pubs.acs.

org/jmc Article

Head-to-Tail Cyclization after Interaction with Trypsin: A Scorpion

Venom Peptide that Resembles Plant Cyclotides
Caroline B. F. Mourão, Guilherme D. Brand, João Paulo C. Fernandes, Maura V. Prates, Carlos Bloch, Jr,
João Alexandre R. G. Barbosa, Sônia M. Freitas, Rita Restano-Cassulini, Lourival D. Possani,
and Elisabeth F. Schwartz*
Cite This: J. Med. Chem. 2020, 63, 9500−9511 Read Online

ACCESS *
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Metrics & More Article Recommendations sı Supporting Information

Downloaded via UNIV OF SOUTH AUSTRALIA on October 6, 2020 at 17:11:45 (UTC).

ABSTRACT: Peptidase inhibitors (PIs) have been broadly studied due to

their wide therapeutic potential for human diseases. A potent trypsin
inhibitor from Tityus obscurus scorpion venom was characterized and named
ToPI1, with 33 amino acid residues and three disulfide bonds. The X-ray
structure of the ToPI1:trypsin complex, in association with the mass
spectrometry data, indicate a sequential set of events: the complex formation
with the inhibitor Lys32 in the trypsin S1 pocket, the inhibitor C-terminal
residue Ser33 cleavage, and the cyclization of ToPI1 via a peptide bond
between residues Ile1 and Lys32. Kinetic and thermodynamic characterization
of the complex was obtained. ToPI1 shares no sequence similarity with other
PIs characterized to date and is the first PI with CS-α/β motif described from
animal venoms. In its cyclic form, it shares structural similarities with plant
cyclotides that also inhibit trypsin. These results bring new insights for
studies with venom compounds, PIs, and drug design.

■ INTRODUCTION
Peptidases (proteases) are involved in a wide range of
biological processes and in many human diseases. Therefore,
peptidase inhibitors (PIs) have been studied due to their wide
therapeutic potential as regulators of these enzymes.1−3 Serine
PIs are the largest superfamily of PIs and widely distributed in
nature, and they can be subdivided into many classes based on
their conserved functional motifs. The Kunitz-type inhibitors
are the best characterized of them and, currently, the most
described from venomous/poisonous animals.4 Among the
other structural motifs found in these animals are the Ascaris-
type, Kazal-type, and Bowman−Birk-type motifs. Moreover,
some of these PIs are also K+ channel blockers; however, the
site of interaction with K+ channels differs from the site of
interaction with peptidases.4
Besides these, naturally occurring cyclic peptides rich in
disulfide bonds have been characterized from plants and are
known as cyclotides, which have approximately 30 amino acid
residues and a cyclic cystine knot (CCK) motif. A minor
cyclotide subfamily is known as the trypsin inhibitors, which
currently contains the peptides MCoTI-I to MCoTI-VIII, from
the bitter gourd Momordica cochinchinensis.5 Head-to-tail
cyclization increases the peptide’s resistance to exopeptidase
digestion, which leads to a higher stability and half-life.
Together with the CCK motif, these features are of great
interest for drug design.5
To date, three types of PIs have been described from
scorpion venoms: (i) Kunitz-type,6−12 with 58−70 amino acid
residues, reticulated by three or four disulfide bonds; (ii)
Ascaris-type,12−16 with about 65 amino acid residues and five
disulfide bonds; and (iii) serpin-type,11,12 only with putative
compounds to date. Here, we present the characterization of
an initially acyclic peptide with 33 amino acid residues, isolated
from Tityus obscurus scorpion venom, found in the Amazonian
region, which evolves into a head-to-tail cyclic peptide in the
presence of trypsin, acquiring structural characteristics like
those of plant cyclotides. ToPI1 features make it unique, with
no similarity with any PI previously described from venomous
animals.
■ RESULTS
Chemical Characterization of Native ToPI1. The
fractionation of T. obscurus venom by RP-HPLC yielded
about 60 chromatographic fractions (Figure 1A), which were
all tested against trypsin by means of a chromogenic assay. The
fraction eluting at 26.3% acetonitrile was the only active
Received: April 24, 2020
Published: July 31, 2020

© 2020 American Chemical Society https://dx.doi.org/10.1021/acs.jmedchem.0c00686

9500 J. Med. Chem. 2020, 63, 9500−9511
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

Figure 1. Purification and primary structure of native peptide ToPI1. (A) Fractionation by RP-HPLC of T. obscurus venom (1.0 mg) in a C18
analytical column. A flow rate of 1.0 mL/min was used, and absorbance was monitored at 216 nm. The dashed line represents the gradient of
acetonitrile (B solution). The component marked with the arrow, eluted at 26.3 min (26.3% acetonitrile), was active against trypsin. (B) The
fraction eluting at 26.3 min was further purified by two subsequent chromatographic steps in a RP-HPLC system on a C18 analytical column. The
acetonitrile gradient of the last chromatographic step was as follows: 5−12% B from 0 to 7 min and 12−22% B from 7 to 57 min, at 1.0 mL/min.
The main fraction (28.5 min, or 16.3% B) corresponds to ToPI1 peptide, which was also active against trypsin. (C) Primary sequence of ToPI1,
including a signal peptide of 23 residues at the N-terminus (in italic), the mature toxin with 33 residues (in bold), and two C-terminal residues
(-GK, highlighted in black). In the formation of the mature toxin, these two C-terminal residues are enzymatically cleaved and the toxin acquires a
C-terminal amidation, already shown for both ToPI1 and α-KTx17.1. Similar scorpion venom toxins or putative toxins are shown in the sequence
alignment. In bold and red are the amino acid residue Lys or Arg, predicted to be one of the key amino acid residues for blockage of potassium
channel currents in scorpion KTxs. Highlighted in red is the P1 residue Lys,32 which interacts with trypsin S1 residue.

fraction. Then, it was purified (Figure 1B), resulting in the
peptide named ToPI1, from Tityus obscurus peptidase inhibitor
1. With a molecular mass of [M + H]+ = 3806.89 Da (Figure
S1A,B), native ToPI1 was active against trypsin in a non-
quantitative assay. Its amino acid sequence was partially
determined by MALDI in-source decay (ISD) method,
resulting in residues 9−29 (Figure S1C), and by the cDNA
library. ToPI1 precursor has 177 nucleotides that encode a 58-
amino acid residue peptide, including a signal peptide of 23
residues at the N-terminus and two C-terminal residues (-GK)
that, when cleaved, result in the mature toxin with 33 residues.
These two residues are enzymatically cleaved in the formation
of the mature toxin, which is amidated at the C-terminus
(Figure 1C), a common post-translational modification in
scorpion toxins.17 As expected, the theoretical molecular mass
of the mature toxin ([M + H]+ = 3806.8930), including three
disulfide bonds and the C-terminal amidation, matches with its
experimental molecular mass ([M + H]+ = 3806.8975) (Figure
S1B), with an accuracy of 1.2 ppm.
ToPI1 presents low sequence similarity with other
sequences from public databases, and all of them are scorpion
venom K+ channel toxins (KTxs) or putative toxins (Figure
1C). Sequence similarity with KTxs was only detected when
considering the precursor sequences since the signal peptides,
besides the cysteine residues, were the main responsible for
sequence similarity. Searches for similarity with ToPI1 mature
9501
peptide with other non-redundant sequences available in
databases did not result in significant similarities.
Chemical Synthesis of ToPI1s and ToPI1s-K21A. Due to
the insufficient amount of ToPI1 in the venom, it was
chemically synthesized by means of the Merrifield technique,
oxidized to form the three disulfide bonds and purified, being
named ToPI1s (Figure S2). This nomenclature was used only
to clarify whether native or synthetic peptide was used, but it is
worth saying that no difference was detected between them
during this study. The yield of the pure folded peptide (>97%
purity) with [M + H] + = 3806.89 Da was about 8%. Since, in
many KTxs, Lys21 is predicted to be one of the key amino acid
residues for blockage of K+ channel currents, the analogue
ToPI1s-K21A was also synthesized, replacing Lys21 by Ala21.
Chromogenic Assays for Serine Peptidase Inhibition.
Such as native ToPI1, ToPI1s also inhibits trypsin, with a 1:1
stoichiometric ratio (Figure 2A). ToPI1s exhibits no inhibitory
effect against chymotrypsin, and it almost completely loses its
trypsin inhibitory activity in its reduced form, with no disulfide
bonds (maximum of 20% inhibition at 3.5 mM) (Figure 2C).
The mutation Lys21 to Ala21 did not have major effects in
trypsin inhibition since ToPI1s-K21A and ToPI1s had similar
inhibition profiles, which were also shared by BPTI, one of the
most active and studied PIs.4,18 The extra sum-of-squares F test
showed no difference between the IC50 values of ToPI1s,
ToPI1s-K21A, and BPTI, which varies from 214.0 to 232.3 nM
https://dx.doi.org/10.1021/acs.jmedchem.0c00686
J. Med. Chem. 2020, 63, 9500−9511
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

Figure 2. Trypsin inhibitory activity of ToPI1s. (A) Dose−response profiles for trypsin (400 nM) inhibition by ToPI1s, ToPI1s-K21A, and BPTI
(control) inhibitors (I), fitted (A) by the Morrison’s quadratic equation and (B) by the four-parameter logistic model. Data represent the mean ±
S.E.M. of three independent experiments. (C) Inhibitory effect of ToPI1s against chymotrypsin (400 nM) and of reduced ToPI1s against trypsin
(400 nM). (D) Binding interaction of ToPI1s to surface-immobilized trypsin by surface plasmon resonance. Binding responses obtained by the
injection of ToPI1s at concentrations ranging from 25 to 0.78 nM over a trypsin derivatized CM5 sensor chip at 25 °C are represented as black
lines. Red lines correspond to the fittings of experimental data to a simple bimolecular model (A + B ↔ AB). The association phase of interaction
was monitored for 4 min, and the dissociation phase was recorded for 25 min. (E) Free energy profiles for the ToPI1s:trypsin complex interaction
at pH 7.4. Plots show ΔG°, ΔH°, and −TΔS°. For each plot, the first point corresponds to ToPI1s and trypsin in their free forms, the second point
corresponds to the theoretical transition state, and the third point corresponds to the ToPI1s:trypsin complex. Continuous red lines are used for
easy viewing. (F) Gibbs free energy of binding of the ToPI1s:trypsin complex and temperature dependence of rate constants. Equilibrium
dissociation constants (KD) acquired for the ToPI1s:trypsin interaction were used to calculate the Gibbs free energy of binding as a function of
temperature. A linear form of the Gibbs−Helmholtz equation was fit to the data (represented by the red line), resulting in the parameters shown in
Table 2 (R2 = 0.9905). (G, H Eyring plots generated for ka and kd, respectively, for the ToPI1s:trypsin complex. Pearson correlation coefficients
were higher than 0.95. Each point represents the mean ± S.E.M. of the duplicates of the concentration series at each temperature (in the replicates,
each concentration consisted of three random injections).

