Divergent modes of enzyme inhibition in a homologous structure-activity series

Article abstract of DOI:10.1021/jm9009229

A docking screen identified reversible, noncovalent inhibitors (e.g., 1) of the parasite cysteine protease cruzain. Chemical optimization of 1 led to a series of oxadiazoles possessing interpretable SAR and potencies as much as 500-fold greater than 1. De

Full text of DOI:10.1021/jm9009229

J. Med. Chem. 2009, 52, 5005–5008 5005
DOI: 10.1021/jm9009229
commercially available leadlike12,13 compounds that could
subsequently be optimized by medicinal chemistry. Publically
available structures of cruzain14,15 were interrogated com-
putationally with half a million compounds from the lead-
like subset of the ZINC database16 using DOCK 3.5.54
(see Supporting Information for details). Following visual
inspection of the 500 top-ranked docked molecules, 17 were
selected for purchase and testing in an enzyme assay. Among
these, glycolamide ester 1 inhibited cruzain with an IC50 of
77 μM (Figure 1).
Divergent Modes of Enzyme Inhibition in
a Homologous Structure-Activity Series
Rafaela S. Ferreira, Clifford Bryant, Kenny K. H. Ang,
James H. McKerrow, Brian K. Shoichet,* and
Adam R. Renslo*
Small Molecule Discovery Center, Sandler Center for Basic Research
in Parasitic Diseases, and Department of Pharmaceutical Chemistry,
University of California, San Francisco, 1700 4th Street,
San Francisco, California 94158
Inhibition of cruzain by 1, though relatively modest, was
independent of incubation time and was competitive, with a
Ki of 32 μM (Figure 2). Both observations were consistent
with a reversible, noncovalent mechanism of inhibition. The
assays were conducted in the presence of 0.01% of the deter-
gent Triton X-100 (a control for colloidal aggregation2,17),
and close congeners of 1 such as 2 and 3 were also competitive
inhibitors of cruzain under similar conditions. Thus, the initial
compounds in this series (1-3) were deemed “clean” as far as
aggregation and reversibility was concerned, and chemistry
was initiated on the series.
Received June 22, 2009
Abstract: A docking screen identified reversible, noncovalent in-
hibitors (e.g., 1) of the parasite cysteine protease cruzain. Chemical
optimization of 1 led to a series of oxadiazoles possessing inter-
pretable SAR and potencies as much as 500-fold greater than 1.
Detailed investigation of the SAR series subsequently revealed that
many members of the oxadiazole class (and surprisingly also 1) act
via divergent modes of inhibition (competitive or via colloidal
aggregation) depending on the assay conditions employed.
Preliminary SAR profiling around the glycolamide scaffold
was carried out with commercially available and synthesized
analogues (Figure 1, Table 1, and Supporting Information).
The dichloroaniline ring in 1 could be replaced by aromatic,
benzylic, or aliphatic ring systems without substantially im-
pacting potency (e.g., 2 and 3). In contrast, the SAR of the
aminopyridine ring was much less forgiving. For example, an
aniline ring could be substituted for aminopyridine (compare
1 and 6, Table 1) but only if the amine was positioned in the
ortho position. Analogues lacking an ortho amino group or
having the amino group in another position on the ring were
essentially inactive (e.g., 4 and 5, Figure 1).
With an aminopyridine (or aniline) ring established as a
putative pharmacophore, we set about replacing the labile
ester function in 1 with more druglike surrogates. The most
straightforward modification (ester to amide) uniformly pro-
duced less potent analogues (e.g., analogues 7 and 8, Table 1).
We next examined 1,2,4-oxadiazole and related heterocycles
as ester bioisosteres.18 Gratifyingly, oxadiazole analogues
(e.g., 9-11) were found to be at least as potent as the esters-
based progenitors 1-3. Both aliphatic and aromatic “C-
rings” were tolerated in the oxadiazole series (e.g., 10 and 11).
With more leadlike scaffolds in hand, we sought to
expand our survey of C-ring variants in the oxadiazole
series. An established method18 for preparing the oxadia-
zole ring system proved well suited for library generation
(see Supporting Information). The end result of the
C-ring survey was ∼50 new analogues, prepared over three
cycles of synthesis, with potencies ranging from >100 μM
to 200 nM and with clear and interpretable SAR (Table 2
and Supporting Information). Hence, analogues bearing
basic, amine containing rings (17, 18) and analogues bear-
ing heterocycles linked by aliphatic chains of more than one
carbon atom (19) were generally without measurable acti-
vity (IC50>100 μM). Consistent with the preliminary SAR
profile, analogues bearing aliphatic, benzylic, or aryl/
heteroaryl C-rings generally possessed mid-low micro-
molar potencies against cruzain. Most exciting was the
Small-molecule aggregation leading to promiscuous inhibi-
tion of enzymes is now well recognized as a common source of
false positives in high-throughput screening.1-4 While these
phenomena are not thoroughly understood at the molecular
scale, common characteristics of aggregates can be useful in
their identification, for example, their capacity to inhibit
unrelated enzymes5 and the ability of nonionic detergents to
disrupt them and reverse enzyme inhibition.6,7 Aggregation is
viewed by many medicinal chemists as an all-or-nothing
phenomenon of primary concern in high-throughput screen-
ing and hit validation. That nonspecific modes of inhibition
could emerge in the course of a standard hit-to-lead optimiza-
tion campaign is not generally appreciated. Even less appre-
ciated is the notion that promiscuous inhibition could be
responsible for multiple logs of apparent (interpretable)
SAR. Recently, we uncovered exactly these effects while
optimizing a novel class of reversible, nonelectrophilic inhibi-
tors of the trypanosome cysteine protease cruzain. Here we
describe aspects of this work that bear consideration by any
group engaged in chemical optimization guided by biochem-
ical assay data.
Cruzain is the major cysteine protease of the protozoan
parasite Trypanosoma cruzi, the causative agent of Chagas’
disease, which affects 16-18 million people in Latin America.
The importance of cruzain as a target has been demonstrated
through the efficacy of inhibitors of this enzyme in mouse8 and
dog9 models of the disease. Several efforts have been conducted
to find cruzain inhibitors, and diverse classes of compounds
have been reported but mostly covalent warhead-based chemo-
types.10,11
Motivated to find novel inhibitors with a reversible, non-
covalent mode of inhibition, we turned to virtual screening of
*To whom correspondence should be addressed. For B.K.S.: phone,
415-514-4126; fax, 415-514-4260; e-mail, shoichet@cgl.ucsf.edu. For
A.R.R.: phone, 415-514-9698; fax, 415-514-4507; e-mail, adam.renslo@
ucsf.edu.
r
2009 American Chemical Society
Published on Web 07/28/2009
pubs.acs.org/jmc

