The
phosphoinositide 3-kinase (PI3K)/mechanistic target of rapamycin
(mTOR) pathway is frequently overactivated in cancer, and drives cell
growth, proliferation, survival, and metastasis. Here, we report a
structure–activity relationship study, which led to the discovery
of a drug-like adenosine 5′-triphosphate-site PI3K/mTOR kinase
inhibitor: (S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triazin-2-yl)pyridin-2-amine
(PQR530, compound 6), which qualifies as a clinical candidate
due to its potency and specificity for PI3K and mTOR kinases, and
its pharmacokinetic properties, including brain penetration. Compound 6 showed excellent selectivity over a wide panel of kinases
and an excellent selectivity against unrelated receptor enzymes and
ion channels. Moreover, compound 6 prevented cell growth
in a cancer cell line panel. The preclinical in vivo characterization
of compound 6 in an OVCAR-3 xenograft model demonstrated
good oral bioavailability, excellent brain penetration, and efficacy.
Initial toxicity studies in rats and dogs qualify 6 for
further development as a therapeutic agent in oncology.
Highlights d Integrated structural model of full-length monomeric nuclear receptor LRH-1 d Model accurately predicts a novel inter-domain charge clamp d DNA binding and transcriptional activation domains dock via conserved helices d Binding of a known transcriptional co-regulator alters LRH-1 inter-domain dynamics
The lipid kinase PI4KB, which generates phosphatidylinositol 4phosphate (PI4P), is a key enzyme in regulating membrane transport and is also hijacked by multiple picornaviruses to mediate viral replication. PI4KB can interact with multiple protein binding partners, which are differentially manipulated by picornaviruses to facilitate replication. The protein c10orf76 is a PI4KB-associated protein that increases PI4P levels at the Golgi and is essential for the viral replication of specific enteroviruses. We used hydrogendeuterium exchange mass spectrometry to characterize the c10orf76-PI4KB complex and reveal that binding is mediated by the kinase linker of PI4KB, with formation of the heterodimeric complex modulated by PKA-dependent phosphorylation. Complexdisrupting mutations demonstrate that PI4KB is required for membrane recruitment of c10orf76 to the Golgi, and that an intact c10orf76-PI4KB complex is required for the replication of c10orf76dependent enteroviruses. Intriguingly, c10orf76 also contributed to proper Arf1 activation at the Golgi, providing a putative mechanism for the c10orf76-dependent increase in PI4P levels at the Golgi.
Lipin/Pah phosphatidic acid phosphatases (PAPs) generate diacylglycerol to regulate triglyceride synthesis and cellular signaling. Inactivating mutations cause rhabdomyolysis, autoinflammatory disease, and aberrant fat storage. Disease-mutations cluster within the conserved N-Lip and CLip regions that are separated by 500-residues in humans. To understand how the N-Lip and CLip combine for PAP function, we determined crystal structures of Tetrahymena thermophila Pah2 (Tt Pah2) that directly fuses the N-Lip and CLip. Tt Pah2 adopts a two-domain architecture where the N-Lip combines with part of the CLip to form an immunoglobulin-like domain and the remaining CLip forms a HAD-like catalytic domain. An N-Lip CLip fusion of mouse lipin-2 is catalytically active, which suggests mammalian lipins function with the same domain architecture as Tt Pah2. HDX-MS identifies an N-terminal amphipathic helix essential for membrane association. Disease-mutations disrupt catalysis or destabilize the protein fold. This illustrates mechanisms for lipin/Pah PAP function, membrane association, and lipin-related pathologies.
The GTPase Rab11 plays key roles in receptor recycling, oogenesis, autophagosome formation, and ciliogenesis. However, investigating Rab11 regulation has been hindered by limited molecular detail describing activation by cognate guanine nucleotide exchange factors (GEFs). Here, we present the structure of Rab11 bound to the GEF SH3BP5, along with detailed characterization of Rab-GEF specificity. The structure of SH3BP5 shows a coiled-coil architecture that mediates exchange through a unique Rab-GEF interaction. Furthermore, it reveals a rearrangement of the switch I region of Rab11 compared with solved Rab-GEF structures, with a constrained conformation when bound to SH3BP5. Mutation of switch I provides insights into the molecular determinants that allow for Rab11 selectivity over evolutionarily similar Rab GTPases present on Rab11-positive organelles. Moreover, we show that GEF-deficient mutants of SH3BP5 show greatly decreased Rab11 activation in cellular assays of active Rab11. Overall, our results give molecular insight into Rab11 regulation, and how Rab-GEF specificity is achieved.
Using a novel chemistry-based assay for identifying electrophilic natural products from unprocessed extracts, we identified the PI3-kinase/mTOR dual inhibitor neolymphostin A from Salinispora arenicola CNY-486. The method further showed that the vinylogous ester substituent on the neolymphostin core was the exact site for enzyme conjugation. Tandem MS/MS experiments on PI3Kα treated with the inhibitor revealed that neolymphostin covalently modified Lys802 with a shift in mass of +306 amu, corresponding to addition of the inhibitor and elimination of methanol. The binding pose of the inhibitor bound to PI3Kα was modelled, and hydrogen-deuterium exchange mass spectrometry experiments supported this model. Against a panel of kinases, neolymphostin showed good selectivity for PI3-kinase and mTOR. In addition, the natural product blocked AKT phosphorylation in live cells with an IC50 of ~3 nM. Taken together, neolymphostin is the first reported example of a covalent kinase inhibitor from the bacterial domain of life.
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