(R2 = 0.9758) [F(2, 130) = 1745, p = 0.18] (Figure 2B). The
concentration−response profiles for trypsin inhibition of these
three inhibitors were well fit by the Morrison’s quadratic
equation (Figure 2A), which is in agreement with tight binding
inhibitors.19
Surface Plasmon Resonance. The ToPI1s interaction
kinetics with trypsin was evaluated by surface plasmon
resonance (SPR) at nanomolar concentrations from 15 to 35
°C (Figure S3). Surface control experiments (Figure S3A,B)
were performed, and they indicated that the ToPI1s:trypsin
interaction is bimolecular, with no obvious intermediate states,
9502
and that mass transport effects are negligible in these
experimental conditions. Hence, further experiments were
performed at a flow rate of 40 μL/min. As shown in Figure 2D,
at 25 °C, ToPI1s (25−0.78 nM) strongly binds to trypsin and
has a slow dissociation rate. Sensorgrams recorded at other
temperatures are shown in Figure S3. The dissociation
equilibrium constant (KD) of the ToPI1s:trypsin complex at
25 °C was 530 ± 76 pM, which was similar in the other tested
temperatures (Table 1). Both the sensorgrams and the kinetic
parameters show that, as temperature increases, association
(ka) and dissociation (kd) rates become faster.
https://dx.doi.org/10.1021/acs.jmedchem.0c00686
J. Med. Chem. 2020, 63, 9500−9511
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

Table 1. Kinetic Parameters of the ToPI1s:Trypsin a slightly higher maximum current reduction in hKv1.1
Interaction as a Function of Temperature Calculated by channels, we chose this subtype to test ToPI1s and ToPI1s-
Adjusting the Data Obtained by SPRa K21A at 20 μM (Figure S4C). While the addition of 5 μM
ToPI1s caused a current reduction by 5.1% (I/Imax = 0.9486 ±
ToPI1s:trypsin
0.006), the addition of 20 μM reduced it by 18.3% (I/Imax =
−1 −1
T (°C) ka × 10 (M ·s )
5
kd × 10−4 (s−1) KD × 10−10 (M) 0.8173 ± 0.022) (Figure S4D). In turn, the mutant analogue
15 4.54 ± 0.60 2.99 ± 0.28 6.58 ± 0.23 ToPI1s-K21A reduced the hKv1.1 current by 3.9% (I/Imax =
20 7.28 ± 2.03 3.74 ± 0.91 5.16 ± 0.20 0.9606 ± 0.014), which is 4.7 times lower than ToPI1s at the
25 9.66 ± 0.15 5.12 ± 0.66 5.30 ± 0.76 same concentration, suggesting that Lys21 may be relevant in
30 13.90 ± 1.13 7.26 ± 0.44 5.24 ± 0.10 the inhibitor interaction with the hKv1.1 channel. However,
35 17.50 ± 0.28 8.27 ± 1.79 4.72 ± 0.95 there was no significant statistical difference between groups
a
Data are presented as the mean ± S.E.M. for two independent (KW = 5.982, p = 0.0502) (Figure S4D). All effects were
concentration series for each temperature. In the series, each reversible after washing the cell with external solution.
concentration was randomly injected in triplicate. Circular Dichroism (CD) Spectroscopy. Native ToPI1
and the synthetic peptides ToPI1s and ToPI1s-K21A were
The equilibrium constants determined for the temperature- analyzed by CD spectroscopy, with spectra recorded from 260
dependent SPR analyses were used to calculate the Gibbs free to 190 nm at 25 °C. All purified peptides dissolved well in
energy (ΔG), the enthalpy (ΔS), and entropy (ΔS) of the water at 0.04 mg/mL. As expected, ToPI1s and ToPI1 had
complex ToPI1s:trypsin binding reaction (Table 2). There was superimposable CD spectra (Figure S5A), confirming the
success of synthesis and peptide folding. The mutation Lys21 to
Table 2. Thermodynamics Parameters of the Formation of Ala21 does not seem to affect the secondary structure of
the ToPI1s:Trypsin Complex ToPI1s-K21A since its CD spectra was also similar to native
ToPI1. Little variation in ToPI1s CD spectra was observed
Gibbs−Helmholtz parameters
with increasing temperature, from 25 to 95 °C. At more
ΔG° (kcal·mol−1)a −12.6 extreme pH values, such as 3.0 and 9.0, this variation was
ΔH (kcal·mol−1) 2.3 ± 0.8 slightly higher than at pH 5.0 and 7.0 (Figure S5B−F). The
ΔS (cal·mol−1·K−1) 49.9 ± 2.8 thermal stability of ToPI1s may also be observed by the
theoretical activation parametersb linearity of the thermal denaturation curve, without reaching
ΔG†a (kcal·mol−1) 9.3 molar ellipticity values close to zero. The results show that,
ΔG†d (kcal·mol−1) 21.9 regardless of pH, in the range of 3.0−9.0, the peptide did not
ΔH†a (kcal·mol−1) 11.2 denature up to 95 °C. In addition, there was no evidence of
ΔH†d (kcal·mol−1) 8.9 protein precipitation at elevated temperatures.
ΔS†a (cal·mol−1·K−1) 6.5 X-ray Crystallography. It was observed that linear ToPI1s
ΔS†d (cal·mol−1·K−1) −43.6 undergoes cleavage and cyclization after its interaction with
a
ΔG obtained from −RT ln(KA). bTheoretical activation parameters trypsin. Cyclization was obtained via the formation of the
were derived from Eyring plots using the kinetic rate constants ToPI1s:trypsin complex, which was verified both by MALDI-
obtained by SPR. The Gibbs free energy of activation at 25 °C (298 TOF MS (Figure S6) and crystallographic analyses (Figure 3).
K) was derived from the relationship ΔG = ΔH − TΔS. It is worth mentioning that the peptide cyclization was only
observed in the presence of trypsin during this study. The
a linear dependency between ΔG and reaction temperature (R2 crystallographic structure of the complex between ToPI1s and
= 0.9905), indicating a negligible heat capacity change (ΔCpvh) trypsin was solved to high resolution, 1.29 Å, by molecular
upon complex formation (Figure 2F). The ToPI1s:trypsin replacement. Data collection, processing, and refinement
complex formation is endothermic (ΔH° = 2.3 ± 0.8 kcal· statistics are listed in Table 3. The structure was solved in a
mol−1), driven by a large entropy increase (ΔS° = 49.9 ± 2.8 P212121 space group with just one molecule of ToPI1s and one
cal·mol−1·K−1), which is similar to other trypsin inhib- of trypsin in the asymmetric unit. As expected from sequence
itors.20−22 Applying transition state theory to the reaction similarity results, the structure of ToPI1s is similar to the
rate constants yields the Eyring plots (Figure 2G,H). The structure of scorpion toxins that act on K+ channels, presenting
activation parameters obtained for the binding of the the cysteine-stabilized α/β (CS-α/β) motif.23 The structure of
ToPI1s:trypsin complex are summarized in Table 2. Theoreti- the ToPI1s:trypsin complex is deposited in the RCSB Protein
cal reaction pathways tracing the lowest energy continuous Data Bank under accession code 6MRQ.
pathway between reactants and product for the ToPI1s:trypsin Confirmation that ToPI1s is bound to bovine trypsin
complex interaction are shown in Figure 2E. The species of forming a complex (Figure 3A) can be checked by the omit
maximum energy is defined as the transition state. map electron density of ToPI1s (Figure 3B). The structure
Electrophysiological Tests. ToPI1s, at 5 μM concen- shows the P1 residue, Lys32, at the S1 site of trypsin, in an
tration, was electrophysiologically tested on six K+ channel interaction similar to canonical inhibitors (S1−P1 interaction).
subtypes (hKv1.1, hKv1.4, EAG1, hERG1, hERG2, and This unambiguously identifies the Lys32 residue as P1 in
hERG3). Considering that this is a high toxin concentration detriment of the other seven lysine and two arginine residues
compared to that of other K+ channel scorpion toxins (KTxs), present in the inhibitor sequence that could be in this position.
the reduction in the current caused by the ToPI1s at 5 μM was This lysine is flanked by two isoleucine residues, Ile31 and Ile1.
minimal or null in the tested subtypes (Figure S4A). There was The covalent bond between Lys32 and Ile1 binds the N- and C-
no statistical difference between the groups treated with 5 μM terminus, establishing a cyclic peptide formed upon loss of a
of ToPI1s by Kruskal−Wallis non-parametric test (KW = serine residue at its C-terminus (Figure 3B and Figure 4). This
5.463, p = 0.3621) (Figure S4B). However, since we observed bond respects the peptide bond planarity and distance, being
9503 https://dx.doi.org/10.1021/acs.jmedchem.0c00686
J. Med. Chem. 2020, 63, 9500−9511
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

