Structure-activity relationships for inhibition of cysteine protease activity and development of Plasmodium falciparum by peptidyl vinyl sulfones

Article abstract of DOI:10.1128/AAC.47.1.154-160.2003

The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 appear to be required for hemoglobin hydrolysis by intraerythrocytic malaria parasites. Previous studies showed that peptidyl vinyl sulfone inhibitors of falcipain-2 blocked the development of P. falciparum in culture and exerted antimalarial effects in vivo. We now report the structure-activity relationships for inhibition of falcipain-2, falcipain-3, and parasite development by 39 new vinyl sulfone, vinyl sulfonate ester, and vinyl sulfonamide cysteine protease inhibitors. Levels of inhibition of falcipain-2 and falcipain-3 were generally similar, and many potent compounds were identified. Optimal antimalarial compounds, which inhibited P. falciparum development at low nanomolar concentrations, were phenyl vinyl sulfones, vinyl sulfonate esters, and vinyl sulfonamides with P₂ leucine moieties. Our results identify independent structural correlates of falcipain inhibition and antiparasitic activity and suggest that peptidyl vinyl sulfones have promise as antimalarial agents.

Full text of DOI:10.1128/AAC.47.1.154-160.2003

Structure-Activity Relationships for
Inhibition of Cysteine Protease Activity and
Development of Plasmodium falciparum by
Peptidyl Vinyl Sulfones
Bhaskar R. Shenai, Belinda J. Lee, Alejandro
Alvarez-Hernandez, Pek Y. Chong, Cory D. Emal, R. Jeffrey
Neitz, William R. Roush and Philip J. Rosenthal
Antimicrob. Agents Chemother. 2003, 47(1):154. DOI:
10.1128/AAC.47.1.154-160.2003.
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 2003, p. 154–160
0066-4804/03/$08.00ϩ0 DOI: 10.1128/AAC.47.1.154–160.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 47, No. 1
Structure-Activity Relationships for Inhibition of Cysteine Protease
Activity and Development of Plasmodium falciparum
by Peptidyl Vinyl Sulfones
Bhaskar R. Shenai,1* Belinda J. Lee,1 Alejandro Alvarez-Hernandez,2 Pek Y. Chong,2
Cory D. Emal,2 R. Jeffrey Neitz,2 William R. Roush,2 and Philip J. Rosenthal1
Department of Medicine, San Francisco General Hospital, University of California, San Francisco, California,1 and
Department of Chemistry, University of Michigan, Ann Arbor, Michigan2
Received 3 June 2002/Returned for modification 28 August 2002/Accepted 24 September 2002
The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 appear to be required for hemo-
globin hydrolysis by intraerythrocytic malaria parasites. Previous studies showed that peptidyl vinyl sulfone
inhibitors of falcipain-2 blocked the development of P. falciparum in culture and exerted antimalarial effects in
vivo. We now report the structure-activity relationships for inhibition of falcipain-2, falcipain-3, and parasite
development by 39 new vinyl sulfone, vinyl sulfonate ester, and vinyl sulfonamide cysteine protease inhibitors.
Levels of inhibition of falcipain-2 and falcipain-3 were generally similar, and many potent compounds were
identified. Optimal antimalarial compounds, which inhibited P. falciparum development at low nanomolar
concentrations, were phenyl vinyl sulfones, vinyl sulfonate esters, and vinyl sulfonamides with P2 leucine
moieties. Our results identify independent structural correlates of falcipain inhibition and antiparasitic
activity and suggest that peptidyl vinyl sulfones have promise as antimalarial agents.
Malaria is one of the most important infectious diseases in
the world. Plasmodium falciparum, the most virulent human
malaria parasite, is estimated to cause over 300 million new
cases and 1 million deaths annually (33). Further complicating
this grim scenario is the emergence of the widespread resis-
tance of P. falciparum to available antimalarial drugs (25). New
drugs to combat malaria are urgently needed.
