Leveraging the cruzain S3 subsite to increase affinity for reversible covalent inhibitors

Article abstract of DOI:10.1016/j.bioorg.2018.04.006

Cruzain is the major cysteine protease of Trypanosoma cruzi, the etiological agent of Chagas disease. Reversible covalent cruzain inhibitors can block the steps of cell differentiation in the parasite and kill the organism. To this end, the description of

Full text of DOI:10.1016/j.bioorg.2018.04.006

Accepted Manuscript
Leveraging the Cruzain S3 Subsite to Increase Affinity for Reversible Covalent
Inhibitors
Lorenzo Cianni, Geraldo Sartori, Fabiana Rosini, Daniela De Vita, Gabriel
Pires, Bianca Rebelo Lopes, Andrei Leitão, Antonio C.B. Burtoloso, Carlos A.
Montanari
PII:
S0045-2068(17)30781-2
DOI:
https://doi.org/10.1016/j.bioorg.2018.04.006
YBIOO 2329
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 October 2017
12 April 2018
12 April 2018
Please cite this article as: L. Cianni, G. Sartori, F. Rosini, D. De Vita, G. Pires, B. Rebelo Lopes, A. Leitão, A.C.B.
Burtoloso, C.A. Montanari, Leveraging the Cruzain S3 Subsite to Increase Affinity for Reversible Covalent
Inhibitors, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.04.006
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Leveraging the Cruzain S3 Subsite to Increase Affinity for Reversible
Covalent Inhibitors
Lorenzo Cianni†*, Geraldo Sartori†, Fabiana Rosini†, Daniela De Vita†,
Gabriel Pires, Bianca Rebelo Lopes†, Andrei Leitão†*, Antonio C. B.
Burtoloso††, Carlos A. Montanari†
†Grupo de Química Medicinal do IQSC/USP, Instituto de Química de São Carlos,
Universidade de São Paulo, Av. Trab. Sancarlense, 400 - Parque Arnold Schimidt, São
Carlos - SP, 13566-590, São Paulo, Brazil
††Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, São
Paulo, Brasil
*corresponding authors: andleitao@iqsc.usp.br (Andrei Leitão). +55 (16) 3373-9943 /
+55 (16) 3373-9985. lorenzo.cianni@usp.br (Lorenzo Cianni). +55 (16) 3373-8060
1

Leveraging the Cruzain S3 Subsite to Increase Affinity for Reversible
Covalent Inhibitors
Cruzain is the major cysteine protease of Trypanosoma cruzi, the etiological agent
of Chagas disease. Reversible covalent cruzain inhibitors can block the steps of cell
differentiation in the parasite and kill the organism. To this end, the description of how
inhibitors modified at the P2/P3 positions lead to analogs with greater cruzain affinity to
the S2/S3 subsites is of fundamental importance. Albeit many efforts are being
employed in the characterization of the interaction processes with S2 subsite, little is
known about the cruzain S3 subsite. In this work, we show a brief but consistent study
to identify favorable substitutions in P3 of dipeptidyl nitriles that increase cruzain
affinity. Using molecular dynamics simulations, we have identified some dipeptidyl
nitrile analogs with modifications at P3 position that had higher cruzain inhibition than
the original unsubstituted compound. A matched molecular pair analysis shows the
importance of including a chlorine atom in the P3-meta position. The modifications
implemented in P3 are confirmed when profiling the thermodynamic parameters via
isothermal titration calorimetry. The classical enthalpy-entropy compensation
phenomenon, in which enthalpy changes are counterbalanced by entropy results in a
small modification of G. The inclusion of the chlorine atom in the P3-meta position
results in the highest reduction of the detrimental entropic contribution observed in P3.
Keywords: Cruzain inhibitors; Dipeptidyl nitriles; Molecular Dynamics Simulation;
Matched Molecular Pair Analysis; Isothermal Titration Calorimetry
2

1. Introduction
Chagas disease, whose etiological agent is the protozoan parasite Trypanosoma cruzi, is
a serious health and social problem for people living in Latin America and areas
previously considered non-endemic such as Japan, East Europe and North America [1].
More than 300,000 new cases are reported every year in 21 countries around the world
with an average of one million people currently infected with T. cruzi [2-4]. At present,
there is no vaccine available to prevent Chagas disease or effective chemotherapies. The
only two existing drugs in the market, Benznidazole and Nifurtimox, show high side
toxic effects and inefficiency in the chronic stage of the disease [5,6]. Beyond doubt,
new drugs that are safe and efficacious are therefore critically needed. One approach
consists in the discovery and development of cruzain (Cz) inhibitors, the major T. cruzi
cysteine protease, responsible for the survival and propagation of the protozoan parasite
[7]. Consequently, its inhibition is an important strategy for the treatment of this
disease. Three-dimensional (3D) structures of the enzyme with a variety of ligands have
already been resolved [8], thereby prompting us to apply target based molecular
designing with the aim of finding new high-affinity Cz inhibitors. In particular, our
recent studies [9] showed key interactions of P1, P2 and P3 portions of dipeptidyl nitrile
ligands with the respective subsites of the enzyme (S1, S2, S3). Specifically, it is
already known the importance of Cz inhibition via hydrogen bond between the ligand
and residues Asp16 and Gly66 located at S2; and His162 in S1 subsite [10].
The importance of the S3 region still needs to be better understood due to its
open-flat water accessible surface area. However, relevant data and information are
available for the hydrogen bonding interaction of the Ser61 residue with the
corresponding P3 region of different inhibitors co-crystallized with Cz (for instance, see
3

PDB code 1ME3). Herein, we report the molecular design and synthesis of a new series
of high Cz affinity dipeptidyl nitrile inhibitors, based on the structure of substance 5
(Scheme 1), to study and evaluate the regioselective interaction with residue Ser61 via
in silico studies, kinetics and thermodynamic data.
Scheme 1. Synthetic route for dipeptidyl-nitrile derivatives. Conditions for the reaction
steps: (a) HATU, DIPEA, DMF, Argon atmosphere, 16 h, RT; (b) HCOOH, 16 h, RT;
(c, d) HATU, DIPEA, DMF, Argon atmosphere, 16 h, RT.
4

2. Methods
2.1 Molecular Dynamics Simulation
All ligands in this work were considered covalently bonded to the catalytic Cys25
residue from Cz. Initially, we generated the modified cysteine amino acid cross-linked
with the inhibitor and capped at N and C terminal with ACE (acetyl) and NME
(methylamine) groups, respectively. The formal charge of the compound was predicted
by LigPrep [11] considering a pH of 5.8. Electrostatic potential of each modified
cysteine was calculated at HF/6-31G+ level without geometry optimization in
Gaussian09 to a subsequent RESP [12] partial atomic charge derivation using RED-
vIII.4 [13]. In this step, we attributed the partial charge of main chain N, H, C and O
atoms as -0.4157, 0.2719, 0.5973 and -0.5679, respectively (as per standard AMBER
amino acids) and defined total charge of ACE and NME groups as zero. Then, the
library and topological parameters of each nonstandard cysteine were generated using
Antechamber package implemented in AmberTools14 considering FF14SB [14] and
gaff (general amber force field) [15] to treat the original cysteine residue and ligand
atoms, respectively [16]. The missing parameters at the interface of gaff-FF14SB atoms
were estimated based on comparable connectivity in gaff force field.
Cruzain structure was retrieved from Protein Data Bank (PDB code 1ME3) [17].
All water molecules were removed, histidine residues were renamed according to their
protonation and tautomeric states, and Asp60 side chain was considered in its
protonated state. Cross-linked adducts prepared in the previous section replaced the
standard Cys25 amino acid. Complexes were parametrized with FF14SB force field to
amino acid atoms and gaff force field for ligand atoms in tleap. Sodium atoms were
added to neutralize the system, and a truncated octahedral box of TIP3P water
molecules [18] was created extending at least 10 Å beyond any protein atom.
5