Figure 3. Tridimensional structure of the ToPI1s in complex with trypsin (PDB 6MRQ). (A) The overall structure showing ToPI1s in sticks and
the trypsin molecule in green cartoon. (B) ToPI1s is shown in detail at the region of the Lys32 P1 residue. Residues from P3 to P2′ are covered by a
2Fo-Fc omit map contoured at 1.5σ. The lack of cleaved residue Ser33 and the peptide bond between Lys32 and Ile1 are clear in the electron density.
The cysteine residues are designated as one-letter codes with ToPI1 sequence numbering. Sulfur atoms are in yellow. (C) Stereoview of the
interaction between the molecules ToPI1s, illustrated with gray carbons and residue numbers up to 32, and trypsin, depicted with green carbons
with residue numbers greater than 40. Water molecules are shown as red spheres and hydrogen bonds as dashed lines. (D) Superposition of the
cyclotide MCoTI-II (orange, PDB 4GUX) and ToPI1s (gray).

intact while bound to the active site of trypsin as other PIs

reactive loops. The formation of a cyclic peptidase inhibitor
(cToPI1s) from a linear peptide (ToPI1s) seems to be unique
among the PIs characterized from the venom of animals
available from public databases. In addition, the structure also
shows which cysteines are involved in the three disulfide
bonds, which are disposed between the cysteines at positions 5
and 22, 11 and 27, 15 and 29. Several interactions, mainly
hydrogen bonds, are present in the interface between trypsin
and inhibitor (Figure 3C), most of them are conserved in
similar complexes. In its cyclic form, acquired after interacting
with trypsin, ToPI1s shares some structural similarities with
some plant cyclotides, as shown by the superposition of ToPI1s
and MCoTI-II (Figure 3D).
9504
■ DISCUSSION AND CONCLUSIONS
Amongst the animal’s PIs characterized so far, ToPI1 presents
unique structural features. Based on sequence similarity and
crystallographic structure, ToPI1 is the first PI with CS-α/β
motif described from animal venoms so far, with high trypsin
inhibitory activity but no affinity for chymotrypsin. As
previously reviewed,4,24 the difference between these enzymes’
catalysis and specificity are due to the catalytic triad of residues
(Ser, Asp, and His) and the residues forming the enzymes’
specificity pockets, respectively. Chymotrypsin has greater
affinity for large hydrophobic residues (Phe, Trp, Tyr, Leu, and
Val, although Met, Asn, and His are also reported) at the P1
position of the substrate/inhibitor since the residues Ser189,
Gly216, and Gly226 create a deep hydrophobic pocket in
https://dx.doi.org/10.1021/acs.jmedchem.0c00686
J. Med. Chem. 2020, 63, 9500−9511
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

Table 3. Crystallographic Data Collection and Refinement

Statistics (Molecular Replacement) for ToPI1s in Complex
with Bovine Trypsina
ToPI1s:trypsin
data collection
space group P212121
cell dimensions
a, b, c (Å) 46.81, 59.02, 79.08
α, β, γ (°) 90.00, 90.00, 90.00
resolution (Å) 27.65−1.29 (1.33−1.29)
Rmerge 0.46 (0.703)
I/σI 18.26 (1.22)
completeness (%) 99.20 (92.85)
redundancy 5.3 (2.7)

