The role of N-terminal heterocycles in hydrogen bonding to α-chymotrypsin

Article abstract of DOI:10.1016/j.bmcl.2018.12.032

A series of dipeptide aldehydes containing different N-terminal heterocycles was prepared and assayed in vitro against α-chymotrypsin to ascertain the importance of the heterocycle in maintaining a β-strand geometry while also providing a hydrogen bond do

Full text of DOI:10.1016/j.bmcl.2018.12.032

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The role of N-terminal heterocycles in hydrogen bonding to α-chymotrypsin
a
b
b
a,c,⁎
Nicholas C. Schumann , John Bruning , Andrew C. Marshall , Andrew D. Abell
b
a
School of Chemistry & Physics, The University of Adelaide, North Terrace, Adelaide, SA 5005, Australia
School of Biological Sciences, The University of Adelaide, North Terrace, Adelaide, SA 5005, Australia
c
ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP) and Institute of Photonics and Advanced Sensing (IPAS), The University of Adelaide, Adelaide, South
Australia 5005, Australia
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of dipeptide aldehydes containing different N-terminal heterocycles was prepared and assayed in vitro
against α-chymotrypsin to ascertain the importance of the heterocycle in maintaining a β-strand geometry while
also providing a hydrogen bond donor equivalent to the backbone amide nitrogen of the surrogate amino acid.
The dipeptide containing a pyrrole constraint (10) was the most potent inhibitor, with > 30-fold improved
activity over dipeptides which lacked a nitrogen hydrogen bond donor (namely thiophene 11, furan 12 and
pyridine 13). Molecular docking studies of 10 bound to α-chymotrypsin demonstrates a hydrogen bond between
Chymotrypsin
Protease inhibitors
Heterocycles
β-Strand
Peptidomimetic
Peptidic aldehydes
the pyrrole nitrogen donor and the backbone carbonyl of Gly216 located in the S
critical for overall binding.
3
pocket which is proposed to be
Proteases almost universally bind substrates and peptide-based
peptidomimetic) inhibitors such that their backbones adopt a β-strand
calpain 1 inhibitor 9 which is also thought to adopt a β-strand con-
22
(
formation on binding to calpain. Other aromatic heterocycles (e.g.,
23 24
1
–7
geometry
as defined by specific interactions between amino acid R
tetrazole and quinoline ) and non-aromatic heterocycles (e.g., mor-
25
groups (designated P
n
and Pn′) and corresponding active site subsites
pholines ) have also been incorporated into the backbone of acyclic
inhibitors. Despite the prevalence of heterocycles in the wider area of
peptidomimetics, the optimum heterocycle for use as such a constraint
in protease inhibitors has not been defined. It is proposed that hetero-
8
(
designated S
n
and Sn′). In the case of serine and cysteine proteases,
this binding positions an appropriate C-terminal warhead of an in-
hibitor adjacent to a key active site residue, which then forms a cova-
9
–11
lent and typically reversible linkage.
The peptidic character of these
cyclic constraints that maintain a hydrogen bond donor at the P po-
3
inhibitors can be reduced by incorporating a suitable macrocycle into
the backbone to conformationally define the requisite backbone β-
strand geometry for binding, while improving bioavailability and pro-
sition (as in the case of pyrrole) for hydrogen bonding to the backbone
carbonyl of Gly216 in the S pocket are critical for tight binding to the
active site of α-chymotrypsin.
3
2
6,27
1
2–17
teolytic stability.
For example, CAT811, 1, is a potent inhibitor of
To this end, we now report a series of dipeptide inhibitors of α-
chymotrypsin containing different N-terminal heterocyclic constraints
to verify the importance of this hydrogen bond interaction, as defined
by potency and docking studies. Pyrrole-containing 10 which features a
hydrogen bond donor was selected for comparison to other π-excessive
heterocycle-containing dipeptides that lack this donor, namely thio-
phene 11 and furan 12. Basic pyridine-containing 13 was also in-
vestigated as a potential hydrogen bond acceptor for comparison to the
donor properties of pyrrole 10. The tripeptide 14, with an alanine in
place of the heterocycle, was prepared as a control. α-Chymotrypsin
was selected for study as it is well-characterised, with a number of co-
calpain 2 (IC50 = 30 nM) formed by linking the P
1
and P
Other examples of con-
(2, 3 and 6) and P and P (4
3
residues to
7
,18
define this geometry as shown in Fig. 1.
strained inhibitors which link P
1
and P
3
1
2
and 5) are shown in Fig. 1.