5006 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Ferreira et al.
potency than did small alkyl (e.g., Me, 25) substituents.
Strikingly, methylation of the pyrazole N-H function
completely abrogated activity (analogue 26; IC50 > 100
μM), implying that the enhanced potency of the pyrazole
series might result in part from a hydrogen bonding inter-
action not present in the other aryl and benzylic C-rings
analogues studied.
With ∼50 oxadiazoles synthesized, we were in possession of
three logs of consistent, intepretable SAR and had identified
analogues that were nearly 500-fold more potent than 1. We
were troubled, however, that few of the oxadiazole analogues
exhibited measurable activity against cultured T. cruzi or
Trypanosoma brucei parasites. This discrepancy was initially
rationalized as arising from poor cell permeability or active
efflux from parasite, but a close inspection of the enzyme
dose-response curves, many of which turned out to have
unusually high Hill slopes, suggested a second possibility: that
the inhibitors were acting by superstoichiometric mechan-
isms.1,19 At this juncture we also determined that the analo-
gues from the C-ring survey had inadvertently been assayed at
a 10-fold lower detergent concentration (0.001% Triton
X-100) than was employed in the original profiling of analo-
gues 1-11 (0.01%).
Figure 1. Structures and cruzain inhibition data for original screen-
ing hit 1 and structurally related analogues obtained from commer-
cial sources.
Concerned, we repeated the IC50 determinations for selec-
ted oxadiazole and glycolamide analogues at low and high
detergent concentrations (Table 2, Figure 3).6,7 Significantly,
each of the oxadiazoles examined showed no measurable
dose-response or a significantly higher IC50 when tested at
the higher Triton concentration. Only one analogue (16)
exhibited potency comparable to the early oxadiazole leads
9-11 under high Triton conditions. In contrast, the IC50
values of glycolamide analogues 1-3 were unchanged or only
modestly altered (∼3-fold in the case of1, essentially unaltered
for 2 and 3) at the different detergent concentrations. Also
consistent with aggregation, some oxadiazoles were sensitive
to preincubation with bovine serum albumin (BSAa), which
at high concentration competes with enzyme for colloid
particles (aggregates), preventing or reducing inhibition of
the target enzyme.20 Thus, oxadiazoles 14 and 23 showed,
respectively, 10- and 100-fold increases in IC50 in the presence
of 1 mg/mL BSA, while analogue 16 was unaffected by BSA
pretreatment.
Figure 2. Lineaweaver-Burk plot indicating competitive inhibi-
tion of cruzain by 1 (Ki = 32 μM).
Table 1. SAR for Cruzain Inhibitors Bearing Ester, Amide, or Hetero-
cyclic Linkers
Detergent-reversible inhibition of AmpC β-lactamase is
another marker of promiscuous aggregation, and so this
was examined next. Four of five oxadiazoles tested did indeed
inhibit β-lactamase in a detergent-reversible fashion at rele-
vant compound concentrations, the exception being analogue
25. Quite surprisingly, the original glycolamide lead 1 (but not
2 or 3) was also found to inhibit AmpC, and its inhibition was
reversed by 0.01% Triton. Thus, glycolamide 1 might also act
as an aggregator under certain assay conditions, although
clearly this was not true of 1 in its inhibition of cruzain where
competitive inhibition (at 0.01% Triton) had already been
established (Figure 2).
Next, we sought direct evidence of particle (aggregate)
formation by dynamic light scattering (DLS)21 and flow
cytometry.22 We studied suspected aggregators and analogues
that fail to inhibit enzyme under any conditions, as the latter
compounds presumably do not aggregate (an thus do not
inhibit). At relevant inhibitory concentrations for low deter-
gent conditions (0.001% Triton X-100), suspected oxadiazole
a DiCl-Ph = 2,5-dichlorophenyl. b DiMeOPh = 2,4-dimethoxyphenyl.
finding that substituted pyrazole C-ring analogues inhib-
ited cruzain with submicromolar potencies (e.g., cruzain
IC50 ≈ 200 nM for analogue 21). Further exploration of the
pyrazole series produced additional interpretable SAR.
Hence, aryl (20, 21, 22), heteroaryl (23), or bulky alkyl
(24) substitution on the pyrazole ring conferred superior
a Abbreviations: DLS, dynamic light scattering; BSA, bovine serum
albumin.