refinement
resolution (Å) 27.65−1.29 (1.33−1.29)
no. reflections 294,127 (13,966)
Rwork/Rfree 0.1650/0.1805
no. atoms Figure 4. Comparison of ToPI1 and plant cyclotides and proposed
protein 1897 mechanism for ToPI1 cyclization. (A) Sequence comparison of
ligand/ion 16 cToPI1 and the plant cyclotides MCoTI-I and MCoTI-II. On the
water 297 right is the number of amino acid residues of each sequence. Cys
B factors residues are shown in magenta. The intercysteine loops are numbered
protein 19.98 according to the cyclotides. Loop 1 contains the trypsin binding site
ligand/ion 29.46 of these cyclotides and is shown in bold. Highlighted in red is the P1
water 30.78 residue Lys, which interacts with trypsin S1 residue. Highlighted in
R.m.s. deviations
blue is the Asp residue that is recognized by an asparaginyl
endopeptidase at the propeptide C-terminus of the cyclotide
bond lengths (Å) 0.006
precursor through a transpeptidation reaction. (B) Cyclic backbone
bond angles (°) 1.23 representation of MCoTI-II and cToPI1. The magenta lines indicate
a
One crystal was used. Statistics for the highest-resolution shell are the disulfide bonds. Cys residues are numbered according to the linear
shown in parentheses. precursor sequence, starting with the N-terminal residue. (C)
Proposed mechanism for Ser33 cleavage and head-to-tail cyclization
of ToPI1 through a trypsin-catalyzed transpeptidation reaction. Ser195
chymotrypsin. Otherwise, in trypsin, the enzyme residues attacks the carbonyl of Lys32 to yield a tetrahedral intermediate. The
Asp189, Gly216, and Gly226 create a negatively charged S1 site, oxyanion of the tetrahedral intermediate interacts with the main chain
which results in a higher affinity for substrates/inhibitors NHs of the oxyanion hole within the active site, leading to its
containing positively charged residues (Lys/Arg) at P1, such as stabilization. The intermediate then collapses and forms an acyl-
Lys32 at the P1 position of ToPI1. enzyme intermediate, with the concomitant expulsion of Ser33. Then,
Although ToPI1 shares no sequence similarity with any PI the formation of the peptide bond (aminolysis) between Ile1 and
described to date, after its cleavage and cyclization it shares Lys32 involves the nucleophilic attack of the amine in ToPI1. Finally,
some structural similarities with plant cyclotides, such as the free enzyme is regenerated. The Ser33 residue is shown in the
yellow box. The residues Lys32 and Ile1 are marked with asterisks
MCoTI-I and II (Figure 4), and other acyclic squash trypsin when parts of their atomic formula are also represented in detail.
inhibitors. The loop formed by residues -30MIKILKR4- of
ToPI1 is similar to loop 1 of MCoTI-I and II, represented by
the residues -5PKILKK10- and -5PKILQR10-, respectively differences in the structure are big enough to prevent
(Figure 3D and Figure 4). This reactive site loop forms the conveying that all three disulfides are equivalent.
primary contact with trypsin and the first lysine from this loop We propose that the rearrangement of ToPI1 is caused by
engages the active site.25,26 Besides this expected conserved peptidase-catalyzed transpeptidation through an acyl-enzyme
region, the first and second disulfide bonds are quite intermediate (Figure 4C).27 The acyl-enzyme intermediate
superimposable while the third one, between residues 15 and formed upon proteolysis of the precursor peptide, with the
29, is in a very different position (Cys15 Cα and its sequentially release of Ser33 as the product, would be directly used for the
equivalent atom in Cys21 of MCoTI-II are separated by 6.7 Å nucleophilic attack of the Ile1 amino group forming a cross-link
after superposition). The difference is also observed in the between Ile1 and Lys32 via a peptide bond. Unlike ToPI1,
sequences where six residues are accommodated between where cleavage/cyclization has occurred with the mature
Cys15 and Cys22 in ToPI1 while only one is present between peptide in the presence of trypsin, in cyclotides the cyclization
Cys21 and Cys23 in MCoTI-II, forming a β-sheet. This size occurs simultaneously to the cleavage of the C-terminal
difference is reversed in the region between Cys27 and Cys29 of propeptide from the cyclotide precursor protein. This happens
ToPI1, with only one residue, while between Cys29 and Cys4 of through a transpeptidation reaction mediated by an asparaginyl
MCoTI-II there are eight residues (Figure 3D and Figure 4). endopeptidase (AEP), a cysteine protease commonly found in
The differences in these two cysteine-delimited regions are plants, which recognizes a conserved Asn or Asp residue at the
directly related to the difference found in the position of the C-terminal processing site.28,29 The use of peptidases to form
third disulfide bond. Thus, while there are similarities, peptide bonds in synthetic polypeptides, known as chemo-
9505 https://dx.doi.org/10.1021/acs.jmedchem.0c00686
J. Med. Chem. 2020, 63, 9500−9511
Journal of Medicinal Chemistry
enzymatic approach, has been used as an alternative for
conventional chemical synthesis.30 In this sense, ToPI1 could
be used as a template for the synthesis of cyclic peptides. It is
noteworthy that, although this reaction involves a significant
molecular rearrangement, with ToPI1 cyclization, there was no
kinetically distinguishable intermediate in the reaction, which
appears to occur in a single step, as observed in the linked
reaction test by SPR.
A remarkable feature of ToPI1s is its high stability in its
acyclic form (Figure S5), which is probably enhanced after its
head-to-tail cyclization.5 Regarding high structural stability
under harsh conditions, it is worth mentioning the similarity
with plant cyclotides, which were traditionally extracted via
boiling water.31 Additionally, the reduced activities of ToPI1s
and mainly ToPI1s-K21A against the tested channels suggest
fewer adverse effects from their possible therapeutic
application.32 Due to these characteristics and to their specific
mechanism of interaction with trypsin, these compounds could
be used such as activity based-probes to image trypsin activity
in live animals and tissues. For this, the C-terminal residue
Ser33 should be bound to a tag, such as a fluorophore, that is
detectable once the peptidase cleaves Ser33 with the tag from
the polypeptide.33
As molecular tools, venom-derived peptides have not only
helped to elucidate several critical physiological processes but
also led to the discovery of new drugs and therapeutics for the
treatment of medical conditions including hypertension,
diabetes and chronic pain, as well as potential candidates for
the treatment of cancer, autoimmune diseases, and neuro-
degenerative disorders.34,35 The discovery of a new family of
peptidase inhibitors from scorpion venoms, which will be
better established as other compounds similar to ToPI1 are
described, is quite encouraging since it confirms that venoms
are still a valuable source of new classes of compounds. Thus,
we remain very optimistic regarding the prospecting of venom-
derived peptides for therapeutic application. In order to better
understand the specificity of ToPI1, its activity upon other
peptidases is already under study.
■
EXPERIMENTAL SECTION
General Procedures. Starting materials, unless otherwise
specified, were used as high-grade commercial products. Solvents
were of analytical grade. All solvents and chemicals were used as
purchased without further purification. Chromatographic separations
were performed by reversed-phase (RP) high-performance liquid
chromatography (HPLC). The instrument was a Shimadzu Co.
(Kyoto, Japan) LC10A system equipped with a diode array detector
(SPD-M10A). Three different columns were used: an analytical C18
column (Phenomenex Synergi C18 Fusion-RP 250 × 4.6 mm, 4 μm,
80 Å), a semipreparative C18 column (Phenomenex C18 Jupiter 250
× 10 mm, 5 μm, 300 Å), and a preparative C18 column (Vydac
218TP-1022 C18, 250 × 22 mm, 10 μm, 300 Å). For HPLC, solution
A was 0.12% trifluoroacetic acid (TFA) in water and solution B was
0.1% TFA in acetonitrile. The purity was determined by HPLC, and
the purity of all final compounds was 95% or higher. Molecular mass
analyses of compounds were performed on an Autoflex Speed MALDI
TOF/TOF mass spectrometer (Bruker Daltonics, Germany)
controlled by FlexControl 3.4 software and, in the case of the native
peptide, also by a micrOTOF-Q II MS (Bruker Daltonics, Germany)
equipped with an orthogonal electrospray ionization source (ESI)
operated in the positive mode.
Venom Source. Adult specimens of Tityus obscurus were collected
in the state of Amapá, Brazil, under the Instituto Brasileiro do Meio
Ambiente e dos Recursos Renováveis (IBAMA) license number 048/
2007-CGFAU. They were kept alive in the Arthropod vivarium at the
9506
pubs.acs.org/jmc Article
University of Brası ́lia, and the venom was extracted monthly, treated,
and quantified as previously reported.17
Purification Procedures of Native ToPI1. Aliquots of 1.0 mg of
T. obscurus dried venom were solubilized in 200 μL of deionized
water, centrifuged at 14,000g for 15 min at room temperature, and the
supernatant was submitted to HPLC using the analytical C18 RP
column. The venom molecules were separated using a linear gradient
applied from 100% solution A to 60% solution B during 60 min and
eluted at a flow rate of 1 mL/min, with detection at 216 nm. Fractions
were individually and manually collected, vacuum dried and stored at
−20 °C until use. All fractions were submitted to a trypsin inhibition
assay (n = 3 per fraction), and a pool of the active fraction, eluted at
26.3 min (26.3% solution B), was further fractionated by two
additional chromatographic steps with the same HPLC system and
column, at a flow rate of 1 mL/min, but with the column system at 45
°C. To the first chromatographic step, the following binary gradient
was applied: 10% solution B from 0 to 5 min and 10−25% solution B
from 5 to 50 min. Then, all fractions were analyzed by MALDI-TOF/
TOF MS and an aliquot (20%) of them was tested against trypsin.
The active fraction was submitted to another purification step, with
the following gradient: 5−12% solution B from 0 to 7 min and 12−
22% solution B from 7 to 57 min. The purified peptide, correspondent
to the main chromatographic fraction, was also active against trypsin
after another trypsin chromogenic assay, where 20% of the fully
purified peptide was used. Thus, it was named ToPI1 (Tityus obscurus
peptidase inhibitor 1). Fractions were individually and manually
collected, vacuum dried, quantified, vacuum dried again, and stored at
−20 °C until use. To quantify ToPI1, its absorbance at different
wavelengths (205, 215, and 225 nm) was measured by a
spectrophotometer (Shimadzu UV-1800, Japan) using a 1.0 cm
path length quartz cuvette, and the peptide concentration was
estimated by the following formula:36−38
[(A 215 − A 225)· 144] + (A 205 ·31)
C (μg · mL−1) = · dilution factor
2
Mass Spectrometry and Purity Analysis. Molecular mass
analyses of ToPI1 peptide and the synthetic peptides were performed
on an Autoflex Speed MALDI TOF/TOF MS. Compounds were
reconstituted at 50−300 μL of deionized water and mixed to an α-