Molecular dynamics simulations were carried out using pmemd.cuda script from
Amber14 package. All simulation steps were done using SHAKE method19 to constrain
bonds containing hydrogen atoms and Langevin approach to control temperature
(frequency of 2 s-1). A cutoff of 10 Å was defined for nonbonded interaction
considering a timestep of 2 fs. After, we performed the equilibration protocol consisting
of (i) minimization with positional constraint of 500 kcal.mol-1 for solute atoms; (ii)
minimization without constraints; (iii) system heating from 0 to 310 K in NVT
ensemble and constraint of 10 kcal.mol-1 for solute atoms during 50 ps; (iv) 5 ns
equilibration at NPT ensemble. Then, the whole system was simulated in the NPT
ensemble using three replicates of 100 ns production simulations that were subdivided
into 5 ns sequential simulations. Each simulation is restarted with a new seed for
Langevin simulation and coordinates were saved every 50 ps.
Data regarding the RMSD of the ligand and the distance between the ligand and
protein were calculated for production simulations using the cpptraj as implemented
within the AmberTools14 package. Graphs were generated using Origin software to
perform the analyses.
2.2 Chemistry
All chemicals were purchased as reagent grade and used without further
purification unless otherwise noted. N,N-Dimethylformamide (DMF) was dried over 3
Å activated molecular sieves for 72 hours. All non-aqueous reactions were carried out
under argon atmosphere in oven-dried glassware. Solvents used in high-performance
chromatography (HPLC) were supplied by Tedia and used without further purification
Thin layer chromatography was performed on Fluka Analytical Sigma-Aldrich
silica gel matrix, pre-coated plates with fluorescent indicator 254 nm and/or staining
6

solutions. Flash column chromatography was performed on silica gel (pore size 60 Å,
70-230 mesh).
1H and 13C NMR spectra were recorded on HP – 400 and 500 MHz instruments
in CDCl3 or DMSO-d6. Chemical shifts are referenced to the residual solvent peak, and
J values are given in Hz. The following multiplicity abbreviations are used: (s) singlet,
(d) doublet, (dd) doublet of doublets, (ddd) doublet of doublet of doublets, (dt) doublet
of triplet, (t) triplet, (q) quartet, (m) multiplet, and (br) broad.
Characterization and separation of substances were carried out with a HPLC
system. The analytical HPLC system consisted of a Shimadzu LC (Kyoto, Japan)
equipped with a LC-20AT pump, a LC-20AD pump, a SIL-20A HT autosampler, a
DGU-20A5 degasser, a CBM-20A, SPD-M20A DAD detector and a FRC-10A fraction
collector. Data acquisition was performed using LCsolution software version 1.26 SP5.
The LC system was coupled to an AmaZon SL ion trap mass spectrometer (Bruker
Daltonics, Bremen, Germany) equipped with electrospray ionization (ESI) interface.
Data acquisition was performed with Bruker Daltonics Data Analysis software (version
4.2.383.1). Spectra for all compounds and further details can be found in the
supplementary information.
Solvents were filtered through a 0.45 μm Merck-Millipore membrane before use
and degassed in an ultrasonic bath. In the established HPLC protocol, chiral analysis
and separation were carried out at 32 °C (column oven) where not otherwise specified,
using analytical and semi-preparative cellulose-2 Phenomenex column (Analytical: 5
μm, 250 mm x 4.6 mm I.D, semi-preparative: 5 μm, 250 mm x 10 mm I.D) or Diacel
column (IC-chiralpak: 5 μm, 250 mm x 4.6 mm) via isocratic elution with a flow rate of
0.5 (analytical) and 2.36 mL min-1 (semi-preparative). The most common mobile phase
composition was acetonitrile-water (50:50) (v/v). Volumes of 10 μL (analytical) and
7

1000 μL (semi-preparative) were injected. Quantification was carried out at 200–800
nm and the chromatographic run time varied according to the sample.
Specific rotations ([α]T = 100α/lc, in deg mL g−1 dm−1, but reported herein in
degrees) were observed at the wavelength 589 nm, the D line of a sodium lamp. The
temperature was set to be 25 °C. Samples were weighted using a precision balance
(Sartorius, Model CPA26P) and dissolved in methanol (HPLC grade, Panreac).
Rotations were measured using a Digital Polarimeter (P2000, Jasco): α = observed
rotation in degrees; l = cell path length of 0.1 decimeter; c = concentration in g 100 mL-
1. Values were calculated using 5 measurements for each substance. Melting points
were determined by a Quimica Micro MQAPF-302 apparatus and are uncorrected.
The synthetic route was developed to optimize the set of substituents to be
placed in P1, P2, and P3 that have been defined after the planning and design studies.
All syntheses were started with enantiopure amino acids. Due to the diversity of
building blocks, it was necessary to evaluate different coupling reagents, aiming at the
best yield and lower racemization [9].
The peptide coupling reaction was performed twice; first to bind the amino acid
(enantiomeric pure, amine-Boc-protected) to the nitrile moiety, and secondly (after
removing Boc group) to the wanted benzoyl acids (Scheme 1).
Synthesis of (S)- tert-butyl (1-((cyanomethyl)amino)-1-oxo-3-phenylpropan-2-
yl)carbamate (3)
N, N-diisopropylethylamine (DIPEA, 2.6 Eq) was added to a solution of Boc-L-
phenylalanine (1) (3.77 mmol, 1 Eq), HATU (1.3 Eq) and 1-Amino-1-
cyclopropanecarbonitrile hydrochloride (2) (1.3 Eq.) in DMF (5 mL), under argon
atmosphere. The resulting solution was stirred at room temperature for 16 hours. The
reaction mixture was diluted with ethyl acetate (10 mL) and washed with a saturated
8

NaHCO3 solution (3 x 20 mL) and brine (3 x 20 mL). The organic phase was dried over
MgSO4 and evaporated to give a crude residue that was purified by chromatographic
column on Flash Silica using ethyl acetate: n-hexane (7:3) as the mobile phase to give a
white solid with a good yield.
1
Yield 92%. White solid. Rf= 0.9 (Ethyl Acetate: n-Hexane; 7:3). MP. 146-147 °C. H
NMR (500 MHz,CDCl3) δ 7.27- 7.04 (m, 5H), 4.58 (m, 1H), 3.19 (dd, J =13.8, 5.0 Hz,
1H), 2.88 (dd, J = 9.5, 5.0 Hz, 1H), 1.31 (s, 9H), 1.25 (m, 2H), 1.04 (m, 2H).
13C NMR. (125 MHz, CDCl3) δ 142.88, 131.04, 127.69, 126.21, 120.81, 107.59, 37.36.
Synthesis of (S)-2-amino-N-(1-cyanocyclopropyl)-3-phenylpropanamide (4)
Substance 3 (500 mg) was treated with formic acid (2 mL) at room temperature and
stirred for 18 h. The reaction mixture was evaporated under vacuum to get yellowish
oil. NaOH 1 M was added dropwise to reach pH 9. The product was extracted with
ethyl acetate (4 times) and then washed twice with brine. Then, the organic phase was
evaporated to obtain the colorless oil. The product formation was confirmed by TLC
(ethyl acetate) with ~ 90% yield, and the product was used for the next step without any
further purification.
General procedure for synthesis of substances: 5 -15
Substance (4) (1.3 Eq) was added to a solution with the respective benzoic acid
derivative (1 mmol, 1 Eq), HATU (1.3 Eq) and N,N-diisopropylethylamine (DIPEA, 2.6
Eq) in DMF (5 mL), under argon atmosphere. The resulting solution was stirred at room
temperature for 16 h. This reaction mixture was diluted with ethyl acetate (10 mL) and
washed with a saturated solution of NaHCO3 (3 x 20 mL) and brine (3 x 20 mL). The
organic phase was dried over MgSO4 and evaporated to give a crude residue that was
9

