N. R. Roqué Rosell et al. / Bioorg. Med. Chem. Lett. 24 (2014) 490–494
491
an overview see Ref. 18). This route has led to some potent HRV
presence of the previously described fluorogenic substrate
pro
;
;
3
C
inhibitors: Ruprintrivir (AG7088) is an irreversible inhibitor
DABCYL-APAKQ LLD(EDANS)FDLLK, where marks the position of
with low nM EC50 values.19 Although initial proof of concept was
the scissile bond.
24,25
2
0
achieved,
Rupintrivir did not significantly enhance recovery
Initially, 4 analogues of the inhibitor were synthesized, corre-
sponding to the various lengths of P-side peptide sequence
(Table 1). It was found that sequences which included up to P5
(6) or P4 (7) acted as potent inhibitors of the enzyme with appar-
ent IC50 values in the sub-micromolar range; compound 8, which
extends only to P3 was a significantly poorer inhibitor while
compound 9, which extends only to the P2 position, is essentially
inactive as an inhibitor. These data are consistent with the sub-
levels during a natural infection study and development was
2
1
stopped. Nonetheless, structural aspects of Rupintrivir have been
2
2
used in efforts to inhibit various 3C-like viral cysteine proteases.
These efforts have focused on the design of broad spectrum ani-
tivirals based on the similarities between such viral proteases. For
pro
example the structural relationship between FMDV 3C and HRV
pro
3
C
allows the potent inhibition of the former by Rupintrivir,
22c
pro
designed for the latter. In contrast, the work presented here uses
structural information from our previous studies to design an
strate specificity studies of FMDV 3C , which show that sequence
changes at P4, P2 and P1 result in marked loss of activity.1
5a,c
The
pro
inhibitor specific to FMDV 3C . The optimal substrate for FMDV
presence or absence of a P5 residue was found to have little influ-
ence on activity in this assay. However, it is known that that
residues with very different structures (Ala, Pro, Arg, Asp, Glu
and Leu) are found among the naturally occurring cleavage se-
pro
3
C
has the sequence APAKQ-LLNFD, which corresponds to the
15a,c
VP1/2A junction of the viral polyprotein.
Of this sequence,
the P4-P1 residues (PAKQ) are the most critical for selectivity.
The concept is illustrated in Scheme 1, yielding compound 1 as a
potential inhibitor.
pro
26
quences of FMDV 3C at P5, suggesting that few if any specific
interactions are made with this side chain.
Two different synthetic routes were used to make the inhibitors
described in the present study, as illustrated below for the synthe-
sis of inhibitor 1. In both cases, the unsaturated ester moiety was
achieved by a Wittig or Horner–Wadsworth–Emmons (HWE)
reaction and the peptide sequence through solid phase peptide
synthesis (SPPS). In one route (Scheme 2), glutamine is loaded onto
Wang resin and SPPS carried out. The aldehyde 2 is then achieved
by reductive cleavage of the C-terminus of the peptide from the re-
sin. An HWE reaction to form the Michael Acceptor 3, followed by
deprotection of the side chains completes the synthesis. The other
route (Scheme 3) utilises a Wittig reaction to pre-form a building
block onto which the peptide sequence is subsequently assembled.
A protected glutamic acid 4 was first modified on its C-terminus to
It should be noted that the assays in this study were performed
at an enzyme concentration of 0.6 lM, which was necessary in or-
der to determine the rate of substrate hydrolysis (the activity of
this viral protease is far lower than typical digestive proteases,
necessitating high enzyme concentrations for reliable assay data).
As IC50 cannot be less than 50% enzyme concentration this sets a
lower threshold for what can be observed in these assays. It is
therefore possible that the measured IC50 values for the best
inhibitors are underestimates of the potency at lower enzyme
concentration.
Initially, all compounds were N-acetylated. To examine the ef-
fect of N-terminal modification on inhibitory potency, analogues
of compound 7 were synthesized (Table 2). Compounds 10, 11
and 12 were generated by capping the tetrapeptide with benzoyl,
tosyl and benzyl respectively and 13 by capping the tripeptide with
benzoyl. Compound 1, which lacks the N-terminal acetylation, was
found to be a poorer inhibitor than compound 7. This suggests that
the presence of a charged N-terminus is sub-optimal for inhibition.
23
provide the corresponding Michael acceptor 5. The acid can then
be attached to resin and the peptide sequence added on as before.
Additionally, the N terminus of the peptide can be acetylated
prior to deprotection and cleavage.
Both routes offer advantages and disadvantages. Route A is a
more convenient way to generate a range of different warheads
having the same peptide sequence. Route B is better suited to
production of peptide variants of a particular Michael acceptor
warhead. The latter route is also dependent on attaching the
modified amino acid via the side chain; it is therefore useful for
glutamine variants (as here) and with slight modification could
yield asparagine, glutamate or aspartate sequences. A minor limi-
tation of route B is that the warhead needs to be resistant to the
peptide synthesis conditions (particularly the TFA cleavage). An
advantage is that although this synthetic route is slightly longer
than route A the overall yield is much higher (on average 37% after
pro
The natural substrates for 3C are extended polypeptides, which
the N-acylated version is expected to mimic more closely. All
N-capped versions of compound 1 (7, 10, 11, 12) were found to
have improved activity, reinforcing this view. The best inhibitor
of the series has a benzoyl cap (12). A benzoyl cap is also found
to improve inhibition when the P4 Pro residue is removed (13);
this shorter peptide is more active than the corresponding acety-
lated version (8) although loss of the P4 Pro results in less potent
inhibition overall.
In the co-crystal structure of the enzyme with a peptide
substrate, the P5 position is mostly solvent exposed and lacks
1
5c
1
5 steps compared with 3–6% over 13 steps).
significant enzyme contacts, which is illustrated in Figure 1. This
is consistent with P5 variations having only minor effects on
inhibitor potency.
To characterise the compounds synthesised were tested for
pro
their ability to inhibit recombinant FMDV 3C
in vitro in the
O
NH
2
O
NH
2
+
O
O
O
+
O
O
H
H
N
H
N
H
H
N
2
2
N
O-
N
OEt
N
H
N
H
N
H
N
H
O
O
O
O
1
NH3+
P1
NH3+
P1
P4
P3
P2
P '
P4
P3
P2
MA
1
Scheme 1. FMDV 3Cpro P4–P1 substrate sequence and the first FMDV 3C inhibitor design (1). A tetrapeptide is modified on its N-terminus containing a Michael acceptor
0
pro
0
(
MA) moiety, in this case an
a
,b-unsaturated ester, taking the P1 position of the original substrate.