cyano-4-hydroxycinnamic acid matrix solution (1:3, v:v), spotted in
triplicate onto a MALDI target plate and dried at room temperature
for 15 min. The spectra were acquired at positive reflector mode.
Calibration of the system was performed using the Peptide
Calibration Standard for Mass Spectrometry calibration mixture (up
to 4000 Da mass range, Bruker Daltonics). Spectra were processed
with FlexAnalysis 3.4. In addition to MALDI-TOF/TOF MS, native
ToPI1 was also analyzed in a micrOTOF-Q II MS (Bruker
Daltonics). The sample was diluted in variable concentrations of
1% formic acid in a water/acetonitrile mixture (1:1, v:v) and applied
to the mass spectrometer source by direct infusion.
Amino Acid Sequence Determination of ToPI1. The amino
acid sequence of ToPI1 native peptide was partially determined by
MALDI in-source decay (ISD) using a 1,5-diaminonaphthalene
matrix solution on an Autoflex Speed MALDI TOF/TOF MS.39
Calibration was performed with bovine serum albumin (BSA;
FRESENIUS KABI, Brazil). The sequencing was guided by ToPI1
putative sequence obtained from the cDNA library of T. obscurus
venom gland, performed as previously described.17 Similarity searches
were performed using BLASTp and BLASTn (http://www.ncbi.nlm.
nih.gov/blast) with an e-value cutoff set to <10−5 to identify putative
functions and structures. ClustalO (http://www.ebi.ac.uk/Tools/
msa/clustalo/) was used for multiple sequence alignments and
calculation of the identity percentage between paired sequences. The
theoretical molecular mass of mature ToPI1 peptide was calculated
with the Isotopic Pattern tool, available from Bruker Daltonics
analysis platform. The nucleotide sequence encoding ToPI1 is
available at the European Nucleotide Archive (ENA) under the
accession code LR594010.
https://dx.doi.org/10.1021/acs.jmedchem.0c00686
J. Med. Chem. 2020, 63, 9500−9511
Journal of Medicinal Chemistry
Chemical Synthesis of ToPI1s and ToPI1s-K21A. ToPI1 was
chemically synthesized by a solid-phase method using the Fmoc/t-
butyl strategy40 on a Prelude automated peptide synthesizer (Protein
Technologies, Inc., USA). A Rink amide MBHA resin (100−200
mesh) (Novabiochem) was used to provide the amino group at the C-
terminus of synthetic ToPI1 (named ToPI1s), identical to the native
peptide. Chemical synthesis was performed according to the
manufacturer’s instructions, with a 100 μmol scale and a 4-fold
molar excess of Fmoc amino acid derivatives. Peptide was cleaved
from the resin on the Tribute UV-IR automated peptide synthesizer
(Protein Technologies, Inc., USA) with simultaneous removal of side-
chain protecting groups by treatment with 10 mL of cleavage cocktail
(81.5% trifluoroacetic acid, 5% water, 5% phenol, 5% thioanisole,
2.5% ethanedithiol, and 1% triisopropylsilane) for 2 h under stirring at
room temperature. Then, the cleaved polypeptide was transferred to a
50 mL tube and washed and precipitated with 10 mL aliquots of ice-
cold diisopropyl ether followed by centrifugation at 4000g for 20 min.
The pellet containing the synthetic polypeptide was resuspended in
15−25 mL of water followed by 15−25 mL of a water:acetonitrile
solution (2:1; v/v). Then, it was transferred to a 250 mL round-
bottom glass flask, frozen in liquid nitrogen, and finally lyophilized.
After lyophilization, the material was quantified by dry weight and
kept at −20 °C. The peptide was synthesized in its reduced form and
later oxidized to form the three disulfide bonds. To induce the
disulfide formation, the crude reduced peptide was dissolved in
deionized water (5 mg/mL) and a folding buffer (25:1; v/v) in
reducing conditions (20 mM Na2HPO4; 0.1 mM NaCl; 5 mM
reduced glutathione; 0.5 mM oxidized glutathione) was added
dropwise under constant stirring.41 Then, the pH was adjusted to
7.9 with NaOH and the solution remained at room temperature for 6
h when the pH was adjusted to 3.0 with formic acid. An aliquot of 1.0
μL of the folded peptide was analyzed in an Autoflex Speed MALDI
TOF/TOF mass spectrometer, as previously described, to verify if the
process occurred according to expected. The oxidized sample was
lyophilized, quantified by dry weight, and maintained at −20 °C until
peptide purification.
The analog peptide ToPI1s-K21A was also synthesized, and its
amino acid sequence was suggested from the comparison of ToPI1
with potassium channel blocker scorpion toxins. The steps of
chemical synthesis, cleavage, and oxidation of ToPI1s-K21A were
identical to the steps described above for ToPI1s, replacing the Lys
residue at position 21 for Ala residue.
Purification Procedures of ToPI1s. The synthetic folded peptide
ToPI1s was purified by three successive steps of RP-HPLC
fractionation using C18 columns and the previously described
solutions. In the first step, aliquots of approximately 25 mg of the
synthetic and oxidized sample in deionized water were filtered
(Millex-LCR hydrophilic PFTE membrane filter, 0.45 μm pore size,
Millipore) and chromatographed on the preparative C18 column with
detection at 216 and 280 nm. The following binary gradient was used,
at 10 mL/min flow rate: 5% B from 0 to 5 min; 5−50% B from 5 to
30 min; 50−95% B from 30 to 35 min; 95% B from 35 to 45 min.
Then, the main fractions were analyzed by MALDI-TOF MS, as
described above, and the fraction containing the peptide with 3806.9
Da (corresponding to the folded peptide ToPI1s) was subsequently
chromatographed on the semipreparative C18 column at 45 °C, at 1.5
mL/min flow rate, as follows: 5−16% B from 0 to 10 min; 16−23% B
from 10 to 37 min; 23−95% B from 37 to 39 min; 95% B from 39 to
44 min. After that, the major fraction was analyzed by MALDI-TOF
MS, and aliquots of about 1.0 mg of folded ToPI1s were purified using
the analytical C18 RP column maintained at 45 °C, at 1.0 mL/min
flow rate, as follows: 7−16% B from 0 to 5 min; 16−22% B from 5 to
33 min; 22−95% B from 33 to 35 min; 95% B from 35 to 40 min.
After purification, the main peptide had its molecular mass verified by
MALDI-TOF MS and was subsequently quantified (in the same way
as described for the native peptide), lyophilized, and stored at −20
°C. The identity between synthetic and native peptides was verified
by percentage of acetonitrile in RP-HPLC elution, mass spectrometry,
circular dichroism, and trypsin inhibition.
9507
pubs.acs.org/jmc Article
A small amount of synthetic ToPI1s in its reduced form was also
purified for some comparison assays. Aliquots of approximately 1.5
mg of the material obtained directly from the synthesis in deionized
water were filtered (Millex-LCR hydrophilic PFTE membrane filter,
0.45 μm pore size, Millipore) and chromatographed using the C18
analytical column at 45 °C and 1.0 mL/min flow rate, with detection
at 216 nm. To the first chromatographic step, the following binary
gradient was used: 5−16% B from 0 to 5 min; 16−22% B from 5 to 45
min; 22−95% B from 45 to 50 min; 95% B from 50 to 55 min. After
that, the main fractions were analyzed by MALDI-TOF MS, and
aliquots of about 1.0 mg of the fraction containing the peptide with
3812.9 Da (corresponding to the reduced ToPI1s) were purified using
the same column and system, with the following gradient: 7−17% B
from 0 to 5 min; 17−19% B from 5 to 15 min; 19−22% from 15 to 35
min; 22−95% B from 35 to 40 min; 95% B from 40 to 45 min. After
purification, the molecular mass of the peptide was checked and the
peptide was quantified, lyophilized, and stored at −20 °C until use.
Purification Procedures of ToPI1s-K21A. The synthetic peptide
ToPI1s-K21A was purified similarly to ToPI1s, being the first
purification step, on a preparative C18 column, equal to the previously
performed. Then, the main fractions were analyzed by MALDI-TOF
MS, as described, and the fraction containing the peptide with
3850.35 Da (corresponding to the folded peptide ToPI1s-K21A) was
subsequently chromatographed on the analytical C18 column,
maintained at 45 °C, at 1.0 mL/min flow rate. We used the same
method described for ToPI1s (7−16% B from 0 to 5 min; 16−22% B
from 5 to 33 min; 22−95% B from 33 to 35 min; 95% B from 35 to 40
min) but with two identical chromatographic steps. After purification,
the peptide molecular mass was checked and the compound was
quantified, lyophilized, and stored at −20 °C until use.
Chromogenic Assays for Serine Peptidase Inhibition. While
native ToPI1 was only subjected to the trypsin inhibition assay at a
single non-quantified concentration (20% of the chromatographic
fraction), due to its low availability in the venom, the synthetic
peptide ToPI1s was tested against both trypsin and chymotrypsin by
means of chromogenic assays. The synthetic peptide ToPI1s-K21A as
well as ToPI1s in its reduced form were also tested on trypsin
inhibition chromogenic assays. As control for trypsin inhibition,
BPTI/aprotinin (Sigma-Aldrich) was used at the same concentration
of tested compounds. As control for chymotrypsin inhibition,
phenylmethylsulfonyl fluoride (PMSF) (Roche Applied Science,
USA) was used at 1.0 and 2.0 mM final concentrations (Cf).
The peptidase inhibition was obtained by measuring the hydrolysis
of synthetic chromogenic substrates in the presence of serine
peptidases: trypsin (bovine pancreatic trypsin; EC 3.4.21.4) and
chymotrypsin (bovine pancreatic α-chymotrypsin; EC 3.4.21.1), and
their respective chromogenic substrates Nα-benzoyl-DL-arginine 4-
nitroanilide hydrochloride (BAPNA) and N-glutaryl-L-phenylalanine
p-nitroanilide (GAPNA) (Sigma-Aldrich, USA).42 Protein concen-
trations were determined by spectrophotometry as previously
described.43 Trypsin and chymotrypsin stock solutions were initially
prepared in a 1.0 mM HCl solution (initial concentration of 2.8 μM).
Their respective substrates, BAPNA and GAPNA, were solubilized in
dimethyl sulfoxide (DMSO) at 50 mg/mL, being subsequently
dissolved in trypsin assay buffer (50 mM Tris-HCl, 20 mM CaCl2, pH
8.2) or chymotrypsin assay buffer (50 mM Tris-HCl, 20 mM CaCl2,
pH 7.6), respectively. All tested compounds were dissolved in the
respective assay buffer.
The experiment was performed on flat bottom 96-well polystyrene
plates in a total volume of 280 μL per well. Plate assembly (serial
dilutions of the peptide and pipetting of buffer, enzyme, and
substrate) was done by using the Microlab STARlet robotic platform
(Hamilton Company, USA).
Forty microliters of 2.8 μM enzyme (Cf = 400 nM) were incubated
with 40 μL of peptide at different concentrations (Cf = 1−800 nM for
trypsin or 1−3500 nM for chymotrypsin) at 25 °C for 20 min. Each
peptide concentration was applied in triplicate. Then, 200 μL of
BAPNA (Cf = 0.4 mM) or GAPNA (Cf = 1.0 mM) substrate was
added to the previous solution, and the product release (p-
nitroaniline) was monitored at the FlexStation 3 Benchtop Multi-
https://dx.doi.org/10.1021/acs.jmedchem.0c00686
J. Med. Chem. 2020, 63, 9500−9511
Journal of Medicinal Chemistry
Mode Microplate Reader (Molecular Devices, USA) spectropho-
tometer at λ = 410 nm for 10−30 min at 25 ° C, with records at every
30 s.
The residual enzyme activity at different peptide concentrations
was calculated using the absorbance (A410) given at 10 min for trypsin
or 30 min for chymotrypsin. The positive control of the reaction