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5007
Table 2. Results of Enzyme Inhibition Assays and Secondary Assays of Aggregation for Selected Oxadiazole Analogues and Glycolamide 1
a Cruzain IC50 values were determined in 0.01% or 0.001% Triton, and in the later case with or without preincubation with BSA. b Compounds were tested
against β-lactamase in the absence or in the presence of 0.01% Triton, at the concentrations noted, and classified as aggregators if inhibition was detergent-sensitive.
c Particle formation was evaluated by flow cytometry and in some cases examined and confirmed by DLS. The concentrations noted are the lowest evaluated
concentration at which particle formation was observed.
Figure 3. IC50 curves for original lead compound 1 (top) and oxadiazole 23 (bottom) at various concentrations of Triton X-100 and with or
without preincubation with BSA. The oxadiazole shows a much more significant dose-response shift at low detergent concentrations, likely
owing to the inadvertent optimization of this series under low Trion X-100 conditions.
aggregatorsandalso 1-3 were abovetheircritical aggregation
concentrations (CACs)22 and formed colloidal aggregates
(the exception being 12) of size 250-400 nm. In contrast,
inactive analogues (e.g., 17-19) did not show particle forma-
tion even at high concentration.
These studies provided strong evidence for a nonspecific
mode of inhibition by oxadiazoles under low Triton (0.001%)
assay conditions. Not all the secondary assay data were in
perfect agreement however, and the DLS and AmpC inhibi-
tion data were puzzling, as they suggested that at least some of
the originalglycolamide analogues could also formaggregates
under low Triton conditions. We therefore re-evaluated the
nature of cruzain inhibition at high and low Triton concen-
trations for glycolamide 1 and a representative oxadiazole
(23). Under low Triton conditions, oxadiazole 23 (at 5 μM)
was a noncompetitive inhibitor of cruzain, consistent with
aggregation-based inhibition. In contrast, under high Triton
conditions, 23 (at 50 μM) was a competitive inhibitor of
cruzain. Similarly, glycolamide 1 was found to be noncom-
petitive under low Triton conditions and at a relevant
inhibitor concentration (25 μM). Hence, in glycolamide 1
and oxadiazole 23, divergent modes of enzyme inhibition are
operating under the two different assay conditions. Even so,
there are subtle differences between these two chemotypes.
Members of the oxadiazole class, having been unwittingly
optimized for aggregation (driven by an assay condition
favoring aggregation) can be >100-fold more potent as
aggregators than as competitive inhibitors (e.g., 20-23
Table 2, Figure 3). In contrast, glycolamide 1 confers
similar potencies by the two modes of inhibition. Thus, the