We recently reported macrocyclic picomolar inhibitors of cathepsins
L and S with an N-terminal pyrrole, where this group effectively re-
places a backbone amide bond and the P amino acid as in 7 (see
3
2
1
Fig. 2). A combination of the macrocycle and planar pyrrole unit
defines the backbone into the requisite β-strand, while reducing pep-
tidic character. The X-ray structure of one such inhibitor (8) bound to
its target protease, α-chymotrypsin, clearly confirms these design fea-
2
1
crystal structures having been reported as a basis for docking; and
also since its active site is characteristic of other serine proteases from
the chymotrypsin family.2
2
1
tures with phenylalanine occupying the S pocket. An N-terminal
1
1,22,28,29
pyrrole has also been reported in acyclic inhibitors as exemplified in
⁎
Corresponding author at: School of Chemistry & Physics, The University of Adelaide, North Terrace, Adelaide, SA 5005, Australia.
E-mail address: andrew.abell@adelaide.edu.au (A.D. Abell).
https://doi.org/10.1016/j.bmcl.2018.12.032
Received 31 October 2018; Received in revised form 11 December 2018; Accepted 14 December 2018
0960-894X/ © 2018 Elsevier Ltd. All rights reserved.
Please cite this article as: Schumann, N.C., Bioorganic & Medicinal Chemistry Letters, https://doi.org/10.1016/j.bmcl.2018.12.032

N.C. Schumann et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Table 1
In vitro inhibition data.
Compounds
IC50 (µM)a
P
1
S:R ratiob
1
1
1
1
1
0
1
2
3
4
1.1 ± 0.12
40 ± 4.2
36 ± 7.6
35 ± 5.8
1.4 ± 0.29
94:6
79:21
74:26
87:13
96:4
a
This represents an under estimate of activity because of the presence of
some of the non-natural epimer.
b
Ratio determined by 1H NMR
The target peptidomimetics (10–14) were prepared as detailed in
Scheme 1. N-Boc-leucine was coupled to phenylalaninol in the presence
of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5b]pyridinum
3
-oxid hexafluorophosphate (HATU) to give the corresponding Boc-
protected dipeptide which on treatment with TFA gave the tri-
fluoroacetate salt 15. With this key intermediate in hand, separate
HATU mediated couplings with the carboxylic acids 16–19 gave di-
peptide alcohols 20–23, respectively. Ethyl ester 22 was converted over
two steps to methyl ester 24 to maintain common N-terminal func-
tionality. Hydrolysis of 22 with lithium hydroxide to the carboxylic acid
intermediate (not shown) and subsequent esterification with methyl
iodide gave methyl ester 24. Preparation of the N-Boc alanine deriva-
tive 27 by coupling between 15 and N-Boc-alanine proved challenging
and so it was prepared by firstly coupling N-Boc-alanine and dipeptide
methyl ester 25 to give tripeptide 26 which was subsequently reduced
7
,16
Fig. 1. Macrocyclic inhibitors of cysteine proteases calpain 2 (1–3)
and
1
9
20
cruzain (4 and 5) and the serine protease NS3 (6). Positions designated P -
1
P correspond to groups which interact with the specificity pockets of the
3
corresponding target proteases according to the nomenclature of Schechter and
Berger.8
with LiBH
4
to give the alcohol 27. Oxidation of the primary alcohols 20,
2
1, 23, 24 and 27 with Dess-Martin Periodinane (DMP) gave the de-
sired aldehydes 10–14, with some epimerisation of the P residue oc-
1
curring during oxidation, ranging from 4 to 26% (Table 1). Similar
levels of epimerisation have been reported for related peptidic alde-
3
0
hydes.
Pyrrole 16 was prepared over two steps by Vilsmeier-Haack for-
3
1
mylation of methyl 2-pyrrolecarboxylate to give mixtures of 4- and 5-
3
2
substituted pyrroles and subsequent oxidation of the 5-substituted
Fig. 2. β-Strand constrained heterocyclic protease inhibitors of cathepsins L
and S, 7, α-chymotrypsin, 8, and calpain 1, 9.