Among potential new targets for antimalarial chemotherapy
are enzymes that mediate hemoglobin hydrolysis. Intraeryth-
rocytic P. falciparum trophozoites derive amino acids for pro-
tein synthesis from the hydrolysis of host cell hemoglobin in an
acidic food vacuole (12, 20, 27). Proteases that hydrolyze he-
moglobin in the food vacuole include members of the aspartic
protease (1), cysteine protease (38, 39), and metalloprotease
(9) families. Cysteine protease inhibitors arrested the erythro-
cytic life cycle of P. falciparum (26). Examination of inhibitor-
treated parasites revealed abnormally swollen food vacuoles
filled with undigested hemoglobin, indicating that the block in
parasite development was due to the inhibition of hemoglobin
hydrolysis (26).
P. falciparum contains three fairly typical papain family cys-
teine proteases, known as falcipains (28, 38, 39). Falcipain-2
and falcipain-3 appear to be the principal cysteine protease
hemoglobinases (38, 39). Both of these proteases localize to
vacuolar parasite fractions and readily hydrolyze hemoglobin
under physiological reducing conditions at acidic pHs (37).
Falcipain-2 is considerably more active against small peptide
substrates, but the specificities of the two proteases are similar;
both enzymes display a strong preference for leucine at the P2
position (38, 39). The role of falcipain-1 in hemoglobin hydro-
lysis is unknown.
In earlier studies, peptidyl vinyl sulfones inhibited falci-
pain-2 activity and parasite development at nanomolar concen-
trations and were active in vivo against murine malaria (22, 29,
30). We have now initiated efforts to define structure-activity
relationships (SAR) for the inhibition of falcipain-2, falci-
pain-3, and parasite development by a new series of peptidyl
vinyl sulfones, vinyl sulfonate esters, and vinyl sulfonamides.
We show that SAR for the two proteases are similar and that
multiple compounds are potent inhibitors of the falcipains and
of parasite development. However, the structural correlates for
inhibition of the proteases differ notably from those for the
inhibition of parasite development.
MATERIALS AND METHODS
Synthesis of vinyl sulfones, sulfonamides, and sulfonate esters. All inhibitors
studied had a peptide backbone with multiple substituents at the P3 position, Phe
or Leu at P2, and homoPhe or O-(phenyl)Ser at P1; based on the substituents at
the P1Ј position, the inhibitors were further subclassified into phenyl vinyl sul-
fones, vinyl sulfonamides, or vinyl sulfonate esters (Fig. 1). These inhibitors were
synthesized by using appropriate modifications of previously described methods
(31, 32). The series of phenyl vinyl sulfones was prepared by a Horner-Wads-
worth-Emmons reaction of N-tert-butoxycarbonyl (N-BOC)-homophenylalanal
(Fig. 2, compound labeled 1) (23) with diethyl [(phenylsulfonyl)methyl]phospho-
nate (10) to produce N-BOC-homophenylalanyl phenyl vinyl sulfone (compound
labeled 2). The BOC group was removed with trifluoroacetic acid, and the
resulting amines were coupled to R3 substituents under standard peptide cou-
pling conditions (Fig. 2).
Vinyl sulfonamides [with homoPhe or O-(phenyl)Ser at P1] and vinyl sulfonate
esters were synthesized via the vinyl sulfonyl chlorides labeled 6 and 7 in Fig. 3
by using the general sequence reported by Gennari et al. (13) with appropriate
modifications. Vinyl sulfonamides were synthesized by a Horner-Wadsworth-
Emmons reaction of N-BOC-homophenylalanal (Fig. 3, structure 1) with tri-
ethyl-␣-phosphorylmethanesulfonate (5, 21) to produce the N-BOC-homophe-
nylalanyl vinyl sulfonate ethyl ester labeled 4 in Fig. 3. The sulfonate ester was
dealkylated with tetrabutylammonium iodide and converted to the sulfonyl chlo-
ride labeled 6 in Fig. 3 with triphosgene and catalytic N-dimethylformamide (24).