(100% enzyme activity or 0% inhibition) was performed in the
presence of enzyme and the absence of inhibitor (40 μL of enzyme,
40 μL of buffer, and 200 μL of substrate). The negative control for
baseline subtraction was performed in the absence of enzyme (80 μL
of buffer and 200 μL of substrate). The experiments were performed
on three independent replicates for trypsin and two independent
replicates for chymotrypsin, and the values are presented as the mean
± S.E.M. (n ≥ 3).
By using the software GraphPad Prism version 6.0, the trypsin
inhibition profiles were fit by the Morrison’s quadratic equation,19,44
considering that Km equals to 0.8 mM. The Km value was
experimentally obtained by keeping trypsin concentration at 400
nM while increasing BAPNA concentration (Cf = 0.025−2.4 mM);
data was fit by the Michaelis−Menten equation by nonlinear
regression (data not shown) and was in accordance to previously
calculated values.42,45 The peptide concentration that inhibits 50% of
the enzymatic activity, IC50, was fit by the four-parameter logistic
model (variable slope). Then, the extra sum-of-squares F test was
performed to statistically compare the IC50 values obtained with
different peptides.
In order to check the molecular mass of ToPI1s after interacting
with trypsin, an aliquot of the solution from the chromogenic assay,
with ToPI1s (Cf = 400 nM) and trypsin (Cf = 400 nM) incubated for
1 h, without BAPNA addition, was analyzed by MALDI-TOF/TOF
MS, as previously detailed. As control, an aliquot of ToPI1s and
trypsin assay buffer was also analyzed.
Surface Plasmon Resonance. The kinetics and thermodynamics
of the interaction between ToPI1s and the surface immobilized
trypsin at physiological pH were evaluated by surface plasmon
resonance (SPR) in a BIAcore 3000 biosensor (GE Healthcare, UK),
similar to the methodology previously described.22 HBS-EP buffer,
CM5 sensor chips, amine coupling kits, and immobilization and
regeneration buffers were purchased from GE Healthcare.
Trypsin Immobilization. CM5 sensor chips were docked into the
instrument and hydrated for about 24 h before trypsin immobiliza-
tion. Sequencing grade modified trypsin from Promega (Madison, WI,
USA) was immobilized by standard amine coupling chemistry
(probably in its free N-terminus) using HBS-EP (pH 7.4) as running
buffer, flowed at a rate of 5 μL/min. The enzyme was resuspended in
10 mM sodium acetate buffer (pH 5.5) at a concentration of 5 μg/mL
and injected in the flow cells previously activated by injecting a 35 μL
mixture of 50 mM NHS:200 mM EDC, until immobilization levels of
approximately 2000 resonance units (RU) were achieved. Then, the
remaining surface-activated groups of experimental and control cells
were deactivated with 35 μL of 1.0 M ethanolamine (pH 8.5). All
experiments were conducted using two CM5 sensor chips.
Mass Transport and Linked Reaction Test. In order to identify
any possible mass transfer limitations, the ToPI1s:trypsin interaction
kinetics was evaluated at different flow rates (5, 15, and 75 μL/min).
In addition, to investigate if there is a detectable reaction intermediate
in the ToPI1s:trypsin interaction, the dissociation rate was monitored
while varying the analyte injection time (1, 3, and 20 min). In both
control experiments, a solution of 500 nM ToPI1s (from a stock
solution in water) diluted in HPS-EP buffer (pH 7.4) was used.
ToPI1s Interaction with Trypsin. SPR analyses were performed at
40 μL/min flow rate over surface immobilized trypsin. In all
experiments, a blank surface consisting of an activated and deactivated
flow cell was used as a reference for data subtraction. ToPI1s (from a
stock solution in water) was diluted in HPS-EP buffer (pH 7.4) in
twofold dilution series, from 25 to 0.78 nM. At each evaluated
temperature (15, 20, 25, 30, and 35 °C), each peptide concentration
and double blanks (reference buffer) were randomly injected in
triplicate. The entire experiment was replicated at the different
temperatures to ensure reproducibility. After each temperature change
9508
pubs.acs.org/jmc Article
of the system, and before a new concentration series, the system was
normalized with 70% glycerol. Data corresponding to the association
phase of interaction were acquired for 4 min, and the dissociation
phase was monitored for 25 min due to the slow dissociation kinetics.
Before the next cycle started, the surfaces were regenerated with one
30 s injection of 10 mM glycine-HCl buffer (pH 3.0).
Data Analysis for SPR. The response from an average of the blank
injections was subtracted from all experimental sensorgrams to
remove possible systematic artifacts between experimental and
reference flow cells.46 Then, the corrected response data of the
sensorgrams were fit to a simple bimolecular model (A + B ↔ AB)
using BIAevaluation 3.1. Both association (ka) and dissociation (kd)
rate constants were extrapolated from the data in the sensorgrams of
each concentration series by nonlinear least-squares global data fitting.
The equilibrium dissociation constant KD was determined by the ratio
kd/ka.
Thermodynamic and Transition State Analyses. To obtain the
thermodynamic parameters, equilibrium constants were calculated for
the ToPI1s:trypsin interaction at each evaluated temperature and data
was fit to the linear form of the Gibbs−Helmholtz equation as
previously described22 using OriginPro 8.0 (Origin Lab, MA, USA).
Transition state analysis was carried out using the Eyring equation,
which gives the specific reaction rate for a chemical reaction in terms
of the enthalpy and entropy of activation (ΔH†a and ΔS†a,
respectively) and the temperature.46 This equation assumes an active
state in equilibrium with reactants (A + B ↔ AB* ↔ AB). It was also
calculated considering an average of the kinetic and equilibrium
constants at 25 °C. In the current context, this should be considered
as a semi-empirical model.
Electrophysiological Tests. Cell Culture. Cell lines were
cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) and maintained
at 37 °C in a 5% CO2 atmosphere. For transient expression of hKv1.1,
hKv1.4, EAG1, hERG1, hERG2, and hERG3, Chinese hamster ovary
(CHO) cells were cotransfected with plasmids of interest (∼2.0 μg of
DNA) along with plasmid for green fluorescent protein (GFP) (∼0.2
μg of DNA) by using Lipofectamine 3000 (Thermo Fisher Scientific,
USA) according to the manufacturer’s instructions. Cells were
cultured under standard conditions. Currents were measured 1 to 3
days after transfection.
Solutions. For recording hKv1.1, hKv1.4, and EAG1 currents, the
standard extracellular solution (bath) contained (mM): 130 NaCl, 5
KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, and 5 D-glucose (pH 7.29). The
hERG1, hERG2, and hERG3 currents were recorded in a high K+
extracellular solution, which contained (in mM): 95 NaCl, 40 KCl, 2
CaCl2, 2 MgCl2, 10 HEPES, and 5 D-glucose (pH 7.29). This
experimental condition provided the best signal-to-noise relation for
these channels. For all recordings, the internal solution pipette
contained (in mM): 130 K-aspartate, 10 NaCl, 2 MgCl2, 10 EGTA,
and 10 HEPES (pH 7.29). Tested peptides were diluted in the
extracellular solution from a concentrated stock in deionized water to
the final concentrations of 5.0 or 20.0 μM.
Patch-Clamp Recordings and Data Analysis. Electrophysiological
experiments were performed with patch-clamp technique in a whole-
cell configuration using MultiClamp 700B amplifiers and Digidata
1440A digitizer. For data analysis, the pClamp10 software package
(Molecular Devices, USA) was used. For recording of hKv1.1 and
hKv1.4 currents, the cells were held at −90 mV holding potential,
hyperpolarized to −100 mV for 500 ms, and depolarized to +40 mV
for 500 ms followed by a repolarizing step at −50 mV (500 ms). The
time between pulses was 10 s. For recording of hERG tail currents,
the cells were held at −80 mV holding potential, depolarized at +60
mV for 500 ms to activate the current, and the membrane potential
was stepped back at −120 mV to record the inward tail current, pulses
were delivered every 5 s. The EAG1 currents were evoked by voltage
steps to +60 mV for 1.0 s from a holding potential of −80 mV. Prior
to performing experiments with ToPI1s on each K+ channel subtype,
specific blockers were tested for proper current functioning (data not
shown).
https://dx.doi.org/10.1021/acs.jmedchem.0c00686
J. Med. Chem. 2020, 63, 9500−9511
Journal of Medicinal Chemistry
Glass micropipettes were pulled from borosilicate capillaries, and
after filling with internal solution, pipette resistance was about 2.0−
3.0 MΩ in the bath solution. When necessary, 80−90% of cell
capacitance and series resistance errors were compensated for prior to
each voltage clamp protocol to decrease the voltage errors to less than
5% of the protocol pulse. The effect of peptides in a given
concentration is displayed as remaining current fraction (RF = I/I0,
where I is the current amplitude measured in the presence of the
peptide upon reaching block equilibrium and I0 is the current
amplitude measured in the peptide-free control bath solution). ToPI1s
was tested on hKv1.1 (n = 3), hKv1.4 (n = 5), EAG1 (n = 3), hERG1
(n = 3), hERG2 (n = 2), and hERG3 (n = 3) subtypes at 5.0 μM and
on hKv1.1 (n = 3) also at 20.0 μM. The analogue ToPI1s-K21A was
tested on hKv1.1 (n = 3) at 20.0 μM concentration, and its current
effect was compared to the results elicited by ToPI1s. Data points of
the concentration−response curves are averages of independent
measurements where the error bars represent the S.E.M. Data were
analyzed by Kruskal−Wallis non-parametric test followed by Dunn’s
post-test (α = 0.05). All statistical analyses were performed with the
GraphPad Prism version 6.0 software.
Circular Dichroism (CD) Spectroscopy. In order to compare
the secondary structure of native ToPI1, ToPI1s, and ToPI1s-K21A,
CD measurements were carried out on a JASCO J-815 spectropo-
larimeter (JASCO International Co. Ltd., Tokyo, Japan), equipped
with a Peltier temperature controller (JASCO PTC-4238715, Japan).
Far-UV spectra were recorded at 25 °C between 190 and 260 nm at a
scan speed of 50 nm/min and a resolution of 0.1 nm, using 0.1 cm
path length quartz cells. The peptides were analyzed at a
concentration of 0.04 mg/mL in deionized water. Three consecutive
scans were accumulated for each experiment and the average
spectrum was stored and baseline corrected. Data was collected
from a single experiment.
Thermal denaturation assays were performed with ToPI1s (0.05
mg/mL) in 2 mM of the buffers: Na-acetate pH 3.0, Na-acetate pH
5.0, Tris-HCl pH 7.0, and Tris-HCl pH 9.0. Temperature was
increased from 25 to 95 °C, allowing the temperature to equilibrate
before recording each spectrum. To avoid sample evaporation, 200 μL
of mineral oil was added to the cuvette. Spectra scans were recorded
at increments of 0.1 °C. Thermal denaturation was monitored at 208
nm, and at each 10 °C, three consecutive scans were recorded
between 190 and 260 nm, with a resolution of 0.1 nm. Data was
collected from a single experiment.
Ellipticity values ([Θ]obs) were baseline corrected by subtracting
each water or buffer spectrum, and they were converted to the mean