3
3
pyrrole under Pinnick conditions to give the desired carboxylic acid
1
6. Preparation of thiophene 17 was achieved by initial esterification of
2
,5-thiophenedicarboxylic acid with thionyl chloride in methanol to
give the corresponding diester which was subsequently selectively
mono-hydrolysed with sodium hydroxide to give monoester 17. The
corresponding furan was similarly prepared by Fischer esterification of
2
,5-furandicarboxylic acid to the corresponding diester and subsequent
selective mono-hydrolysis with sodium hydroxide to monoester 18. The
furan was prepared as the ethyl ester due to limited solubility of the
corresponding methyl ester which prevented purification and sub-
sequent amidation with dipeptide 15.
The backbones of peptidic alcohols 20–24 and aldehydes 10–13
1
were defined as β-strand by H NMR spectroscopy, where this geometry
is characterized by coupling constants between each α-hydrogen and its
3
34
3
adjacent amide NeH ( JHα-HN) ≥8 Hz. These dipeptides all gave JHα-
HN ranging from 8.0 to 8.7 Hz, indicating backbone β-strand geometry,
as per the heterocyclic constraint design feature. N-Boc alanine deri-
3
vatives 27 and 14, which lack a N-terminal heterocycle, gave
J
Hα-
HN = < 8 Hz, suggesting the backbone β-strand character of these
compounds is ill-defined.
Peptidic aldehydes 10–14 were assayed against α-chymotrypsin
using a fluorescence-based assay to determine in vitro efficacy, and the
results are summarised in Table 1. As anticipated, pyrrole-containing
10 was the most potent inhibitor with an IC50 value of 1.1 µM, while
dipeptides containing thiophene (11), furan (12) and pyridine (13)
were comparatively weaker, with IC50 values of 40, 36 and 35 µM, re-
spectively. The improved potency of 10 over the other heterocyclic
Scheme 1. Synthesis of aldehyde inhibitors 10–14. Reagents (i) 16, 17 or 18,
HATU, DIPEA, DMF (43–50%); (ii) LiOH, H
2
O, THF, then MeI, K
2
CO , DMF
3
(
75% over two steps); (iii) DMP, CH
DMF (62%); (v) DMP, CH Cl (52%); (vi) N-Boc-alanine, HATU, DIPEA, DMF
82%); (vii) LiBH , THF (100%); (viii) DMP, CH Cl (55%).
2
Cl
2
or THF (20–28%); (iv) HATU, DIPEA,
2
2
(
4
2
2
aldehydes (> 30-fold) is likely due to the pyrrole nitrogen donor hy-
drogen bonding to the protease, as discussed earlier2
6,27,35–37
and
2

N.C. Schumann et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
almost identical fashion, with the pyrrole units closely overlaying. This
suggests that hydrogen bond with Gly216 is critical for overall binding.
Interestingly, this superimposition of 8 and 10 clearly demonstrates
that both bind in the expected extended β-strand conformation, with
their backbones adopting near identical conformations (Fig. 3). Thus, it
appears that this critical geometry can be achieved by inclusion of a
pyrrole without the added need for macrocylisation.
In summary, a series of dipeptide aldehydes with different N-term-
inal heterocycles was prepared and assayed in-vitro against α-chymo-
trypsin to reveal the importance of the heterocycle in maintaining a
backbone β-strand geometry and hydrogen bond donor. Pyrrole-con-
taining 10 was the most potent inhibitor of the series with an IC50 of
Fig. 3. Comparison of a general peptidic substrate of chymotrypsin and in-
hibitor 10.
1
.1 µM, while inhibitors 11–13 were all comparatively weaker
with > 30-fold lower potency. Molecular docking of 10 bound to α-
chymotrypsin suggests a unique hydrogen bond between its NeH and
the backbone carbonyl of Gly216, with the pyrrole nitrogen shown to be
closely positioned to the backbone carbonyl of Gly216 in the S pocket.
3
This interaction is known to be important in the binding of substrates to
α-chymotrypsin and related proteases.3
6,39,40
Acknowledgments
The authors ADA and NCS acknowledge funding support from the
Centre for Nanoscale BioPhotonics (CNBP) through the Australian
Research Council (ARC) CE140100 003. This work was performed in
part at the OptoFab node of the Australian National Foundation Facility
utilizing commonwealth and South Australian State Government
funding.