* Corresponding author. Mailing address: Box 0811, University of
California, San Francisco, San Francisco, CA 94143-0811. Phone:
(415) 206-3352. Fax: (415) 648-8425. E-mail: bshenai@itsa.ucsf.edu.
154

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STRUCTURE-ACTIVITY RELATIONSHIPS FOR P. FALCIPARUM
155
FIG. 1. General structures of the peptidyl vinyl sulfones studied. All compounds except 371 and 373 contained a urea linkage between the R3
substituent and the peptide scaffold. R1 and R2 represent the side chains of the P1 and the P2 amino acids, respectively; R1Ј represents vinyl sulfone,
sulfonamide, or sulfonate ester substituents.
O-Benzylhydroxylamine was added to the sulfonyl chloride labeled 6 (Fig. 3) in
the presence of 2,6-lutidine to form vinyl sulfonamide (compound labeled 8). The
BOC group was removed with trifluoroacetic acid, and the resulting amine was
coupled to R3 substituents under standard peptide coupling conditions. A series
of vinyl sulfonamides and vinyl sulfonate esters (with Leu at the P2 and homoPhe
at the P1 position) was synthesized by analogous procedures via compounds 6
and 7 (Fig. 4 and 5). The O-phenyl serine derivative labeled 3 was synthesized by
the general procedure of Cherney (7) with the appropriate modifications.
Assays of enzyme inhibition. IC50s against falcipain-2 were determined as
described earlier (30). Briefly, equal amounts (ϳ1 nM) of recombinant falci-
pain-2 (38) were incubated with different concentrations of vinyl sulfones (added
from 100ϫ stocks in dimethyl sulfoxide [DMSO]) in 100 mM sodium acetate (pH
5.5)–10 mM dithiothreitol for 30 min at room temperature before addition of the
substrate benzoxycarbonyl-Leu-Arg-7-amino-4-methyl-coumarin (final concen-
tration, 25 ␮M). Fluorescence was continuously monitored for 30 min at room
temperature in a Labsystems Fluoroskan II spectrofluorometer. IC50s were de-
termined from plots of activity over enzyme concentration with GraphPad Prism
software.
For the determination of second-order binding constants, the concentrations
of falcipain-2 and falcipain-3 were determined by active-site titration with ben-
zoxycarbonyl-Phe-Arg-fluoromethyl ketone (39). Inhibitor assays were per-
formed under pseudo-first-order conditions (i.e., the concentration of the inhib-
itor was at least 10-fold higher than that of the enzyme; substrate hydrolysis,
Ͻ5%) by using the progress curve method (41). Falcipain-2 (0.6 nM) or falci-
pain-3 (0.8 to 1.2 nM) was incubated with different inhibitor concentrations in a
solution containing 100 mM sodium acetate buffer (pH 5.5), 10 mM dithiothre-
itol, and the substrate benzoxycarbonyl-Leu-Arg-7-amino-4-methyl-coumarin
(25 ␮M for falcipain-2 and 100 ␮M for falcipain-3). Product formation was
continuously monitored in a Labsystems Fluoroskan II spectrofluorometer for 10
min at room temperature. To determine the observed first-order inactivation
rate constant (kobs), progress curves (fluorescence versus time) were analyzed by
nonlinear-regression analysis (GraphPad Prism software) using the pseudo-first-
FIG. 2. Scheme for the synthesis of vinyl phenyl sulfones with Leu at the P2 position and homoPhe at the P1 position. Et, ethyl; Ph, phenyl; TFA,
trifluoroacetic acid; THF, tetrahydofuran; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; HOBT, 1-hydroxybenzotriazole
hydrate; NMM, N-methylmorpholine; DMF, N-dimethylformamide.