residue ellipticity ([Θ]MRW) in deg·cm2·dmol−1, as previously
described,47 considering the mean molecular mass per residue of
115 Da. The software used to record and process the data were
Spectra Manager (JASCO) and OriginPro 8.0, respectively. The
obtained spectra were analyzed for the intensity and position of
dichroic bands.
X-ray Crystallography. The co-crystallization of ToPI1s with
bovine trypsin was undertaken by using a 2:1 molar ratio mixture of
ToPI1s and 60 mg/mL bovine trypsin, 1 mM HCl, and 20 mM CaCl2
pH 7.6. The search for proper conditions was carried out using
hanging drop vapor diffusion and Crystal Screen and Crystal Screen 2
(Hampton Research) in 96-well plates with the Mosquito robot (TTP
LabTech). Optimization of the initial conditions leads to a final
crystallization solution of 0.1 M HEPES sodium pH 7.5, 1.5 M
lithium sulfate monohydrate in a vapor diffusion sitting drop
procedure. Diffraction data collection took place at the MX-2
beamline of the Laboratório Nacional de Luz Sı ́ncrotron (LNLS,
Campinas, Brazil) at 100 K temperature with a wavelength of 1.20 Å.
Data processing was performed with the XDS program.48 The
PHASER program49 was used for structure determination by
molecular replacement with the bovine trypsin (PDB 4I8H) as the
search model. The initial phases were good enough to build the
peptide using COOT throughout the modeling process.50 The
PHENIX program was used for refinement,51 showing 98.01% of
Ramachandran favored, 1.99% of Ramachandran allowed, and 0% of
Ramachandran outliers.
9509
■
pubs.acs.org/jmc Article
ASSOCIATED CONTENT
* Supporting Information
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00686.
Molecular mass and partial amino acid sequencing of
native ToPI1 (Figure S1), purification and molecular
mass analysis of ToPI1s (Figure S2), kinetics of the
interaction between ToPI1s and trypsin by SPR (Figure
S3), effect of ToPI1s on different subtypes of Kv
channels (Figure S4), circular dichroism spectra analyses
(Figure S5), and molecular mass analyses of ToPI1s
alone and ToPI1s after interacting with trypsin (Figure
S6) (PDF)
PDB ID code 6MRQ: ToPI1s in complex with trypsin
(PDB)
■
AUTHOR INFORMATION
Corresponding Author
Elisabeth F. Schwartz − Neuropharma Lab, Departamento de
Ciências Fisiológicas, Instituto de Ciências Biológicas,
Universidade de Brası ́lia, 70910-900, Brazil; orcid.org/
0000-0001-8239-4055; Email: efschwa@unb.br
Authors
Caroline B. F. Mourão − Neuropharma Lab, Departamento de
Ciências Fisiológicas, Instituto de Ciências Biológicas,
Universidade de Brası ́lia, 70910-900, Brazil; Instituto Federal
de Brası ́lia, Brası ́lia-DF 72220-260, Brazil
Guilherme D. Brand − Laboratório de Sı ́ntese e Análise de
Biomoléculas, LSAB, Instituto de Quı ́mica, Universidade de
Brası ́lia, Brası ́lia-DF 70910-900, Brazil; orcid.org/0000-
0002-1615-0009
João Paulo C. Fernandes − Laboratório de Biofı ́sica Molecular,
Departamento de Biologia Celular, Instituto de Ciências
Biológicas, Universidade de Brası ́lia, Brası ́lia-DF 70910-900,
Brazil
Maura V. Prates − Laboratório de Espectrometria de Massa,
EMBRAPA Recursos Genéticos e Biotecnologia, Brası ́lia-DF
70770-917, Brazil
Carlos Bloch, Jr − Laboratório de Espectrometria de Massa,
EMBRAPA Recursos Genéticos e Biotecnologia, Brası ́lia-DF
70770-917, Brazil
João Alexandre R. G. Barbosa − Laboratório de Biofı ́sica
Molecular, Departamento de Biologia Celular, Instituto de
Ciências Biológicas, Universidade de Brası ́lia, Brası ́lia-DF
70910-900, Brazil
Sônia M. Freitas − Laboratório de Biofı ́sica Molecular,
Departamento de Biologia Celular, Instituto de Ciências
Biológicas, Universidade de Brası ́lia, Brası ́lia-DF 70910-900,
Brazil
Rita Restano-Cassulini − Instituto de Biotecnologı ́a,
Universidad Nacional Autónoma de México, Cuernavaca,
Morelos 62210, Mexico
Lourival D. Possani − Instituto de Biotecnologı ́a, Universidad
Nacional Autónoma de México, Cuernavaca, Morelos 62210,
Mexico
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c00686
https://dx.doi.org/10.1021/acs.jmedchem.0c00686
J. Med. Chem. 2020, 63, 9500−9511
Journal of Medicinal Chemistry
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
Authors will release the atomic coordinates and experimental
data upon article publication.
The nucleotide sequence encoding ToPI1 is available at the
European Nucleotide Archive (ENA) under the accession code
LR594010.
■
ACKNOWLEDGMENTS
This work was supported by the Brazilian National Council for
Scientific and Technological Development - CNPq (407625/
2013-5 and 490447/2013-9). C.B.F.M., E.F.S. and J.P.C.F.
were supported by CNPq. The authors also thank Adolfo C. de
Souza for his help with some mass spectrometry analysis.
■
ABBREVIATIONS USED
CCK, cyclic cystine knot motif; ISD, in-source decay; KTx,
scorpion venom K+ channel toxin; PI, peptidase inhibitor; SPR,
surface plasmon resonance
■ REFERENCES
(1) Eatemadi, A.; Aiyelabegan, H. T.; Negahdari, B.; Mazlomi, M.
A.; Daraee, H.; Daraee, N.; Eatemadi, R.; Sadroddiny, E. Role of
Protease and Protease Inhibitors in Cancer Pathogenesis and
Treatment. Biomed. Pharmacother. 2017, 86, 221−231.
(2) Liang, G.; Bowen, J. P. Development of Trypsin-like Serine
Protease Inhibitors as Therapeutic Agents: Opportunities, Challenges,
and Their Unique Structure-Based Rationales. Curr. Top. Med. Chem.
2016, 16, 1506−1529.
(3) Deu, E.; Verdoes, M.; Bogyo, M. New Approaches for Dissecting
Protease Functions to Improve Probe Development and Drug
Discovery. Nat. Struct. Mol. Biol. 2012, 19, 9−16.
(4) Mourão, C. B. F.; Schwartz, E. F. Protease Inhibitors from
Marine Venomous Animals and Their Counterparts in Terrestrial
Venomous Animals. Mar. Drugs 2013, 11, 2069−2112.
(5) Zhang, R.-Y.; Thapa, P.; Espiritu, M. J.; Menon, V.; Bingham, J.-
P. From Nature to Creation: Going around in Circles, the Art of
Peptide Cyclization. Bioorg. Med. Chem. 2018, 26, 1135−1150.
(6) Zhao, R.; Dai, H.; Qiu, S.; Li, T.; He, Y.; Ma, Y.; Chen, Z.; Wu,
Y.; Li, W.; Cao, Z. SdPI, the First Functionally Characterized Kunitz-
Type Trypsin Inhibitor from Scorpion Venom. PLoS One 2011, 6,
No. e27548.
(7) Ding, L.; Wang, X.; Liu, H.; San, M.; Xu, Y.; Li, J.; Li, S.; Cao, Z.;
Li, W.; Wu, Y.; Chen, Z. A New Kunitz-Type Plasmin Inhibitor from
Scorpion Venom. Toxicon 2015, 106, 7−13.
(8) Chen, Z.-Y.; Hu, Y.-T.; Yang, W.-S.; He, Y.-W.; Feng, J.; Wang,
B.; Zhao, R.-M.; Ding, J.-P.; Cao, Z.-J.; Li, W.-X.; Wu, Y.-L. Hg1,
Novel Peptide Inhibitor Specific for Kv1.3 Channels from First
Scorpion Kunitz-Type Potassium Channel Toxin Family. J. Biol.
Chem. 2012, 287, 13813−13821.
(9) Chen, Z.; Cao, Z.; Li, W.; Wu, Y. Cloning and Characterization
of a Novel Kunitz-Type Inhibitor from Scorpion with Unique
Cysteine Framework. Toxicon 2013, 72, 5−10.
(10) Ma, H.; Xiao-Peng, T.; Yang, S.-L.; Lu, Q.-M.; Lai, R. Protease
Inhibitor in Scorpion (Mesobuthus eupeus) Venom Prolongs the
Biological Activities of the Crude Venom. Chin. J. Nat. Med. 2016, 14,
607−614.
(11) Romero-Gutierrez, T.; Peguero-Sanchez, E.; Cevallos, M. A.;
Batista, C. V. F.; Ortiz, E.; Possani, L. D. A Deeper Examination of
Thorellius atrox Scorpion Venom Components with Omic Techonol-
ogies. Toxins 2017, 9, 399.
9510
pubs.acs.org/jmc Article
(12) Santibáñez-López, C. E.; Cid-Uribe, J. I.; Zamudio, F. Z.;
Batista, C. V. F.; Ortiz, E.; Possani, L. D. Venom Gland
Transcriptomic and Venom Proteomic Analyses of the Scorpion
́
Megacormus gertschi Diaz-Najera, 1966 (Scorpiones: Euscorpiidae:
Megacorminae). Toxicon 2017, 133, 95−109.
(13) Chen, Z.; Wang, B.; Hu, J.; Yang, W.; Cao, Z.; Zhuo, R.; Li, W.;
Wu, Y. SjAPI, the First Functionally Characterized Ascaris-Type
Protease Inhibitor from Animal Venoms. PLoS One 2013, 8,
No. e57529.
(14) Song, Y.; Gong, K.; Yan, H.; Hong, W.; Wang, L.; Wu, Y.; Li,
W.; Li, W.; Cao, Z. Sj7170, a Unique Dual-Function Peptide with a
Specific α-Chymotrypsin Inhibitory Activity and a Potent Tumor-
Activating Effect from Scorpion Venom. J. Biol. Chem. 2014, 289,
11667−11680.
(15) Liu, H.; Chen, J.; Wang, X.; Yan, S.; Xu, Y.; San, M.; Tang, W.;
Yang, F.; Cao, Z.; Li, W.; Wu, Y.; Chen, Z. Functional Character-
ization of a New Non-Kunitz Serine Protease Inhibitor from the
Scorpion Lychas mucronatus. Int. J. Biol. Macromol. 2015, 72, 158−
162.
(16) Romero-Gutiérrez, M. T.; Santibáñez-López, C. E.; Jiménez-
Vargas, J. M.; Batista, C. V. F.; Ortiz, E.; Possani, L. D.
Transcriptomic and Proteomic Analyses Reveal the Diversity of
Venom Components from the Vaejovid Scorpion Serradigitus gertschi.
Toxins 2018, 10, 359.
(17) Guerrero-Vargas, J. A.; Mourão, C. B. F.; Quintero-Hernández,
V.; Possani, L. D.; Schwartz, E. F. Identification and Phylogenetic
Analysis of Tityus pachyurus and Tityus obscurus Novel Putative Na+-
Channel Scorpion Toxins. PLoS One 2012, 7, No. e30478.
(18) Ascenzi, P.; Bocedi, A.; Bolognesi, M.; Spallarossa, A.; Coletta,
M.; Cristofaro, R.; Menegatti, E. The Bovine Basic Pancreatic Trypsin
Inhibitor (Kunitz Inhibitor): A Milestone Protein. Curr. Protein Pept.
Sci. 2003, 4, 231−251.
(19) Copeland, R. A. Evaluation of Enzyme Inhibitors in Drug
Discovery : A Guide for Medicinal Chemists and Pharmacologists; 2nd
ed.; John Wiley & Sons, Inc.: New Jersey, 2013, DOI: 10.1002/
9781118540398.
(20) Ascenzi, P.; Ruoppolo, M.; Amoresano, A.; Pucci, P.; Consonni,
R.; Zetta, L.; Pascarella, S.; Bortolotti, F.; Menegatti, E. Character-
ization of Low-Molecular-Mass Trypsin Isoinhibitors from Oil-Rape
(Brassica napus var. oleifera) Seed. Eur. J. Biochem. 1999, 261, 275−
284.
(21) Baugh, R. J.; Trowbridge, C. G. Calorimetry of Some Trypsin-
Trypsin Inhibitor Reactions. J. Biol. Chem. 1972, 247, 7498−7501.
(22) Brand, G. D.; Pires, D. A. T.; Furtado, J. R., Jr.; Cooper, A.;
Freitas, S. M.; Bloch, C., Jr. Oligomerization Affects the Kinetics and
Thermodynamics of the Interaction of a Bowman-Birk Inhibitor with
Proteases. Arch. Biochem. Biophys. 2017, 618, 9−14.
(23) de la Vega, R. C. R.; Possani, L. D. Current Views on Scorpion
Toxins Specific for K+-Channels. Toxicon 2004, 43, 865−875.
(24) Hedstrom, L. Serine Protease Mechanism and Specificity.
Chem. Rev. 2002, 102, 4501−4524.
(25) Daly, N. L.; Thorstholm, L.; Greenwood, K. P.; King, G. J.;
Rosengren, K. J.; Heras, B.; Martin, J. L.; Craik, D. J. Structural
Insights into the Role of the Cyclic Backbone in a Squash Trypsin
Inhibitor. J. Biol. Chem. 2013, 288, 36141−36148.
(26) Jones, P. M.; George, A. M. Computational Analysis of the
MCoTI-II Plant Defence Knottin Reveals a Novel Intermediate
Conformation That Facilitates Trypsin Binding. Sci. Rep. 2016, 6,
23174.
(27) Conlan, B. F.; Gillon, A. D.; Craik, D. J.; Anderson, M. A.
Circular Proteins and Mechanisms of Cyclization. Biopolymers 2010,
94, 573−583.
(28) Craik, D. J.; Malik, U. Cyclotide Biosynthesis. Curr. Opin.
Chem. Biol. 2013, 17, 546−554.
(29) Du, J.; Yap, K.; Chan, L. Y.; Rehm, F. B. H.; Looi, F. Y.; Poth,
A. G.; Gilding, E. K.; Kaas, Q.; Durek, T.; Craik, D. J. A Bifunctional
Asparaginyl Endopeptidase Efficiently Catalyzes Both Cleavage and
Cyclization of Cyclic Trypsin Inhibitors. Nat. Commun. 2020, 11,
1575.
https://dx.doi.org/10.1021/acs.jmedchem.0c00686
J. Med. Chem. 2020, 63, 9500−9511
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