Fig. 4. Representative pose of 10 docked to α-chymotrypsin (grey), overlaid
with the crystal structure of a macrocyclic inhibitor 5BF (8) bound to α-chy-
motrypsin (yellow).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2018.12.032.
further detailed below in the docking studies. The heterocycle het-
eroatom of 10–13 (Fig. 3) is assumed to replace the backbone amide
NeH at the P position in a natural peptidic substrate of α-chymo-
3
References
trypsin. This amide NeH is known to form a hydrogen bond with the
backbone carbonyl of Gly216 in chymotrypsin’s S subsite. This hy-
drogen bond reorientates Gly216 in the S pocket to facilitate proteolytic
cleavage of its substrate. This change in the orientation of Gly216 assists
3
1
2
3
4
.
.
.
.
Tyndall DA, Nall T, Fairlie DP. Proteases universally recognize beta strands in their
active sites. Chem Rev. 2005;105:973–999. https://doi.org/10.1021/cr040669e.
Loughlin WA, Tyndall JD, Glenn MP, Fairlie DP. Beta-strand mimetics. Chem Rev.
3
2
004;104:6085–6117. https://doi.org/10.1021/cr040648k.
2
6,27
in orienting the substrates scissile bond at P
1
for cleavage
and en-
Leung D, Abbenante G, Fairlie DP. Protease inhibitors: current status and future
prospects. J Med Chem. 2000;43:305–341. https://doi.org/10.1021/jm990412m.
Reid RC, Kelso MJ, Scanlon MJ, Fairlie DP. Conformationally constrained macro-
cycles that mimic tripeptide β-strands in water and aprotic solvents. J Am Chem Soc.
3
5–37
hances discrimination of residues binding at P .
1
The pyrrole 10 is
then able to similarly form a hydrogen bond with the backbone car-
bonyl of Gly216, which would then account for its significantly im-
proved activity over aldehydes 11–13. This interaction thus appears to
be critical for binding. In support, the π-deficient pyridine-containing
2
002;124:5673–5683. https://doi.org/10.1021/ja0256461.
5. Hill TA, Shepherd NE, Diness F, Fairlie DP. Constraining cyclic peptides to mimic
protein structure motifs. Angew Chem. 2014;53:13020–13041. https://doi.org/10.
1
002/anie.201401058.
1
3 with a potential hydrogen bond acceptor, gave diminished activity,
6
7
.
.
Tyndall JA, Fairlie DP. Conformational homogeneity in molecular recognition by
comparable to aldehydes 11 and 12. N-Boc alanine derivative 14
IC50 = 1.4 µM) has comparable activity to 10, where both have a
backbone amide nitrogen donor. Interestingly, 14 lacks backbone β-
proteolytic enzymes. J Mol Recognit. 1999;12:363–370. https://doi.org/10.1002/
(
SICI)1099-1352(199911/12)12:6<363::AID-JMR478>3.0.CO;2-M.
(
Abell AD, Jones MA, Coxon JM, et al. Molecular modeling, synthesis, and biological
evaluation of macrocyclic calpain inhibitors. Angew Chem. 2009;48:1455–1458.
https://doi.org/10.1002/anie.200805014.
strand character. This incorporation of a pyrrole at P retains potency
3
while highlighting the need for a backbone nitrogen donor at this po-
sition. As would be expected, the corresponding alcohols 20–24 and 27
were inactive against α-chymotrypsin at the highest concentration
tested (500 μM).
8. Schechter I, Berger A. On the size of the active site in proteases. I. Papain. Biochem
Biophys Res Commun. 1967;27:157–162. https://doi.org/10.1016/j.bbrc.2012.08.
0
15.
9
.
Powers JC, Asgian JL, Ekici OD, James KE. Irreversible inhibitors of serine, cysteine,
and threonine proteases. Chem Rev. 2002;102:4639–4750. https://doi.org/10.1021/
cr010182v.