156
SHENAI ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 3. Scheme for the synthesis of vinyl sulfonamides with Phe at the P2 position and homoPhe or O-(phenyl)Ser at the P1 position. Bn, benzyl;
Bu, butyl. For all other abbreviations, see the legend to Fig. 2.
order rate equation y ϭ A ϫ (1 Ϫ eϪk ⅐ t) ϩ B, where y is the fluorescence at
time t, A is the amplitude of the reaction, and B is the offset. Plots of kobs versus
the inhibitor concentration ([I]) were then used to determine uncorrected sec-
ond-order rate constants (6), and for each inhibitor, the corrected second-order
rate constant (kass, the association rate constant) was determined with the equa-
tion kass ϭ (kobs/[I]) ϫ (1 ϩ [S]/Km) (4), where [S] is the substrate concentration.
Assay of parasite development. Effects of inhibitors on parasite development
were determined as described earlier (30). Briefly, synchronized W2 strain P.
falciparum parasites (18) were cultured with vinyl sulfones (added from 1,000ϫ
stocks in DMSO) for 48 h beginning at the ring stage. The medium was changed
after 24 h, with maintenance of the appropriate inhibitor concentration. Giemsa-
stained smears were made after 48 h, when control cultures contained nearly all
ring-stage parasites. The number of new ring forms per 500 erythrocytes was
counted, and counts were compared with those of controls cultured in 0.1%
DMSO. IC50s for growth inhibition were determined with GraphPad Prism
software from plots of percentages of the level of parasitemia of the control
relative to inhibitor concentration.
obs
RESULTS
Inhibition of falcipain-2 by vinyl sulfones. Our strategy was
to build on initial results that demonstrated potent inhibition
of falcipain-2 by peptidyl vinyl sulfones (30). Limited prior
FIG. 4. Scheme for the synthesis of vinyl sulfonamides with Leu at the P2 position and Phe at the P1 position. For all other abbreviations, see
the legend to Fig. 2.

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STRUCTURE-ACTIVITY RELATIONSHIPS FOR P. FALCIPARUM
157
FIG. 5. Scheme for the synthesis of vinyl sulfonate esters with Leu at the P2 position and homoPhe at the P1 position. DBU, 1,8-
diazabicyclo[5.4.0]undec-7-ene; OMe, O-methyl. For all other abbreviations, see the legend to Fig. 2.
studies showed that the Leu-homoPhe peptide had strong ac-
tivity and that potency was imparted by alterations of amino
(P3)- and carboxy (P1Ј)-terminal constituents of vinyl sulfone
inhibitors (Fig. 1). Consistent with prior results, compounds
with the core sequence Phe-homoPhe offered modest activity
against falcipain-2 (Table 1); alterations at position P3 in these
compounds had relatively little impact on activity. Compounds
with the core sequence Phe-O-(phenyl)Ser had similar activi-
ties, with IC50s for the inhibition of falcipain-2 in the mid- to
high-nanomolar range (Table 1).
We next tested an additional 30 compounds, all of which
contained the core sequence Leu-homoPhe and which differed
at the P3 and P1Ј positions. Three different constituents at P1Ј
generated phenyl vinyl sulfones, vinyl sulfonamides, and vinyl
sulfonate esters. The Leu-homoPhe compounds were generally
very active against falcipain-2, with IC50s mostly in the high-
picomolar to low-nanomolar range (Table 2). Considering
compounds that were identical except for the P1Ј substituent,
the general rank order of activity against falcipain-2 was vinyl
sulfonate esters Ͼ vinyl sulfonamides Ͼ phenyl vinyl sulfones
(e.g., compare compounds 365, 367, or 369 with 339 and 324,
or compare compounds 364, 341, and 328).
