Full Papers
Intriguingly, N-BPs have been found to exhibit a surprising
variety of other pharmacological activities that include in-
creased longevity in a mouse model of progeria,[5] as well as
antiviral[6] and antiparasitic[7] effects. In the clinic, antitumor ef-
fects have been observed in breast cancer[8] and multiple mye-
loma[9] patients. The exact mechanisms underlying the antitu-
mor activity are not yet fully understood. They may include
apoptosis of tumor cells due to increased demand for prenyla-
tion in rapidly proliferating cells, and activation of gd Tcells by
accumulating and presenting phospho-antigens such as iso-
pentenyl pyrophosphate (IPP), one of the FPPS substrates.[10,11]
Both mechanisms involve FPPS inhibition, therefore pointing
to FPPS as a potential novel drug target for direct antitumor
therapy.
Herein we describe two additional chemical lead series of al-
losteric FPPS inhibitors that were discovered during the course
of our integrated lead finding efforts. These two series, salicylic
acid and quinoline derivatives, are fivefold more potent in
vitro than our first lead series represented by the benzoindole
derivative 2, and are equally potent to the best bisphospho-
nates. They are again devoid of significant affinity toward bone
mineral, and therefore represent interesting lead molecules for
the treatment of non-bone-related diseases as anticancer or
anti-infective agents.
In an accompanying report, we describe how such lead mol-
ecules can also be optimized for bone indications by fine-
tuning their affinity toward bone mineral through covalent at-
tachment of a suitable bone-affinity tag, while simultaneously
retaining desirable properties such as oral bioavailability.[22]
N-BPs have unusual pharmacokinetic (PK) properties charac-
terized by rapid and strong binding to bone mineral, associat-
ed with low plasma drug concentration soon after administra-
tion, low permeability into non-endocytic cells, and low oral Results and Discussion
bioavailability.[2] While this peculiar PK profile is a blessing for
Discovery of the salicylic acid class of allosteric FPPS
inhibitors
the treatment of bone diseases and contributes to the excep-
tional safety and potency of this class of drugs, it severely
compromises the efficacy of N-BPs as antitumor drugs, for
which high cell permeability and high plasma levels over an
extended period of time are required.
The fragment screen by NMR and fragment optimization work
were performed so rapidly that compound 2 had already been
discovered[18] by the time the high-throughput screen was run.
The HTS was based on a scintillation proximity assay (SPA) that
Crystallographic analyses of human FPPS complexes with
marketed N-BPs[12,13] have revealed how these drugs mimic
one of the FPPS substrates, dimethylallyl pyrophosphate
(DMAPP), and explained why early attempts to move away
from the bisphosphonate chemotype were largely unsuccess-
ful.[14] Currently marketed N-BPs are very small in size but are
highly efficient inhibitors. The geminal bisphosphonate moiety
of these drugs fills the site normally occupied by the pyro-
phosphate group of DMAPP, and plays a major role in recogni-
tion and binding through strong electrostatic interactions with
a trinuclear Mg2+ center and three basic side chains: Arg112,
Lys200, and Lys257. As all bisphosphonate oxygen atoms make
important contributions to these interactions, modifying or re-
placing the bisphosphonate moiety by a less polar isostere
long seemed virtually impossible, leaving the central carbon
atom as the only available position for chemical elaboration of
novel inhibitors. In particular, N-BPs with extended, lipophilic
side chains exploiting the GPP/FPP pocket have been investi-
gated, with the goal to expand the clinical utility of FPPS inhib-
itors.[15–17]
3
monitored the incorporation of H-labeled IPP into [3H]farnesyl
pyrophosphate ([3H]FPP) by capturing the latter onto a phos-
phatidylserine-coated scintillating microtiter plate (“flash-
plate”).[23] In total, 850000 compounds were screened at a con-
centration of 10 mm. The best non-bisphosphonate hits had
IC50 values in the single-digit to double-digit micromolar range.
Of particular interest was compound 3a (Scheme 1, R=H),
a salicylic acid derivative with an IC50 value of 54 mm in the SPA
identified by a similarity search from a salicylic acid fragment
identified by NMR. Although the activity of 3a was close to
the detection limit, we noticed that this hit shared some sub-
structures (naphthyl and benzyloxy moiety, carboxylic acid
functionality) with other NMR fragment hits or optimized allo-
steric inhibitors, such as 2 and 4 (Scheme 1). As this compound
was also deemed chemically attractive, it was selected for
follow-up validation and characterization studies.
FPPS was co-crystallized with 3a, and the X-ray structure of
the complex was determined. Crystallographic analysis con-
firmed that this hit was a genuine allosteric inhibitor of FPPS,
binding to the same allosteric pocket as the previously identi-
fied NMR fragment hits, with the enzyme in the open state
and the flexible C-terminal tail disordered (Figure 1A).[12] This
allosteric pocket is mainly lined by a-helices aC, aG, aH, and aJ
and also involves Tyr10 from helix aA as well as Lys57 from the
loop connecting helices aB and aC. The naphthyl group binds
to the hydrophobic base of the pocket, making close contacts
to Tyr10, Phe206, Phe239, Leu344, Thr63, and to the alkyl por-
tion of the side chains of Lys347, Arg60, and Asn59 (Figure 1B).
The side chain amide of Asn59 is in a parallel orientation with
respect to the naphthyl group and contributes amide–p stack-
ing interactions. The salicylic acid ring forms a face-to-edge ar-
omatic–aromatic interaction with Phe239, while one carboxylic
With the same goal in mind, we initiated a drug discovery
project aimed at novel FPPS inhibitors based on an integrated
lead finding approach combining fragment-based screening
(FBS), high-throughput screening (HTS), in silico screening,
SAR-by-inventory, biophysical validation of hits, and hit-to-lead
chemistry. The fragment-based screen rapidly identified the
first allosteric non-bisphosphonate inhibitors of FPPS, and
quickly led to the benzoindole derivative
2 (Scheme 1),
a novel, potent (IC50 =80 nm) FPPS inhibitor devoid of avidity
toward bone mineral.[18] This part of our work has already been
described in detail elsewhere.[18] Building on these results,
other research groups have recently reported further examples
of non-bisphosphonate allosteric FPPS inhibitors.[19–21]
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