Pyrrole-containing 10 was docked into α-chymotrypsin (PDB
1
0. Hedstrom L. Serine protease mechanism and specificity. Chem Rev.
2
1
38
4
q2k) using ICM in order to further define the hydrogen bond be-
2
002;102:4501–4523. https://doi.org/10.1021/cr000033x.
tween the pyrrole nitrogen donor and the backbone carbonyl of Gly216
11. Siklos M, BenAissa M, Thatcher GR. Cysteine proteases as therapeutic targets: does
selectivity matter? A systematic review of calpain and cathepsin inhibitors. Acta
Pharm. Sin. B. 2015;5:506–519. https://doi.org/10.1016/j.apsb.2015.08.001.
in the S subsite. ICM allows for covalent bonding between the reactive
3
C-terminal aldehyde and the active site Ser195 hydroxyl, and so 10 was
modelled bound to α-chymotrypsin as a reversible hemiacetal adduct.
This pose was then overlaid onto the crystal structure of a pyrrole-
1
1
2. Craik DJ, Fairlie DP, Liras S, Price D. The future of peptide-based drugs. Chem Biol
Drug Des. 2013;81:136–147. https://doi.org/10.1111/cbdd.12055.
3. Adessi C, Soto C. Converting a peptide into a drug: strategies to improve stability and
bioavailability. Curr Med Chem. 2002;9:963–978. https://doi.org/10.2174/
2
1
containing macrocyclic chymotrypsin inhibitor (8, 5BF, PDB 4q2k)
0
929867024606731.
bound to α-chymotrypsin in an extended β-strand geometry, as shown
in Fig. 4. As predicted, the pyrrole nitrogen of 10 lies in close proximity
to the backbone carbonyl of Gly216 at a 1.9 Å separation, indicating a
strong hydrogen bond. Both 10 and previously reported 8 bind in an
14. Smith AB, Hirschmann R, Pasternak A, et al. Design and synthesis of peptidomimetic
inhibitors of HIV-1 protease and renin. Evidence for improved transport. J Med Chem.
1
994;37:215–218. https://doi.org/10.1021/jm00028a001.
1
5. Pehere AD, Zhang X, Abell AD. Macrocyclic peptidomimetics prepared by ring-
closing metathesis and azide-alkyne cycloaddition. Aust J Chem. 2017;70:2. https://
3

N.C. Schumann et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
doi.org/10.1071/ch16532.
28. Dong KH. Design of protease inhibitors on the basis of substrate stereospecificity.
Pept Sci. 1999;51:3–8. https://doi.org/10.1002/(SICI)1097-0282(1999)
51:1<3::AID-BIP2>3.0.CO;2-Q.
1
1
6. Pehere AD, Abell AD. New β-strand templates constrained by Huisgen cycloaddition.
Org Lett. 2012;14:1330–1333. https://doi.org/10.1021/ol3002199.
7. Zhang X, Bruning JB, George JH, Abell AD. A mechanistic study on the inhibition of
alpha-chymotrypsin by a macrocyclic peptidomimetic aldehyde. Org Biomol Chem.
29. Rawlings ND, Salvesen G. Handbook of Proteolytic Enzymes. Academic Press; 2013.
30. Ganneau C, Moulin A, Demange L, Martinez J, Fehrentz JA. The epimerization of
peptide aldehydes–a systematic study. J Pept Sci. 2006;12:497–501. https://doi.org/
10.1002/psc.751.
2
016;14:6970–6978. https://doi.org/10.1039/c6ob01159d.
1
1
2
2
2
8. Morton JD, Lee HY, McDermott JD, et al. A macrocyclic calpain inhibitor slows the
development of inherited cortical cataracts in a sheep model. Invest Ophthalmol Vis
Sci. 2013;54:389–395. https://doi.org/10.1167/iovs.12-11088.
31. Vilsmeier A, Haack A. Über die Einwirkung von Halogenphosphor auf Alkyl-for-
manilide. Eine neue Methode zur Darstellung sekundärer und tertiärer p-Alkylamino-
benzaldehyde. Ber Dtsch Chem Ges. 1927;60:119–122. https://doi.org/10.1002/cber.
19270600118.
9. Chen YT, Lira R, Hansell E, McKerrow JH, Roush WR. Synthesis of macrocyclic
trypanosomal cysteine protease inhibitors. Bioorg Med Chem Lett.
2
008;18:5860–5863. https://doi.org/10.1016/j.bmcl.2008.06.012.
32. Hong F, Zaidi J, Pang Y-P, Cusack B, Richelson E. Design, synthesis and pharmaco-
logical evaluation of active pyrrole based, nonpeptidic analogues of neurotensin
(8–13). Perkin Trans. 1997;20:2997–3004. https://doi.org/10.1039/A703319B.
