Designing for selectivity: A combination of protein crystal‐structure analysis, virtual screening, and synthetic chemistry has been used to develop noncytotoxic inhibitors of RabGGTase (IC50: 42 nM for the example shown; red O, blue N, yellow S) that are selective over FTase and GGTase I. Furthermore, the inhibitors display cellular activity and inhibit cancer cell proliferation.
Post-translational attachment of geranylgeranyl isoprenoids
to
Rab GTPases, the key organizers of intracellular vesicular transport,
is essential for their function. Rab geranylgeranyl transferase (RabGGTase)
is responsible for prenylation of Rab proteins. Recently, RabGGTase
inhibitors have been proposed to be potential therapeutics for treatment
of cancer and osteoporosis. However, the development of RabGGTase
selective inhibitors is complicated by its structural and functional
similarity to other protein prenyltransferases. Herein we report identification
of the natural product psoromic acid (PA) that potently and selectively
inhibits RabGGTase with an IC50 of 1.3 μM. Structure–activity
relationship analysis suggested a minimal structure involving the
depsidone core with a 3-hydroxyl and 4-aldehyde motif for binding
to RabGGTase. Analysis of the crystal structure of the RabGGTase:PA
complex revealed that PA forms largely hydrophobic interactions with
the isoprenoid binding site of RabGGTase and that it attaches covalently
to the N-terminus of the α subunit. We found that in contrast
to other protein prenyltransferases, RabGGTase is autoinhibited through
N-terminal αHis2 coordination with the catalytic
zinc ion. Mutation of αHis dramatically enhances
the reaction rate, indicating that the activity of RabGGTase is likely
regulated in vivo. The covalent binding of PA to the N-terminus of
the RabGGTase α subunit seems to potentiate its interaction
with the active site and explains the selectivity of PA for RabGGTase.
Therefore, psoromic acid provides a new starting point for the development
of selective RabGGTase inhibitors.
Members of the Ras superfamily of small GTPases are frequently mutated in cancer. Therefore, inhibitors have been developed to address the acitivity of these GTPases by inhibiting their prenylating enzymes FTase, GGTase I, and RabGGTase. In contrast to FTase and GGTase I, only a handful of RabGGTase inhibitors have been developed. The most active RabGGTase inhibitor known until recently was an FTase inhibitor which hit RabGGTase as an off-target. We recently reported our efforts to tune the selectivity of these inhibitors toward RabGGTase. Here we describe an extended set of selective inhibitors. The requirements for selective RabGGTase inhibitors are described in detail, guided by multiple crystal structures. In order to relate in vitro and cellular activity, a high-throughput assay system to detect the attachment of [(3)H]geranylgeranyl groups to Rab was used. Selective RabGGTase inhibition allows the establishment of novel drug discovery programs aimed at the development of anticancer therapeutics.
Gezielter Entwurf von Selektivität: Eine Kombination aus Proteinkristallstrukturanalyse, virtuellem Screening und Synthesechemie wurde genutzt, um nicht‐zytotoxische Inhibitoren der RabGGTase zu entwerfen (IC50: 42 nM für das gezeigte Beispiel; rot O, blau N, gelb S), die selektiv gegenüber FTase und GGTase I sind. Die Inhibitoren sind außerdem zellaktiv und hemmen die Proliferation von Krebszellen.
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