5008 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Ferreira et al.
differences in detergent effects (Figure 3) and initially
surprising results concerning AmpC inhibition by 1 can be
reconciled.
Triton for the full set of ∼50 oxadiazole analogues synthesized,
retrospective comparison of DOCK scores and biochemical
inhibition data for oxadiazole analogues. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
Two important conclusions can be drawn from the studies
described here. First, divergent modes of inhibition can exist
in a homologous SAR series, and these divergent mechanisms
are only discernible by careful consideration of assay condi-
tions. We argue that such a possibility should be considered as
the default position, since the migration from competitive to
noncompetitive regimes will not usually be obvious from
structure alone. The ester bioisostere approach described here
is a standard one and could not have been predicted a priori to
have led to a series of aggregators with nanomolar potencies.
That this in fact occurred owes to the use of an assay condition
favoring the noncompetitive mode of inhibition.
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Acknowledgment. This work was generously supported by
funding from the Sandler Foundation (to A.R.R.) and the
QB3-Malaysia Program (K.K.H.A.) and by Grant GM71630
(to B.K.S.). We thank BD Biosciences for a long-term loan of
a BD solubility scanner for flow cytometry of aggregates and
Ana Lazic for providing the cruzain construct. We acknowl-
edge Priyadarshini Jaishankar and DongMei Zhou for experi-
mental contributions in the early stages of this work. We
thank Drs. Michelle Arkin and Jim Wells for helpful sugges-
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manuscript.
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Supporting Information Available: Experimental details for
the DOCK screen (including docking pose for 1) and for all
biochemical assays and secondary assays of aggregation, syn-
thetic procedures and spectroscopic characterization data for all
new compounds, table of cruzain inhibition data at 0.001%