purified by chromatographic column on Flash Silica using as mobile phase ethyl
acetate: hexane (7:3) to give a white solid with 65-80% yield.
(2S)-N-(1-cyanocyclopropyl)-3-phenyl-2-(phenylformamido)propanamide (5)
70% yield. White solid. MP 189-190°C. NMR 1H (500 MHz, DMSO-d6) δ 9.02 (s,
1H), 8.64 (d, J = 8.1 Hz, 1H), 7.82 (d, J = 7.2 Hz, 2H), 7.52 (t, J = 7.3 Hz, 1H), 7.44
(t, J = 7.5 Hz, 2H), 7.30 (d, J = 7.2 Hz, 2H), 7.25 (t, J = 7.5 Hz, 2H), 7.17 (t, J = 7.2 Hz,
1H), 4.60 (m, 1H), 3.03 (dd, J = 23.4, 13.6, 7.6 Hz, 2H), 1.46 (d, J = 2.7 Hz, 2H), 1.04
(d, J = 9.7 Hz, 2H). NMR 13C (125 MHz, DMSO-d6) δ 173.18, 166.71, 138.29, 134.26,
131.81, 129.61, 128.61, 128.55, 127.93, 126.80, 121.18, 55.05, 37.39, 20.19, 16.15,
16.13. HRMS (ESI (+)) Calcd. [C20H20N3O2] 334.1550, Observed: 334.1550 [M+H].
HPLC (50:50 ACN:Water): tR(min)= 12.01. Purity: 97.73 %.
(S)-2-chloro-N-(1-((1-cyanocyclopropyl)amino)-1-oxo-3-phenylpropan-2-yl)benzamide
(6)
5
80% yield. White solid. MP 175- 176 °C [α]D = -45.3° ± 0.5 (CH3OH, C=0.1 ). FT-IR
1
(KBr, cm-1) 3300, 3284, 3030, 2247, 1663, 1650, 1525, 1502, 1329, 1053, 744, 698. H
NMR (500 MHz, DMSO-d6) δ 8.95 (s, 1H), 8.71 (d, J= 10.0 Hz, 1H), 7.42 (m, 2H),
7.35 (td, J= 9.5, 3.0 Hz, 1H), 7.28-7.17 (m, 5H), 4.57 (m, 1H), 3.06 (dd, J= 13.5, 5.0
Hz, 1H), 2.98 (dd, J= 19.0, 13.5 Hz, 1H), 1.44 (m, 2H), 1.02 (m, 2H).13C NMR (125
MHz, DMSO-d6) δ 172.21, 166.27, 137.53, 136.38, 131.05, 130.21, 129.75, 129.42,
129.17, 128.28, 127.07, 126.62, 120.81, 54.30, 39.52, 37.22, 19.89, 15.89. ESI-MS (-)
Calc. for [C20H18ClN3O2] 367.83, found: 366.25 [M-H+]- .HPLC (50:50 ACN:Water):
tR(min)= 12.01. Purity: 96.39 %.
10

((S)-3-chloro-N-(1-((1-cyanocyclopropyl)amino)-1-oxo-3-phenylpropan-2-
yl)benzamide (7)
5
75% yield. White solid. MP 214-215 °C [α]D = -47.3 ° ± 0.2 (CH3OH, C=0.1 ). FT-IR
1
(KBr, cm-1) 3302, 2924, 2243, 1672, 1649, 1537, 1525, 1319, 1076, 750, 696, 686. H
NMR (500 MHz, DMSO-d6) δ 9.03 (s, 1H), 8.83 (d, J= 10.0 Hz, 1H), 7.87 (t, J= 2 Hz,
1H), 7.77 (dt, J= 7.5, 1.5 Hz, 1H), 7.59 (ddd, J= 8.0, 2.0, 1.0 Hz, 1H), 7.48 (t, J= 8 Hz,
1H), 7.29-7.23 (m, 4H), 7.17 (td, J= 7.5 , 1.5 Hz, 1H), 4.59 (m, 1H), 3.06 (dd, J= 13.5,
5.0 Hz, 1H), 2.98 (dd, J= 19.0, 13.5 Hz, 1H), 1.24 (m, 2H), 1.03 (m, 2H).13C NMR
(125MHz, DMSO-d6) δ 172.95, 165.31, 138.21, 136.21, 133.49, 131.66, 130.68,
129.57, 128.56, 127.73, 126.84, 126.71, 121.16, 55.12, 37.32, 20.20, 16.14, 16.12. ESI-
MS (-) Calc. for [C20H18ClN3O2] 367.83, found: 366.25 [M-H+]-. HPLC (50:50
ACN:Water): tR (min)= 14.7. Purity: 94.57 %.
(S)-4-chloro-N-(1-((1-cyanocyclopropyl)amino)-1-oxo-3-phenylpropan-2-yl)benzamide
(8)
5
75% yield. White solid. MP 218 -219 °C. [α]D = -48.0° ± 1 (CH3OH, C=0.1). FT-IR
1
(KBr, cm-1) 3300, 3284, 3030, 2247, 1663, 1649, 1525, 1502, 1329, 1053, 744, 698. H
NMR (500 MHz, DMSO-d6) δ 9.03 (s, 1H), 8.78 (d, J= 10.0 Hz, 1H), 7.83 (dt, J = 10.5,
2.5 Hz, 2H), 7.52 (dt, J= 10.5, 2.5 Hz, 2H), 7.28-7.22 (m, 4H), 7.16 (tt, J = 7, 1.5 Hz,
1H), 4.58 (m, 1H), 3.05 (dd, J= 13.5, 5.0 Hz, 1H), 2.96 (dd, J= 19.0, 13.5 Hz, 1H), 1.45
(m, 2H), 1.01 (m, 2H). 13C NMR (125 MHz, DMSO-d6) δ 172.74, 165.38, 137.92,
136.35, 132.69, 129.58, 129.29, 128.43, 128.26, 126.53, 120.87, 54.81, 39.52, 37.06,
19.89, 15.85, 15.82. ESI-MS (-) Calc. for [C20H18ClN3O2] 367.83, found: 402.22
[M+Cl-]-. HPLC (50:50 ACN:Water): tR (min)= 16.11. Purity: 99.99 %.
11