screened for activity against cultured malaria parasites. Ring-
stage parasites were treated with different concentrations of
inhibitors, and after 48 h, the numbers of new ring-stage par-
asites in both treated and control cultures were compared. In
this assay, the phenyl vinyl sulfones were the most potent
inhibitors, with most tested compounds yielding IC50s for the
inhibition of parasite development in the low-nanomolar range
TABLE 2. Effects of R3-Leu-homoPhe-vinyl sulfonyl-RЈ peptidyl
vinyl sulfonyl compounds on the activity of falcipain-2 and
development of P. falciparuma
FP-2 IC50
(nM)
Culture IC50
(nM)
Compound
R1Ј
322
324
325
326
327
328
344
345
371
290
291
336
337
338
339
340
341
373
348
349
351
354
355
357
362
363
364
365
367
369
Sulfone
35
27
200
1,800
4.5
Sulfone
Sulfone
8.7
9.2
3.5
6.9
9.9
6.7
140
2.3
2.2
25
Sulfone
15
Sulfone
22
Sulfone
3.9
Sulfone
21
Sulfone
1.6
Sulfone
ND
4.4
Sulfonamide
Sulfonamide
Sulfonamide
Sulfonamide
Sulfonamide
Sulfonamide
Sulfonamide
Sulfonamide
Sulfonamide
Sulfonate ester
Sulfonate ester
Sulfonate ester
Sulfonate ester
Sulfonate ester
Sulfonate ester
Sulfonate ester
Sulfonate ester
Sulfonate ester
Sulfonate ester
Sulfonate ester
Sulfonate ester
Effects of vinyl sulfones on cultured malaria parasites. Of
the 30 compounds with the core sequence Leu-homoPhe, 28
had IC50s against falcipain-2 below 100 nM, and these were
46
Ͼ10,000
1,500
2,000
220
22
14
16
3.6
2.5
2.2
16
0.7
1.9
0.9
0.7
21
0.9
0.7
350
0.7
0.8
0.9
0.9
TABLE 1. Effects of R3-Phe-homoPhe-vinyl sulfonamide-
N-benzyloxy and R3-Phe-O-(phenyl)Ser-vinyl sulfonamide-
N-benzyloxy sulfonamides on the activity of falcipain-2a
1.6
3,300
14
35
Compound
R1
R1Ј
IC50 (nM)
9.7
84
1,100
120
49
ND
42
390
394
220
308
309
310
311
312
313
314
315
316
HomoPhe
Sulfonamide
Sulfonamide
Sulfonamide
Sulfonamide
Sulfonamide
Sulfonamide
Sulfonamide
Sulfonamide
Sulfonamide
110
120
71
120
120
56
290
230
130
HomoPhe
HomoPhe
HomoPhe
O-(phenyl)Ser
O-(phenyl)Ser
O-(phenyl)Ser
O-(phenyl)Ser
O-(phenyl)Ser
a For structures, see Fig. 3.
a For structures, see Fig. 2, 4, and 5. FP-2, falcipain-2; ND, not done.

158
SHENAI ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 3. Second-order binding constants for binding of peptidyl
vinyl sulfonyl compounds to falcipain-2 and falcipain-3
DISCUSSION
Earlier studies showed that peptidyl vinyl sulfones were po-
tent inhibitors of falcipain-2 (30) and that one compound
cured Plasmodium vinckei-infected mice (22). To better char-
acterize SAR for both enzyme and parasite inhibition, we syn-
thesized and evaluated 39 new peptidyl vinyl sulfones, vinyl
sulfonate esters, and vinyl sulfonamides. Consistent with prior
results, we showed that inhibitors with the core sequence Leu-
homoPhe provided potent inhibition of falcipain-2, falcipain-3,
and cultured malaria parasites. Additional evaluations of com-
pounds with the Leu-homoPhe core highlighted key SAR de-
terminants at the P3 and P1Ј constituents of the inhibitors.
It is noteworthy that second-order rate constants for inhibi-
tion of falcipain-2 and falcipain-3 were consistently quite sim-
ilar. Prior studies with peptide substrates have shown the en-
zymes to have similar specificities (with marked preference for
substrates with Leu at P2), but falcipain-3 was much less active
than falcipain-2 against these substrates. It is not clear why this
difference is not seen with peptidyl inhibitors. Rather, second-
order rate constants for the inhibition of falcipain-3 were gen-
erally somewhat higher than those for falcipain-2, though in
most cases the differences between the two proteases were
fairly small. This finding is important for two reasons. First, it
suggests that our prior screens of compounds against falci-
pain-2 were probably adequate in predicting inhibition of both
principal P. falciparum cysteine protease hemoglobinases. Sec-
ond, it argues that drug discovery against these two enzymes
will likely not be complicated by the need to develop separate
inhibitors of each protease.
