it is highly upregulated
at sites of stratum corneum rupture and at the junction of the
stratum granulosum with the stratum corneum, thus providing
evidence for in situ activation of the KLK7 target protease.
To assess the theranostic value of Boc-FFP and biotin-FFP,
first, we examined their in vivo safety. No signs of dermal
irritation or corrosion were observed following their application
on the skin at 1 mM (Fig. S7, ESI†). The assay was conducted
with the standard protocol for testing chemicals (https://ntp.
with the mouse Klk7 as well. To test whether biotin-FFP can niehs.nih.gov/iccvam/suppdocs/feddocsoecd/oecdtg404.pdf)
indeed bind to mouse Klk7, skin extracts from Spink5À/À with a slight modification in that the substances were not
Klk5À/À mice obtained on P3 were incubated with 50 mM removed after application for 4 hours. Also, we observed no
biotin-FFP and analyzed by Western blotting. On P3, signs of skin irritation or corrosion in the epidermis of Klk5À/À
Spink5À/ÀKlk5À/À mice develop symptoms of desquamation mice. The rationale behind testing the compounds on the
and inflammation, associated with increased epidermal Klk7 Klk5À/À background is that for the successful treatment of NS
expression and chymotrypsin activity15 and about 70% of them cocktails of KLK5 and KLK7 inhibitors will likely be required.6
succumb 7–8 days after birth. As shown in (Fig. 2A), Klk7 is The KLK7-ABP-inhibitor was applied on the epidermis of
highly expressed in Spink5À/ÀKlk5À/À skin, as expected. No Spink5À/ÀKlk5À/À mice, which allowed validation of the effect
other bands were detected indicating that biotin-FFP success- of Klk7 inhibition following ablation of Klk5. Previous studies
fully labels active Klk7 without binding to other endogenous have established that KLK7 elimination alone is not sufficient
proteins. This further supports the specificity of the ABP to rescue the lethal phenotype of Spink5À/À but its combined
(Fig. 2B). The biotin-FFP was used to detect and spatially inhibition with KLK5 is required for a sustained normalization
localize by activography the active Klk7 induced by TPA in of NS skin.6
mouse skin16 (Fig. 2C–E). Consequently, Boc-FFP and biotin-
FFP can be exploited for validation of the inhibition of Klk7 Boc-FFP inhibitor from day 3 onwards when macroscopic signs
activity in Spink5À/ÀKlk5À/À mice.
of desquamation appeared. Fig. 4A and B show significant
Spink5À/ÀKlk5À/À mice were treated daily with 1 mM of
Using activography we also tested whether the biotin-FFP reduction of desquamation upon application of Boc-FFP. We
could be used to detect the active endogenous KLK7 in skin investigated the expression of the proinflammatory cytokines
biopsy cryosections obtained from a healthy human donor and Tslp, Tnfa, Il-1b, Il-18, Il-17a, and Il-23, which are known to be
two NS patients. Fig. 3 shows that, in a healthy epidermis, constitutively induced in the epidermis of Spink5À/À mice and,
active KLK7 is detectable only at the stratum corneum, as also, were shown to be reactivated when delayed desquamation
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preclinical mouse model of NS, which reinforces the use of
ABPs as theranostic compounds.
We thank M. Valari, MD/PhD (Aghia Sofia Children’s Hos-
pital, Athens, Greece), for providing the human biopsies, Dr D.
ˆ
Darmoul (INSERM, Hopital Saint Louis, Paris, France) for a
sample of bacterial recombinant KLK7, and E. Tsiaousi for
technical assistance. We also thank Prof. A. McKenzie (MRC
LMB, Cambridge, UK) for kindly providing the Spink5À/À mice.
We acknowledge support of this work by the project ‘‘BIOLU-
MINPD; code T1EDK-03884’’, which is implemented under the
call ‘‘Research-create-innovate’’ funded by the Operational Pro-
gram ‘‘Competitiveness, Entrepreneurship and Innovation’’:
(NSRF 2014–2020) and co-financed by Greece and the European
Union (European Regional Development Fund).
Conflicts of interest
There are no conflicts to declare.
Fig. 4 (A) Topical application of the Boc-FFP on the epidermis of
Spink5À/ÀKlk5À/À mice led to delayed desquamation rates as shown in
this representative image. The area treated is marked with a black line.
(B) Quantification of desquamation. Application of Boc-FFP results in
almost 50% suppression of desquamation. (C) Application of the KLK7
inhibitor results in a strong suppression of proinflammatory cytokines
in Spink5À/ÀKlk5À/À skin. The cytokine expression was quantified by
RT-qPCR. Data shown are median Æ s.e.m. (n = 4 mice, per genotype).
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6510 | Chem. Commun., 2021, 57, 6507–6510
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