(S)-N-(1-((1-cyanocyclopropyl)amino)-1-oxo-3-phenylpropan-2-yl)-3-methylbenzamide
(9)
73% yield. White solid. MP 140 – 141 °C [α]D5 = -51.3° ± 0.7 (CH3OH, C=0.1). FT-IR
1
(KBr, cm-1) 3303, 3285, 3030, 2247, 1663, 1650, 1525, 1502, 1329, 1053, 744, 698. H
NMR (500 MHz, DMSO-d6) δ 8.87 (s, 1H), 8.38 (d, J= 10.0 Hz, 1H), 7.61 (m, 2H),
7.29 – 7.12 (m, 7H), 4.62 (m, 1H), 3.06 (dd, J= 13.5, 5.0 Hz, 1H), 2.99 (dd, J= 19.0,
13.5 Hz, 1H), 2.33 (s, 3H) 1.37 (m, 2H), 1.04 (m, 2H). 13C NMR (125 MHz, DMSO-d6)
δ 173.00, 166.93, 137.90, 137.67, 134.24, 133.47, 132.09, 130.08, 129.49, 128.38,
128.31, 126.78, 124.92, 120.71, 79.34, 78.68, 54.77, 37.56, 21.36, 20.13, 16.13. ESI-
MS (-) Calc. for [C21H21N3O2] 347.42, found: 346.31 [M-H+]-. HPLC (50:50
ACN:Water): tR (min)= 13.5. Purity: 95.78 %.
(S)-N-(1-((1-cyanocyclopropyl)amino)-1-oxo-3-phenylpropan-2-yl)-3-
hydroxybenzamide (10)
5
65% yield. White solid. MP 228 - 229 °C. [α]D = -46.7° ± 0.6 (CH3OH, C=0.1). FT-IR
(KBr, cm-1) 3416, 3324, 3305, 2246, 1673, 1649, 1525, 1284, 1027, 706. 1H NMR (500
MHz, DMSO-d6) δ 9.62 (s, 1H), 8.98 (s, 1H), 8.52 (d, J= 10 Hz, 1H), 8.12 (m, 1H),
7.66 (m, 2H), 7.26-7.13(m, 5H), 6.91 (td, J= 7.5, 1.5 Hz, 1H), 4.57 (m, 1H), 3.02 (dd,
J= 13.5, 5.0 Hz, 1H), 2.69 (dd, J= 19.0, 13.5 Hz, 1H),1.46 (m, 2H), 1.03 (m, 2H). 13C
NMR (125 MHz, DMSO-d6) δ 175.47, 167.64, 156.77, 137.59, 135.53, 129.87, 129.42,
129.42, 129.07, 129.07, 127.17, 121.62, 121.57, 120.20, 114.53, 56.87, 38.46, 19.09,
19.09. ESI-MS (-) Calc. for [C20H19N3O3] 349.39. found: 348.23 [M-H+]-.The substance
10 was subjected to semi-preparative chromatography using the following conditions to
afford 20 mg: C18 column (250x 10 mm; 10 m; 100 A; LUNA); isocratic elution
with ACN/water, 30:70; flow rate 2.5 mL/min; injection volume 100 L; stock solution
12

of substance 10 was 10.0 mg/mL in mobile phase. Analyses were carried out on a mass
spectrometer operating in the scan mode (enhanced resolution; 100 - 1200 m/z) under
positive ionization with detection of [M+H]+ 350.16 m/z or [M+Na]+ 372.15 m/z. The
ESI parameters were as follows: capillary voltage – 4500 V; end plate voltage – 500 V;
dry gas – 9 L/min; nebulizer – 40 psi; dry temperature – 300 C. HPLC (Analytic)
(50:50 ACN:Water): tR (min)= 14.53. Purity: 98.61 %.
(S)-N-(1-((1-cyanocyclopropyl)amino)-1-oxo-3-phenylpropan-2-yl)nicotinamide (11)
65% yield. White solid. MP 180 - 181 °C [α]D5 = -63.2 ° ± 0.4 (CH3OH). FT-IR (KBr,
cm-1) 3561.30, 3332.46, 2246.20, 1674.28, 1649.50, 1535.40, 1524.40, 1484.95,
1027.14, 705.0. 1H NMR (500 MHz, DMSO-d6) δ 9.11 (s, 1H), 8.97- 8.94 (m, 2H),
8.70(dd, J= 10.5, 5.5 Hz, 1H), 8.15 (dt, J= 10.5, 5.5 Hz , 2H), 7.49 (ddd, J= 15.5, 10,5
5.0 Hz, 1H), 7.29 (m, 4H), 7.18 (tt, J= 10.0, 3.2 Hz, 1H), 7.48 (m, 1H), 4.62 (m, 1H),
3.11 (dd, J= 13.5, 5.0 Hz, 1H), 3.10 (dd, J= 19.0, 13.5 Hz, 1H), 1.47 (m, 2H), 1.05 (m,
2H).13C NMR (125 MHz, DMSO-d6) δ 172.91, 165.31, 152.40, 149.04, 138.11, 135.61,
129.80, 129.60, 128.55, 126.83, 123.77, 121.16, 55.07, 39.77, 37.40, 20.19, 16.14,
16.09. ESI-MS (-) Calc. for [C19H18N4O2] 334.38, found: 333.21 [M-H+]-. HPLC (50:50
ACN:Water): tR (min)= 8.27. Purity: 98.03 %.
(S)-N-(1-((1-cyanocyclopropyl)amino)-1-oxo-3-phenylpropan-2-yl)-3-fluorobenzamide
(12)
75% yield. White solid. MP 219 - 220 °C [α]D5 = - 41.4 ° ± 0.6 (CH3OH). FT-IR (KBr,
cm-1) 3271, 3064, 3026, 2297, 2237, 1676, 1664, 1587, 1541, 1523, 1485, 1435, 1377,
1325, 1300, 1288, 1271, 1224, 1215, 1180, 750, 702. 1H NMR (500 MHz, DMSO-d6) δ
9.02 (s, 1H), 8.76 (d, J = 6.5 Hz, 1H), 7.68 - 7.67 (m, 1H), 7.63 - 7.59 (m, 1H), 7.50 (dt,
13