(30) Yazawa, K.; Numata, K. Recent Advances in Chemoenzymatic Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R.
Peptide Syntheses. Molecules 2014, 19, 13755−13774. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P.
(31) Weidmann, J.; Craik, D. J. Discovery, Structure, Function, and H. PHENIX: A Comprehensive Python-Based System for Macro-
Applications of Cyclotides: Circular Proteins from Plants. J. Exp. Bot. molecular Structure Solution. Acta Crystallogr., Sect. D: Struct. Biol.
2016, 67, 4801−4812. 2010, 66, 213−221.
(32) Vandenberg, J. I.; Perry, M. D.; Perrin, M. J.; Mann, S. A.; Ke,
Y.; Hill, A. P. HERG K+ Channels: Structure, Function, and Clinical
Significance. Physiol. Rev. 2012, 92, 1393−1478.
(33) Vizovišek, M.; Vidmar, R.; Drag, M.; Fonović, M.; Salvesen, G.
S.; Turk, B. Protease Specificity: Towards in vivo Imaging
Applications and Biomarker Discovery. Trends Biochem. Sci. 2018,
43, 829−844.
(34) Pennington, M. W.; Czerwinski, A.; Norton, R. S. Peptide
Therapeutics from Venom: Current Status and Potential. Bioorg. Med.
Chem. 2018, 26, 2738−2758.
(35) Yang, X.; Wang, Y.; Wu, C.; Ling, E.-A. Animal Venom
Peptides as a Treasure Trove for New Therapeutics Against
Neurodegenerative Disorders. Curr. Med. Chem. 2019, 26, 4749−
4774.
(36) Aitken, A.; Learmonth, M. P. Protein Determination by UV
Absorption. In The Protein Protocols Handbook; Humana Press:
Totowa, NJ, 2009, pp 3−6, DOI: 10.1007/978-1-59745-198-7_1.
(37) Stoscheck, C. M. [6] Quantitation of Protein. Methods Enzymol.
1990, 182, 50−68.
(38) Waddell, W. J. A Simple Ultraviolet Spectrophotometric
Method for the Determination of Protein. J. Lab. Clin. Med. 1956, 48,
311−314.
(39) Debois, D.; Smargiasso, N.; Demeure, K.; Asakawa, D.;
Zimmerman, T. A.; Quinton, L.; De Pauw, E. MALDI In-Source
Decay, from Sequencing to Imaging. Top. Curr. Chem. 2012, 331,
117−141.
(40) Pires, D. A. T.; Bemquerer, M. P.; do Nascimento, C. J. Some
Mechanistic Aspects on Fmoc Solid Phase Peptide Synthesis. Int. J.
Pept. Res. Ther. 2014, 20, 53−69.
(41) Stricher, F.; Martin, L.; Vita, C. Design of Miniproteins by the
Transfer of Active Sites onto Small-Size Scaffolds. Methods Mol. Biol.
2006, 340, 113−149.
(42) Erlanger, B. F.; Kokowsky, N.; Cohen, W. The Preparation and
Properties of Two New Chromogenic Substrates of Trypsin. Arch.
Biochem. Biophys. 1961, 95, 271−278.
(43) Barbosa, J. A. R. G.; Silva, L. P.; Teles, R. C. L.; Esteves, G. F.;
Azevedo, R. B.; Ventura, M. M.; de Freitas, S. M. Crystal Structure of
the Bowman-Birk Inhibitor from Vigna unguiculata Seeds in Complex
with β-Trypsin at 1.55 Å Resolution and its Structural Properties in
Association with Proteinases. Biophys. J. 2007, 92, 1638−1650.
(44) Morrison, J. F. Kinetics of the Reversible Inhibition of Enzyme-
Catalysed Reactions by Tight-Binding Inhibitors. Biochim. Biophys.
Acta 1969, 185, 269−286.
(45) Ohta, K.; Makinen, K. K.; Loesche, W. J. Purification and
Characterization of an Enzyme Produced by Treponema denticola
Capable of Hydrolyzing Synthetic Trypsin Substrates. Infect. Immun.
1986, 53, 213−220.
(46) Day, Y. S. N.; Baird, C. L.; Rich, R. L.; Myszka, D. G. Direct
Comparison of Binding Equilibrium, Thermodynamic, and Rate
Constants Determined by Surface- and Solution-Based Biophysical
Methods. Protein Sci. 2002, 11, 1017−1025.
(47) Kelly, S. M.; Jess, T. J.; Price, N. C. How to Study Proteins by
Circular Dichroism. Biochim. Biophys. Acta, Proteins Proteomics 2005,
1751, 119−139.
(48) Kabsch, W. XDS. Acta Crystallogr., Sect. D: Struct. Biol. 2010,
66, 125−132.
(49) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M.
D.; Storoni, L. C.; Read, R. J. Phaser Crystallographic Software. J.
Appl. Crystallogr. 2007, 40, 658−674.
(50) Emsley, P.; Cowtan, K. Coot: Model-Building Tools for
Molecular Graphics. Acta Crystallogr., Sect. D: Struct. Biol. 2004, 60,
2126−2132.
(51) Adams, P. D.; Afonine, P. V.; Bunkóczi, G.; Chen, V. B.; Davis,
I. W.; Echols, N.; Headd, J. J.; Hung, L.-W.; Kapral, G. J.; Grosse-

9511 https://dx.doi.org/10.1021/acs.jmedchem.0c00686
J. Med. Chem. 2020, 63, 9500−9511