Considering SAR for enzyme inhibition, vinyl sulfonate es-
ters, and vinyl sulfonamides offered improved potency over
phenyl vinyl sulfones. Little is known about the “prime-side”
specificities of cysteine proteases, but in the case of the falci-
pains, it appears that larger P1Ј substituents improve inhibitor
binding. For example, it was previously noted that a naphthyl
vinyl sulfone was more active both against falcipain-2 and
against cultured P. falciparum parasites than was the corre-
sponding phenyl vinyl sulfone (22). P3 substituents also had an
impact on activity against falcipain-2 and falcipain-3, although
for compounds with otherwise identical structures, the second-
order rate constants varied by less than an order of magnitude
among compounds with different P3 substituents. Available
methodologies for evaluating substrate specificities of cysteine
proteases, including peptidyl substrates bound to fluorogenic
reporter molecules, are mostly limited to analyses of “non-
prime” specificity. However, newer combinatorial approaches
to enzyme evaluation (15), including methods for screening
prime-side specificity (36), should improve our ability to char-
acterize the specificities of proteases and thereby predict op-
timal inhibitor interactions.
kass (MϪ1 sϪ1
)
Compound
R1Ј
Falcipain-2
Falcipain-3
325
326
327
328
344
345
290
291
340
341
348
349
351
354
364
Sulfone
21,600
29,700
78,600
111,000
54,600
102,000
74,700
411,000
120,000
245,000
Ͼ106
39,600
31,300
57,100
127,000
61,700
55,700
147,000
407,000
179,000
711,000
Ͼ106
Sulfone
Sulfone
Sulfone
Sulfone
Sulfone
Sulfonamide
Sulfonamide
Sulfonamide
Sulfonamide
Sulfonate ester
Sulfonate ester
Sulfonate ester
Sulfonate ester
Sulfonate ester
288,000
160,000
Ͼ106
Ͼ106
Ͼ106
Ͼ106
Ͼ106
Ͼ106
(Table 2). Inhibition of parasite development was consistently
accompanied by the appearance of darkly stained, swollen food
vacuoles, which are indicative of a block in hemoglobin hydro-
lysis, confirming that the inhibitors exerted their antimalarial
effects by blocking food vacuole hemoglobinases. The other
tested compounds were generally somewhat less active against
cultured parasites, despite their having greater activity against
falcipain-2. With compounds with otherwise identical struc-
tures, the rank order of antiparasitic activity was generally
phenyl vinyl sulfones Ϸ vinyl sulfonamides Ͼ vinyl sulfonate
esters, the opposite of the order seen for activity against falci-
pain-2. Within each compound class, there were fairly good
correlations between inhibition of falcipain-2 and antiparasitic
activity; these correlations were quite strong after the elimina-
tion of two compounds with unusually poor inhibitory activity
against cultured parasites, presumably due to limited access to
intracellular protease targets (r2 was 0.943 for the sulfones,
excluding compound 324; r2 was 0.993 for the sulfonamides,
excluding compound 373; r2 was 0.802 for the sulfonate esters).