J = 10.0, 7.5 Hz, 1H), 7.40 – 7.35 (m, 1H), 7.30 – 7.23 (m, 4H), 7.16 (tt, J = 6.0, 1.5 Hz,
1H), 4.62 – 4.56 (m, 1H), 3.08 (dd, , J = 17.0, 16.5 Hz, 1H), 2.98 (dd, , J = 17.0, 12.5
Hz, 1H), 1.47 - 1.44 (m, 2H), 1.04 – 1.01 (m, 2H).13C NMR (125 MHz, DMSO-d6) δ
172.95, 165.38, 165.36, 163.49, 161.06, 138.17, 136.59, 136.53, 130.86, 130.78,
129.57, 128.55, 126.83, 124.12, 124.09, 121.15, 118.82, 118.61, 114.80, 114.57, 55.11,
40.58, 40.38, 40.17, 39.96, 39.75, 39.54, 39.33, 37.34, 20.17, 16.13. ESI-MS (-) Calc.
for [C20H18FN3O2] 351.14, found: 352.03 [M+H]+. HPLC (50:50 ACN:Water): tR
(min)= 12.41. Purity: 96.8 %.
(S)-3-bromo-N-(1-((1-cyanocyclopropyl)amino)-1-oxo-3-phenylpropan-2-yl)benzamide
(13)
72% yield. White solid. MP 229 - 230 °C [α]D5 = - 66.9 ° ± 0.6 (CH3OH). FT-IR (KBr,
cm-1) 3302, 3029, 3024, 2243, 1672, 1649, 1539, 1525, 1377, 1319, 1303, 1219, 1064,
896, 848, 746, 700, 684. 1H NMR (500 MHz, DMSO-d6) δ 9.05 (s, 1H), 8.86 (d, J = 8.0
Hz, 1H), 8.04 (t, J = 1.5 Hz, 1H), 7.84 - 7.82 (m, 1H), 7.76 – 7.74 (m, 1H), 7.46 (t, J =
8.0 Hz, 1H), 7.32 – 7.26 (m, 4H), 7.21 (tt, J = 6.0, 1.5 Hz, 1H), 4.64 – 4.60 (m, 1H),
3.15 (dd, , J = 17.0, 16.5 Hz, 1H), 3.01 (dd, , J = 17.0, 12.5 Hz, 1H), 1.50 - 1.47 (m,
2H), 1.08 – 1.03 (m, 2H).13C NMR (125 MHz, DMSO-d6) δ 172.95, 165.23, 138.19,
136.41, 134.54, 130.94, 130.60, 129.57, 128.56, 128.01, 127.09, 126.84, 121.97,
121.16, 55.11, 40.58, 40.49, 40.41, 40.32, 40.24, 40.15, 40.08, 39.99, 39.91, 39.82,
39.65, 39.48, 37.32, 20.18, 16.12. ESI-MS (-) Calc. for [C20H18BrN3O2] 412.29, found:
413.50 [M+H]+. HPLC (50:50 ACN:Water): tR (min)= 18.33. Purity: 99.5 %.
(S)-N-(1-((1-cyanocyclopropyl)amino)-1-oxo-3-phenylpropan-2-yl)-3-iodobenzamide
(14)
14

86% yield. White solid. MP 205 - 207 °C [α]D5 = - 44.3 ° ± 0.5 (CH3OH). FT-IR (KBr,
cm-1) 3286, 3029, 3022, 2241, 1672, 1649, 1522, 1317, 1284, 1299, 1219, 1066, 883,
792, 744, 684. 1H NMR (500 MHz, DMSO-d6) δ 9.00 (s, 1H), 8.78 (d, J = 8.0 Hz, 1H),
8.17 (t, J = 1.5 Hz, 1H), 7.88 - 7.86 (m, 1H), 7.28 – 7.22 (m, 5H), 7.16 (tt, J = 7.0, 2.0
Hz, 1H), 4.60 – 4.55 (m, 1H), 3.05 (dd, J = 15.0, 5.0 Hz, 1H), 2.96 (dd, , J = 14.0, 10.0
Hz, 1H), 1.47 - 1.42 (m, 2H), 1.04 – 1.00 (m, 2H).13C NMR (125 MHz, DMSO-d6) δ
172.97, 165.23, 140.32, 138.19, 136.34, 136.26, 130.85, 129.57, 128.55, 127.41,
126.83, 121.16, 94.96, 55.06, 40.58, 40.49, 40.41, 40.32, 40.24, 40.15, 40.08, 39.99,
39.91, 39.82, 39.65, 39.49, 37.32, 20.18, 16.12.. ESI-MS (-) Calc. for [C20H18IN3O2]
460.39, found: 461.32 [M+H]+. HPLC (50:50 ACN:Water): tR (min)= 22.26 Purity:
99.1 %.
(S)-N-(1-((1-cyanocyclopropyl)amino)-1-oxo-3-phenylpropan-2-yl)-3-
(trifluoromethyl)benzamide (15)
62% yield. White solid. MP 193 - 195 °C [α]D5 = - 65.7 ° ± 0.2 (CH3OH). FT-IR (KBr,
cm-1) 3273, 3025, 2296, 2243, 1683, 1674, 1645, 1539, 1489, 1440, 1325, 1303, 1282,
1170, 1124, 1097, 1072, 748, 696, 684. 1H NMR (500 MHz, DMSO-d6) δ 9.04 (s, 1H),
8.98 (d, J = 8.0 Hz, 1H), 8.15 (s, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 7.5 Hz, 1H),
7.70 (t, J = 8.0 Hz, 1H), 7.29 - 7.22 (m, 4H), 7.19 (t, J = 7.5, Hz, 1H), 4.64 – 4.60 (m,
1H), 3.09 (dd, J = 13.5, 5.0 Hz, 1H), 2.98 (dd, J = 13.5, 10.0 Hz, 1H), 1.47 - 1.44 (m,
2H), 1.04 – 1.03 (m, 2H).13C NMR (125 MHz, DMSO-d6) δ 172.91, 165.32, 138.13,
135.13, 132.06, 130.01, 129.58, 129.33, 128.56, 128.39, 126.86, 125.51, 124.57,
121.15, 55.12, 40.55, 40.46, 40.39, 40.30, 40.22, 40.13, 40.05, 39.96, 39.89, 39.79,
39.63, 39.46, 37.37, 20.19, 16.14. ESI-MS (-) Calc. for [C21H18F3N3O2] 401.14, found:
402.15 [M+H]+. HPLC (50:50 ACN:Water): tR (min)= 14.75 Purity: 97.9 %.
15

2.3 Enzymatic inhibition studies
The recombinant cruzain, consisting of the catalytic domain of cruzipain but
excluding the carboxy-terminal extension, was expressed and purified as previously
described. [10] The enzymatic activity was evaluated by fluorometric assays (Biotek
Synergy HT) monitoring the hydrolysis rate of the fluorogenic substrate Z-Phe-Arg-7-
amido-4-methylcoumarin (Z-FR-MCA, Sigma-Aldrich) with fluorescence emission at
460 nm (excitation at 355 nm) at 37 °C. The reaction was followed over 5 minutes.
Enzyme kinetic assays were carried out in a Corning 96-well black flat bottom
microplates containing 200 L of a solution constituted by 100 mM acetate buffer pH
5.5, 300 mM NaCl, 5 mM DTT (dithiothreitol), 5% v/v DMSO (dimethyl sulfoxide),
0.01 % v/v Triton X-100 and 0.15 nM Cz. The enzyme stock aliquot was rapidly
thawed at 37 °C and kept on ice until activation, in which it was incubated for 20 min in
the assay buffer (100 mM acetate pH 5.5 and 5 mM DTT) followed by additional 2 min
with inhibitors before the addition of the substrate to trigger the reaction.
Visual inspection and a pre-reading of plate wells were performed to check for
possible precipitation and background fluorescence, respectively. None of the
substances displayed a significant fluorescence signal around 460 nm; the emission
wavelength used to monitor reaction kinetics. Thus, potential inner-filter effects did not
have to be taken into account in our experiments. Analysis and manipulation of the data
were performed with Sigma Plot 10.
Each experiment was performed in triplicate for each substance. Initial velocities
of the substrate hydrolysis under the first-order reaction were calculated using Gen5TM
Biotek software. The apparent inhibition constant Ki’ was determined by non-linear
regression using equation Vs = Vo/(1+[I]/Ki’), where Vs is the steady-state rate, Vo is the
rate in the absence of inhibitor, and [I] is the inhibitor concentration. The true inhibition
16

