Clear cell Renal Cell Carcinoma (ccRCC) is characterized by
VHL inactivation1,2. Because no
other gene is mutated as frequently, and VHL mutations are
truncal3,
VHL inactivation is regarded as the governing
event4.
VHL loss activates HIF-2, and constitutive HIF-2 restores
tumorigenesis in VHL-reconstituted ccRCC cells5. HIF-2 is implicated in
angiogenesis and multiple other processes6–9, but
angiogenesis is the main target of drugs like sunitinib10. HIF-2, a transcription
factor, has been regarded as undruggable11. A structure-based design approach identified a
selective HIF-2 antagonist (PT2399) that we evaluate using a tumorgraft (TG)/PDX
platform12,13. PT2399 dissociated HIF-2 (an
obligatory heterodimer [HIF-2α/HIF-1β])14 in human ccRCC suppressing
tumorigenesis in 56% (10/18) lines. PT2399 had greater activity than
sunitinib, was active in sunitinib-progressing tumors, and was better tolerated.
Unexpectedly, some VHL-mutant ccRCCs were resistant. Resistance
occurred despite HIF-2 dissociation in tumors and evidence of Hif-2 inhibition
in the mouse as determined by suppression of circulating erythropoietin, a HIF-2
target15 and possible
pharmacodynamic marker. We identified a HIF-2-dependent gene signature in
sensitive tumors. Illustrating drug specificity, gene expression was largely
unaffected by PT2399 in resistant tumors. Sensitive tumors exhibited a
distinguishing gene expression signature, and generally higher HIF-2α
levels. Prolonged PT2399 treatment led to resistance. We identified a binding
site and second site suppressor mutation in HIF-2α and HIF-1β
respectively. Both mutations preserved HIF-2 dimers despite treatment with
PT2399. Finally, an extensively pretreated patient with a sensitive TG had
disease control for >11 months with the close analogue PT2385. We
validate HIF-2 as a target in ccRCC, show that some ccRCC are, unexpectedly,
HIF-2 independent, and set the stage for biomarker-driven clinical trials.
Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is usually linked to inactivation of the pVHL tumor suppressor protein and consequent accumulation of the HIF2α transcription factor 1. Here we show that a small molecule (PT2399) that directly inhibits HIF2α causes tumor regression in preclinical models of primary and metastatic pVHL-defective ccRCC in an on-target fashion. pVHL-defective ccRCC cell lines display unexpectedly variable sensitivity to PT2399, however, suggesting the need for predictive biomarkers.
The architecture of the pore-region of a voltage-gated K+ channel, Kv1.3, was probed using four high affinity scorpion toxins as molecular calipers. We established the structural relatedness of these toxins by solving the structures of kaliotoxin and margatoxin and comparing them with the published structure of charybdotoxin; a homology model of noxiustoxin was then developed. Complementary mutagenesis of Kv1.3 and these toxins, combined with electrostatic compliance and thermodynamic mutant cycle analyses, allowed us to identify multiple toxin-channel interactions. Our analyses reveal the existence of a shallow vestibule at the external entrance to the pore. This vestibule is approximately 28-32 A wide at its outer margin, approximately 28-34 A wide at its base, and approximately 4-8 A deep. The pore is 9-14 A wide at its external entrance and tapers to a width of 4-5 A at a depth of approximately 5-7 A from the vestibule. This structural information should directly aid in developing topological models of the pores of related ion channels and facilitate therapeutic drug design.
More than 90% of clear cell renal cell carcinomas (ccRCC) exhibit inactivation of the von Hippel-Lindau (pVHL) tumor suppressor, establishing it as the major underlying cause of this malignancy. pVHL inactivation results in stabilization of the hypoxia-inducible transcription factors, HIF1a and HIF2a, leading to expression of a genetic program essential for the initiation and progression of ccRCC. Herein, we describe the potent, selective, and orally active small-molecule inhibitor PT2385 as a specific antagonist of HIF2a that allosterically blocks its dimerization with the
Several docking programs are now available that can reproduce the bound conformation of a ligand in an active site, for a wide variety of experimentally determined complexes. However, these programs generally perform less well at ranking multiple possible ligands in one site. Since accurate identification of potential ligands is a prerequisite for many aspects of structure-based drug design, this is a serious limitation. We have tested the ability of two docking programs, FlexX and Gold, to match ligands and active sites for multiple complexes. We show that none of the docking scores from either program are able to match consistently ligands and active sites in our tests. We propose a simple statistical correction, the multiple active site correction (MASC), which greatly ameliorates this problem. We have also tested the correction method against an extended set of 63 cocrystals and in a virtual screening experiment. In all cases, MASC significantly improves the results of the docking experiments.
The hypoxia-inducible
factor 2α (HIF-2α) is a key oncogenic
driver in clear cell renal cell carcinoma (ccRCC). Our first HIF-2α
inhibitor PT2385 demonstrated promising proof of concept clinical
activity in heavily pretreated advanced ccRCC patients. However, PT2385
was restricted by variable and dose-limited pharmacokinetics resulting
from extensive metabolism of PT2385 to its glucuronide metabolite.
Herein we describe the discovery of second-generation HIF-2α
inhibitor PT2977 with increased potency and improved pharmacokinetic
profile achieved by reduction of phase 2 metabolism. Structural modification
by changing the geminal difluoro group in PT2385 to a vicinal difluoro
group resulted in enhanced potency, decreased lipophilicity, and significantly
improved pharmacokinetic properties. In a phase 1 dose-escalation
study, the clinical pharmacokinetics for PT2977 supports the hypothesis
that attenuating the rate of glucuronidation would improve exposure
and reduce variability in patients. Early evidence of clinical activity
shows promise for PT2977 in the treatment of ccRCC.
HIF-2α, a member of the HIF family of transcription factors, is a key oncogenic driver in cancers such as clear cell renal cell carcinoma (ccRCC). A signature feature of these cancers is the overaccumulation of HIF-2α protein, often by inactivation of the E3 ligase VHL (von Hippel−Lindau). Herein we disclose our structure based drug design (SBDD) approach that culminated in the identification of PT2385, the first HIF-2α antagonist to enter clinical trials. Highlights include the use of a putative n → π* Ar interaction to guide early analog design, the conformational restriction of an essential hydroxyl moiety, and the remarkable impact of fluorination near the hydroxyl group. Evaluation of select compounds from two structural classes in a sequence of PK/PD, efficacy, PK, and metabolite profiling identified 10i (PT2385, luciferase EC 50 = 27 nM) as the clinical candidate. Finally, a retrospective crystallographic analysis describes the structural perturbations necessary for efficient antagonism.
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