Second-order rate constants for inhibition of falcipain-2
and falcipain-3. Fifteen vinyl sulfones that were low-nanomo-
lar inhibitors of both falcipain-2 activity and parasite develop-
ment were further analyzed for inhibition of falcipain-2 and the
related P. falciparum protease falcipain-3. The second-order
rate constants (kass) for inhibition of falcipain-2 or falcipain-3
by the selected vinyl sulfonyl inhibitors were determined by the
progress curve method under pseudo-first-order conditions
(41). Inhibition rate constants for falcipain-2 and falcipain-3
were generally similar. As seen with IC50 determinations, the
vinyl sulfonate esters were the most potent inhibitors, with kass
values against both proteases being Ͼ105 for all five tested
compounds (Table 3). The second-order rate constants for the
phenyl vinyl sulfones and vinyl sulfonamides were lower, but
they were consistently Ͼ104. P3 substituents also had a marked
impact on inhibitory kinetics, but a general trend was not
apparent. In some cases (e.g., with compounds 328, 341, and
345) compounds containing P3 groups with an extension at the
4 position yielded higher second-order rate constants than
those lacking such a moiety (e.g., compounds 290 and 325).
Further exploration at that position may be warranted.
Considering SAR for the inhibition of parasite development,
it is noteworthy that the potency of inhibition of falcipain-2 and
falcipain-3 is not an ideal predictor of activity against parasites.
Clearly, the cysteine proteases must be inhibited for a vinyl
sulfone to be active against the parasites, and much more
potent inhibitors are generally better antimalarials (e.g., mor-
pholine urea-Leu-homoPhe-phenyl vinyl sulfone was about an
order of magnitude more active against both falcipain-2 and
cultured parasites than was the corresponding Phe-homoPhe

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compound) (30). However, among very potent inhibitors, ad-
ditional determinants of SAR come into play. Considering our
results, it appears that vinyl sulfonamides and vinyl sulfones
tend to be more active than the vinyl sulfonate esters against
the parasites. Regarding substitution of the aryl ring of vinyl
sulfonate esters, there is a clear pattern of activity against the
parasite cultures in rank order (-OMe Ͼ -H Ͼ -F, contrary to
results of the enzymatic assays. Concerning the P3 substituents,
of the six compounds with the highest antiparasitic activities,
three contained an isonipecotic ester moiety (compounds 328,
341, and 351) while the other three contained a tertiary amine
(compounds 290, 325, and 345). Indeed, compounds contain-
ing the isonipecotic ester moiety were particularly effective
against both the enzymes and cultured parasites. However,
other compounds that exhibited high activity against the en-
zymes did not retain potency against the parasites. For exam-
ple, those compounds containing a prolinol substituent at P3
(compounds 324, 339, 365, 367, and 369) were typically very
potent against the enzymes but showed relatively poor anti-
parasitic activity. The origin of these preferences is not yet clear.
Peptidyl vinyl sulfones were originally developed as inhibi-
tors of papain (14, 19) and were later optimized as specific
inhibitors of human cysteine proteases (3, 23). Related phenyl
vinyl sulfones also inhibit cruzain, a cysteine protease of
Trypanosoma cruzi (31, 32, 35), and one of these compounds is
currently undergoing preclinical studies for the treatment of
Chagas’ disease (34). While the clinical use of peptidyl pro-
tease inhibitors is potentially problematic due to limited bio-
availability or poor pharmacokinetics, there are numerous re-
cent reports of peptidyl inhibitors of renin (17), thrombin (11),
leukocyte elastase (42), neutrophil elastase (8), and human
immunodeficiency virus type 1 protease (2, 16, 40) that are
biologically active after oral administration. Considering these
data, the reported in vivo efficacy of an orally administered
vinyl sulfone against murine malaria (22), and our demonstra-
tion here of marked antimalarial potency, additional evalua-
tion of vinyl sulfonyl derivatives as potential antimalarial cys-
teine protease inhibitors seems appropriate. More broadly, our
data provide the most detailed assessment to date of the SAR
for inhibition of the falcipains and of parasite development and
thereby should aid the progress of peptide- and nonpeptide-
based drug discovery efforts directed against plasmodial cys-
teine proteases.
ACKNOWLEDGMENTS
This work was supported by the National Institutes of Health (grants
AI 35800 and AI 35707) and the Medicines for Malaria Venture.
P.Y.C. was supported by a postdoctoral fellowship from the Susan G.
Komen Breast Cancer Foundation.
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