33. Lindegren BO, Nilsson T. Preparation of carboxylic acids from aldehydes (including
hydroxylated benzaldehydes) by oxidation with chlorite. Acta Chem Scand.
1973;27:888–890. https://doi.org/10.3891/acta.chem.scand.27-0888.
34. Carulla N, Woodward C, Barany G. BetaCore, a designed water soluble four-stranded
antiparallel beta-sheet protein. Protein Sci. 2002;11:1539–1551. https://doi.org/10.
1110/ps.4440102.
0. Seiwert SD, Andrews SW, Jiang Y, et al. Preclinical characteristics of the hepatitis C
virus NS3/4A protease inhibitor ITMN-191 (R7227). Antimicrob Agents Chemother.
2
008;52:4432–4441. https://doi.org/10.1128/AAC.00699-08.
1. Chua KC, Pietsch M, Zhang X, et al. Macrocyclic protease inhibitors with reduced
peptide character. Angew Chem. 2014;53:7828–7831. https://doi.org/10.1002/anie.
2
01404301.
2. Jones MA, Morton JD, Coxon JM, et al. Synthesis, biological evaluation and mole-
cular modelling of N-heterocyclic dipeptide aldehydes as selective calpain inhibitors.
Bioorg Med Chem Lett. 2008;16:6911–6923. https://doi.org/10.1016/j.bmc.2008.05.
35. Schellenberger V, Braune K, Hofmann HJ, Jakubke HD. The specificity of chymo-
trypsin. A statistical analysis of hydrolysis data. Eur J Biochem. 1991;199:623–636.
https://doi.org/10.1111/j.1432-1033.1991.tb16163.x.
0
48.
2
2
2
2
3. Abell AD, Foulds GJ. Synthesis of a cis-conformationally restricted peptide bond
isostere and its application to the inhibition of the HIV-1 protease. J Chem Soc, Perkin
Trans 1. 1997:2475–2482. https://doi.org/10.1039/A702458D.
36. Ma W, Tang C, Lai L. Specificity of trypsin and chymotrypsin: loop-motion-controlled
dynamic correlation as a determinant. Biophys J. 2005;89:1183–1193. https://doi.
org/10.1529/biophysj.104.057158.
4. Nam DH, Lee KS, Kim SH, et al. Design and synthesis of 4-quinolinone 2-carbox-
amides as calpain inhibitors. Bioorg Med Chem Lett. 2008;18:205–209. https://doi.
org/10.1016/j.bmcl.2007.10.097.
37. Coombs GS, Rao MS, Olson AJ, Dawson PE, Madison EL. Revisiting catalysis by
chymotrypsin family serine proteases using peptide substrates and inhibitors with
unnatural main chains. J Biol Chem. 1999;274:24074–24079. https://doi.org/10.
1074/jbc.274.34.24074.
5. Tully DC, Liu H, Chatterjee AK, et al. Synthesis and SAR of arylaminoethyl amides as
noncovalent inhibitors of cathepsin S: P3 cyclic ethers. Bioorg Med Chem Lett.
2
006;16:5112–5117. https://doi.org/10.1016/j.bmcl.2006.07.033.
38. Molsoft ICM, 3.8-6a; San Diego, California 2018.
6. Batra J, Szabo A, Caulfield TR, Soares AS, Sahin-Toth M, Radisky ES. Long-range
electrostatic complementarity governs substrate recognition by human chymotrypsin
C, a key regulator of digestive enzyme activation. J Biol Chem. 2013;288:9848–9859.
https://doi.org/10.1074/jbc.M113.457382.
39. Bode W, Huber R. Natural protein proteinase inhibitors and their interaction with
proteinases. Eur J Biochem. 1992;204:433–451. https://doi.org/10.1111/j.1432-
1033.1992.tb16654.x.
40. Solivan S, Selwood T, Wang Z-M, Schechter NM. Evidence for diversity of substrate
specificity among members of the chymase family of serine proteases. FEBS Lett.
2002;512:133–138. https://doi.org/10.1016/S0014-5793(02)02242-1.
2
7. Knowles JR. Enzyme specificity: α-chymotrypsin. J Theor Biol. 1965;9:213–228.
https://doi.org/10.1016/0022-5193(65)90108-6.
4