constant Ki was calculated by the correction of Ki’ according to Ki = Ki’/(1+[S]/KM),
where [S] is the substrate concentration and Km is the Michaelis constant. A control
measurement with the covalent reversible fast-binding inhibitor Neq0570 was done for
each setup plate. All inhibitors were evaluated at seven different concentrations that
were chosen based on a previous screening (percent inhibition) at 2.5 µM substrate
concentration (~KM) [20]. Further details can be found in the supplementary
information.
2.4 Isothermal Titration Calorimetry (ITC)
An aliquot of the protein was taken, and a new dialysis was performed using the
last buffer employed in this process during the purification of the protein. Dialysis and
concentration were performed over 30 to 60 min period on Amicon ultra 10 kDa
membranes (Merck Millipore). The inhibitor was placed in the syringe and the protein
in the cell. 0.001% Triton-X 100 was added to the solution of the syringe and the cell to
prevent the protein from aggregation. All thermodynamic parameters were calculated
with Microcal PEAQ-ITC Analysis Software, and experiments were performed at least
in duplicate. All graphs and experimental conditions can be found in the supplementary
information.
3. Results and Discussion
To describe the influence of P3 substitution in the phenyl ring of the known dipeptidyl
nitrile inhibitor (5) acting on Cz [9], we performed three replicates of 100 ns molecular
dynamics (MD) simulations for the covalent nitrile-enzyme complexes. We observed a
better interaction pattern between the Ser61 hydroxyl group and the chlorine atom at the
17

meta-position (7) in relation to orto (6) or para (8) derivatives (described below). We
considered the unsubstituted phenyl ring in P3 (compound 5) for comparative purpose
(Figure 1). The MD for substance 7 showed two well-defined distributions for the
distance between H3 (bound to carbon at the meta position of the aromatic ring) and the
hydroxyl group of Ser61 (dm-Phe_Ser61), centered at 3 and 6.8 Å (Figure S2). The para-
chlorophenyl compound (8) did not bring any further stability, showing free rotation of
the phenyl ring in P3 and no preference between parallel or perpendicular conformation
in respect to Gly65 and Gly66 main chains. On the other hand, we observed that
contacts between orto-chlorophenyl substance and protein prevent the ring rotation
during MD. This was confirmed by the high stability of the initial conformation
throughout the simulation, with chlorine pointed respectively to Gly65 (5.1 Å) and
Leu67 (8.3 Å). Regarding the flexibility, the molecule containing meta-chlorophenyl in
P3 lays in the midrange when compared with orto-chlorophenyl derivative (highly
constrained) and para-chlorophenyl (highly flexible), once the ring is free to rotate but
stay during 75 % of simulation time with dm-Phe_Ser61 less than 5.5 Å (Figure 1). From
these frames, we observed that the angle and distance of chlorine atom from the ligand
and Ser61 hydroxyl group fluctuate between 90 and 170 degrees and 3 and 5.5 Å,
respectively. This pattern is in agreement with what Ibrahim [11] has proposed for a
halogen bond in MD simulations using the Amber force field without extra-point
correction. Finally, for substance 11, it was not observed any interaction pattern with
residue Ser61 (not shown) as for substance 5.
It is known that the Cz Ser61 residue interacts with pyridine and triazole groups
via hydrogen bond, as shown in structures of PDB code 1ME3 [17] and 4KLB [8]. To
understand the possibility of halogen and hydrogen bond interaction of meta-substituted
phenyl derivatives with Ser61, we also carried out MD simulations for m-methyl and m-
18

hydroxyl derivatives (Figure 1). The analysis of the P3 subsite interactions reveals the
ability of meta-hydroxyphenyl moiety (10) to perform weak hydrogen bond with Ser61,
once the interaction amid hydroxyl groups of ligand and Ser61 could be retrieved from
the simulation timeframes leading to a frequency of 14 % (not shown). Also, we
observed that meta-methyl phenyl substituent in (9) has a higher tendency to move out
from the initial position, reaching values as higher as 4 Å, while the other two meta-
substituted molecules present a maximum deviation of 3 Å. The dm-Phe_Ser61 of 3.75 Å is
similar to 3.5 Å observed to meta-chlorophenyl derivative (7) that could be explained
by similar van der Waals radius. Both values are considerably higher than the dm-Phe_Ser61
of 2.9 Å observed for meta-hydroxyphenyl analog (10). Simulations showed the
preference of meta-position substitution on the phenyl ring at the P3 portion of the
substance as well as indicate a possible hydrogen bond interaction between hydroxyl
and chlorophenyl derivatives with Ser61 residue. To better understand this hypothesis
we also evaluated the influence of different halogen atoms in meta-position of P3 ring.
The fluorine derivative molecule (compound 12) showed high rotational flexibility of
ring, similar to that found for the molecule 5. It is an indication of the interaction lost
between S3 subsite and P3 ring and thus, between the fluorine atom and Ser61 side
chain. On the other hand, for bromine-derivative molecule (compound 13), we observed
more sampling for smaller dm-phe_Ser61 described by 75% of simulation frames with dm-
Phe_Ser61 below 5.5 Å and maximum peak at 3.5 Å, which are similar with those found for
compound 7. Based on these simulations, probably the molecules 7, 10 and 13 can
perform a hydrogen bond interaction with residue Ser61 of the cruzain S3 cavity.
19

Figure 1. Histograms (A-I) with the occurrence of molecular dynamics simulation
frames binned by the distance between HO-Ser61 and the atom in meta position of the
ring in P3 subsite and (H) labeling of the dm-Phe_Ser61 distance using PDB ID 4QH6.
Derivatives are displayed according to the substituents at the P3 phenyl ring: A) Phenyl
(5), B) o-chlorophenyl-conf1 (6), C) o-chlorophenyl-conf2 (6), D) p-chlorophenyl (8),
E) m-chlorophenyl (7), F) m-hydroxyphenyl (10), G) m-methyl phenyl (9), H) m-
20

fluorophenyl (12), I) m-iodophenyl (14) groups in complex with cruzain. Conf1:
conformation 1. Conf2: conformation 2.
To better evaluate such a preference and to confirm our hypothesis of possible
interaction with Ser61 in S3 subsite, we carried out an in-depth Cz inhibition study for
substances 5-15. Using pKi affinity values [21] from Table 1, we performed a Matched
Molecular Pair Analysis (MMPA) that provides the opportunity to evaluate molecular
modifications for significant structure-activity relationships (SAR). The analysis was
performed in molecular pairs that differ only in positions where there is a promising
chemical transformation. In this approach, transformations occur by identifying the
change from one functional group to another in which its regiochemistry is also taken
into account. MMPA may be useful in interpreting the bimolecular recognition process
and the individual contributions of each transformation in that process. Our major
interest was focused on P3 modifications considering that substitutions at this position
impact the S3 subsite of the cruzain in a manner that is not well elucidated. This
analysis shows that the pKi median of 6.6 spanned in the range 5.9-7.2, and it displays
some significant transformations occurring among the following pairs: p-Cl/m-Cl, m-
H/m-Cl, m-Cl/m-OH, m-H/m-OH, m-Me/m-Cl, m-Me/m-OH/, m-H/m-Br, m-Cl/m-Br,
m-H/m-I, m-Cl/m-CF3, and p-Cl/o-Cl.7 The most substantial difference in the observed
pKi values is found in the m-H/m-I substitution (5 vs. 14) with the value of 0.8 log units
in favor of the iodine atom in meta-position. Moreover, the inclusion of the chlorine
atom in the meta-position resulted in the second major difference observed in the series
with affinity gain about 0.7 log units (5 vs. 7). This result is corroborated by the average
pose obtained after the molecular modeling, where the P3 phenyl ring occupies the S3
subsite of the cruzain in which the meta position is pointing toward the hydroxyl group
21

of Ser61 (Figure 2). Instead, the exchange of the hydrogen atom by a chlorine atom in
para position (5 vs. 8) resulted in affinity loss of only 0.07 log units, which is not
significant due to experimental uncertainty. Quite surprisingly but not completely
unexpected, the m-Cl/m-OH substitution (7 vs. 10) resulted in an affinity loss of 0.10
log units, while the inclusion of the hydroxyl group in meta showed a gain of 0.61 log
units (10 vs. 5). The same gain in affinity was observed for the m-Me/m-Cl derivatives
(7 vs. 9) or m-H/m-Br derivatives (5 vs 13). A minor gain of 0.2 log units was observed
for transformation o-Cl/p-Cl (6 vs. 8). As we expected, the replacement of chlorine,
bromine or iodine atom by a trifluoromethyl substituent resulted in a loss of affinity. In
particular, this effect is observed for the pair m-Br/m-CF3 with a loss of 0.6 log units;
while we pictured a small loss in affinity (0.1 log units) for the substitution m-H/m-F (5
vs, 12). The most considerable loss in affinity (-1.3 log units) occurred by the
transformation observed between substances 7 and 11, where the inclusion of the
nitrogen heteroatom for the formation of the pyridine moiety did not represent a good
approach.
Figure 2. Average pose obtained after the molecular dynamic simulation with substance
7 covalently bond with cruzain (PDB code 1ME3). Colour for substance 7: Carbon
chain is gray; Chlorine is green; Oxygen is red. Protein is represented as a polar surface;
22

from white to red there is an increment of polarity. The catalytic residue of Cysteine is
spotted with yellow color. P1, P2 and P3 represent the substituents of the compound
that interact with their respective subsites (S1, S2 and S3) at the enzyme.
In summary, these results point out the direct influence of the Cl, Br and I
regiochemistry in the P3 phenyl ring as an adequate strategy to gather the halogen bond
with the hydroxy group from Ser61. Known hydrogen bond donors and acceptors (m-
phenol and 3-pyridine) in comparison with the m-Cl derivative led to less potent cruzain
inhibitors. It corroborates the existence of the halogen bond intermediating the
bimolecular recognition process.
The inclusion of halogen atoms in ligands with the aim of improving interaction
with targets is considered an adequate strategy in molecular design [22]. The attractive
interaction between the electrophilic region of the chlorine, bromine or iodine (the so-
called -hole) and that nucleophile of another molecular fragment may represent a good
approximation for the inclusion of one of these atoms in the phenyl meta-position of P3.
Furthermore, even if bromine and iodine have a greater ability to form halogen bond
than chlorine, their atomic radius could influence their ability to fit properly in the S3
region next to Ser61 residue. To better evaluate this trend we employed isothermal
titration calorimetry to determine thermodynamic parameters of cruzain–dipeptidyl
nitrile interactions (Table 1).
23

Table 1. Affinity (pKi) values for substances 5-15 against cruzain.
G (kcal mol-1) H (kcal mol-1) -TS (kcal mol-1)
ID
pKia
Kd (nM)
6.4 (0.026)
6.6 (0.058)
7.2 (0.020)
6.4 (0.018)
252 (24.4)
ND
-9.00
ND
-14.6
ND
5.60
ND
5
6
121 (17.5)
ND
-9.43
ND
-10.7
ND
1.32
ND
7
8
6.6 (0.0099) 155 (12.6)
-9.29
-9.22
-8.83
-9.11
-9.58
-9.76
-9.45
-12.8
-11.9
-12.1
-13.9
-11.3
-12.0
-12.7
3.55
2.64
3.30
5.96
1.77
2.28
3.29
9
7.0 (0.019)
5.9 (0.017)
6.3 (0.023)
175 (9.51)
338 (91.7)
211 (2.50)
10
11
12
13
14
15
6.9 (0.032) 93.1 (1.30)
7.2 (0.02)
6.6 (0.02)
70.2 (4.50)
125 (4.0)
a pKi = -log(Ki/M); Uncertainty = desvpad/(Ki*ln(10))
ND – Not Determined. Standard deviations are reported in brackets. Deviations for ΔG are lower than
0.4%; ΔH < 4.5%; -TΔS < 15% (except for compound 13, with 22%).
As already described in our recent study, [23] isothermal titration calorimetry
(ITC) is a technique used for the thermodynamic characterization of interactions
between biomolecules and small molecules, which is vital for understanding the process
of molecular recognition. Hence, we studied the thermodynamic profiles of substances
5, 7, and 9-15 to better estimate our halogen-bonding hypothesis. The values of Kd
(Table 1) showed the same trend as depicted by pKi, with substance 14 having the
highest affinity for Cz, followed by substances 13, 7 and 10. This ultimately
corroborates the idea of the meta-halogen derivative (7, 14 and 13) having the required
positioning for a putative interaction with Ser61.
24

The thermodynamic signatures gathered from ITC data reveal that binding of
cruzain inhibitors was driven by favorable enthalpy contributions, with detrimental
entropy for all study substances.
Figure 3 shows the thermodynamic signatures of the interactions that have,
mostly, the same free energy (ΔG) of interaction. The interaction energy is related to the
affinity, which is a combination between enthalpy (ΔH) and entropy (ΔS). Classically,
enthalpy reflects the strength of the interactions due to the formation of hydrogen bonds
and van der Waals, while the binding entropy is a combination of the change in the
desolvation and conformational changes after the formation of the bimolecular ligand-
cruzain complex [25].
A plot (not shown) of enthalpy (H), versus the entropy contribution (-TS)
unveils a quasi-perfect omnipresent phenomenon of enthalpy-entropy compensation
(Hintrinsic = -9.4(± 0.25), -TS = 0.9(± 0.071), R2: 0.982, Adjusted R2: 0.976, F-
statistic: 167.2, p-value: 0.001). Interestingly, the effect of the m-chlorophenyl moiety
on binding affinity decreases both binding enthalpy and entropy contributions (see
compounds 5 and 7 in Fig. 3). For substance 7, the decrease in entropy is more efficient
for the enthalpy-entropy compensation process than that observed for the decrease in
enthalpy change, which leads to a lower Gibb’s free energy than 5.
As mentioned, G values are quite steady over the series. This effect could be
due to an enthalpy-entropy compensation effect [24], where solvent reorganization
could play a central role as reported [25]. The substitution of m-H (-TS = 5.6 kcal mol-
1) by m-CH3 (-TS = 3.5 kcal mol-1), m-OH (-TS = 2.6 kcal mol-1) or m-Cl (-TS =
1.3 kcal mol-1) groups results in a decrease of both enthalpy and entropy (-TS) from
5.6 to 1.3 kcal mol-1 (except for substance 12). This yields an entropic difference (-
TS) gain of 4.3 kcal mol-1 (Table 1, Figure 3). This trend is also followed for m-Br (-
25

TS = 1.8 kcal mol-1) and m-I (-TS = 2.3 kcal mol-1). In particular, there is a small but
consistent gain in enthalpy from m-Cl to m-Br (H = -0.6 kcal mol-1) as well as from
m-Cl to m-Br (H = -1.3 kcal mol-1). Furthermore, the enthalpy-entropy compensation
effect is visible also for m-F and m-CF3, where both improve the enthalpy as from the
m-Br, but with a stronger loss in entropy.
8
6
4
2
0
-2
 G
-4
 H
-6
-T  S
-8
-1 0
-1 2
-1 4
-1 6
5
7
9
0
1
2
3
4
5
1
1
1
1
1
1
C o m p o u n d s
Figure 3. Thermodynamic profiling signatures for the set of inhibitors interacting with
cruzain.
4. Conclusions
Previous studies have shown that dipeptidyl nitriles are potent cruzain inhibitors and
may be as potent as benznidazole [9] in in vitro parasite killing. To better understand
and characterize cruzain inhibition, we synthesized key derivatives that allowed us to
establish critical structure-activity relationships at the P3 ring consistent with an
increase in cruzain affinity. From our studies of molecular dynamics simulation, we
have shown that the m-chlorophenyl, m-bromophenyl, and m-hydroxyphenyl moieties
have the right orientation inside P3 to interact with the oxygen of Ser61 residue. So we
26

synthesized 11 new cruzain inhibitors with different substituents in P3 to better evaluate
this trend. The results are in good agreement with the observed MD simulations, once
the best compounds are precisely those with a lower fluctuation of P3 ring. Indeed the
substitution of a hydrogen atom to an iodine atom in meta position in P3 increased the
pKi of 0.8 log unit. Moreover, our ITC studies pictured that the mechanism of binding
of cruzain inhibitors is driven by favorable enthalpy contributions with quasi-perfect
omnipresent phenomenon of enthalpy-entropy compensation. This tendency is well
visible when we screened the 17th group of the periodic table as possible substituents of
the hydrogen atom in meta position.
Overall, our studies provide further details of the SAR regarding the physical-
chemical profiling of cruzain inhibitors that could also be extended to other cysteine
proteases. This may ultimately aid advancing molecular designing efforts directed to
reversible covalent inhibitors.
5. Acknowledgments
We are grateful to São Paulo Research Foundation (FAPESP) for funding this research
(grant number: 2013/18009-4). Authors declare no conflict of interest.
References
[1]
http://www.who.int/mediacentre/factsheets/fs340/en; (Accessed 08 August 2017).
[2] Y. Jackson, A. Angheben, B. Carrilero Fernandez, J.M. Jansa i Lopez del
WHO - Chagas disease (American trypanosomiasis).
Vallado, J.G. Jannin, P. Albajar-Viñas, Management of Chagas disease in Europe.
Experiences and challenges in Spain, Switzerland and Italy, Bull. Soc. Pathol. Exot. 102
(1990) 326-329.
27

[3]
C. Chagas, Nova tripanozomiaze humana: estudos sobre a morfolojia e o ciclo
evolutivo do Schizotrypanum cruzi n. gen., n. sp., ajente etiolojico de nova entidade
morbida do homem, Mem. Inst. Oswaldo Cruz 1 (1909) 159-218.
[4]
A. Rassi Jr., A. Rassi, J.A. Marin-Neto, Chagas disease, Lancet 375 (2010)
1388-1402.
[5]
J.R. Coura, P.A. Vinas, Chagas disease: a new worldwide challenge, Nature 465
(2010) S6-S7.
[6]
J.A. Castro, M.M. de Mecca, L.C. Bartel, Toxic side effects of drugs used to
treat Chagas disease (American trypanosomiasis), Hum. Exp. Toxicol. 25 (2006) 471-
479.
[7]
target validation to the clinic. Adv. Exp. Med. Biol. 712 (2011) 100-115.
[8] H.J. Wiggers, J.R. Rocha, W.B. Fernandes, R. Sesti-Costa, Z.A. Carneiro, J.
M. Sajid, S.A. Robertson, L.S. Brinen, J.H. McKerrow, Cruzain: the path from
Cheleski, A.B. da Silva, L. Juliano, M.H. Cezari, J.S. Silva, J.H. McKerrow, C.A.
Montanari, Non-peptidic Cruzain Inhibitors with Trypanocidal Activity Discovered by
Virtual Screening and In Vitro Assay, PLoS Negl. Trop. Dis. 7 (2013) e2370.
[9]
L.A. Avelar, C.D. Camilo, S. de Albuquerque, W.B. Fernandes, C. Gonçalez,
P.W. Kenny, A. Leitão, J.H. McKerrow, C.A. Montanari, E.V. Orozco, J.F. Ribeiro,
J.R. Rocha, F. Rosini, M.E. Saidel, Molecular Design, Synthesis and Trypanocidal
Activity of Dipeptidyl Nitriles as Cruzain Inhibitors, PLoS Negl. Trop. Dis. 9 (2015)
e0003916.
[10] H.J. Wiggers, J.R. Rocha, W.B. Fernandes, R. Sesti-Costa, Z.A. Carneiro, J.
Cheleski, A.B. da Silva, L. Juliano, M.H. Cezari, J.S. Silva, J.H. McKerrow, C.A.
Montanari, Non-peptidic cruzain inhibitors with trypanocidal activity discovered by
virtual screening and in vitro assay, PLoS Negl. Trop. Dis. 7 (2013) e2370.
28

[11] M.A. Ibrahim, Molecular mechanical study of halogen bonding in drug
discovery, J. Comput. Chem. 32 (2011) 2564-2574.
[12] W.D. Cornell, P. Cieplak, C.I. Bayly, P.A. Kollman, Application of resp charges
to calculate conformational energies, hydrogen-bond energies, and free-energies of
solvation, J. Am. Chem. Soc. 115 (1993) 9620-9631.
[13] F.Y. Dupradeau, A. Pigache, T. Zaffran, C. Savineau, R. Lelong, N. Grivel, D.
Lelong, W. Rosanski, P. Cieplak, The R.E.D. tools: Advances in RESP and ESP charge
derivation and force field library building, Phys. Chem. 12 (2010) 7821-7839.
[14] J.A. Maier, C. Martinez, K. Kasavajhala, L. Wickstrom, K.E. Hauser, C.
Simmerling, ff14SB: Improving the accuracy of protein side chain and backbone
parameters from ff99sb, J. Chem. Theory Comput. 11 (2015) 3696-3713.
[15] J.M. Wang, R.M. Wolf, J.W. Caldwell, P.A. Kollman, D.A. Case, Development
and testing of a general amber force field, J. Comput. Chem. 25 (2004) 1157-1174.
[16] D.A. Case, V. Babin, J.T. Berryman, R.M. Betz, Q. Cai, D.S. Cerutti, T.E.
Cheatham III, T.A. Darden, R.E. Duke, H. Gohlke, A.W. Goetz, S. Gusarov, N.
Homeyer, P. Janowski, J. Kaus, I. Kolossváry, A. Kovalenko, T.S. Lee, S. LeGrand, T.
Luchko, R. Luo, B. Madej, K.M. Merz, F. Paesani, D.R. Roe, A. Roitberg, C. Sagui, R.
Salomon-Ferrer, G. Seabra, C.L. Simmerling, W. Smith, J. Swails, R.C. Walker, J.
Wang, R.M. Wolf, X. Wu, P.A. Kollman, AMBER 14, University of California, San
Francisco, 2014.
[17] L. Huang, L.S. Brinen, J.A. Ellman, Crystal structures of reversible ketone-
Based inhibitors of the cysteine protease cruzain, Bioorg. Med. Chem. 11 (2003) 21-29.
[18] W.D. Cornell, P. Cieplak, C.I. Bayly, P.A. Kollman, Application of RESP
Charges to Calculate Conformational Energies, Hydrogen Bond Energies, and Free
Energies of Solvation, J. Am. Chem. Soc. 115 (1993) 9620-9631.
29