Structure-Based Design and Evaluation of Reversible KRAS G13D Inhibitors.

Oncogenic KRAS mutations were identified decades ago, yet the selective inhibition of specific KRAS mutant proteins represents an ongoing challenge. Recent progress has been made in targeting certain P-loop mutant proteins, in particular KRAS G12C, for which the covalent inhibition of the GDP state via the Switch II pocket is now a clinically validated strategy. Inhibition of other KRAS mutant proteins such as KRAS G13D, on the other hand, still requires clinical validation. The remoteness of the D13 residue relative to the Switch II pocket in combination with the solvent exposure and conformational flexibility of the D13 side chain, as well as the difficulties of targeting carboxylate residues covalently, renders this specific protein particularly challenging to target selectively. In this report, we describe the design and evaluation of potent and KRAS G13D-selective reversible inhibitors. Subnanomolar binding to the GDP state Switch II pocket and biochemical selectivity over WT KRAS are achieved by leveraging a salt bridge with D13.

Discovery of GDC-0077 (Inavolisib), a Highly Selective Inhibitor and Degrader of Mutant PI3Kα.

Small molecule inhibitors that target the phosphatidylinositol 3-kinase (PI3K) signaling pathway have received significant interest for the treatment of cancers. The class I isoform PI3Kα is most commonly associated with solid tumors via gene amplification or activating mutations. However, inhibitors demonstrating both PI3K isoform and mutant specificity have remained elusive. Herein, we describe the optimization and characterization of a series of benzoxazepin-oxazolidinone ATP-competitive inhibitors of PI3Kα which also induce the selective degradation of the mutant p110α protein, the catalytic subunit of PI3Kα. Structure-based design informed isoform-specific interactions within the binding site, leading to potent inhibitors with greater than 300-fold selectivity over the other Class I PI3K isoforms. Further optimization of pharmacokinetic properties led to excellent in vivo exposure and efficacy and the identification of clinical candidate GDC-0077 (inavolisib, 32), which is now under evaluation in a Phase III clinical trial as a treatment for patients with PIK3CA-mutant breast cancer.

High-Throughput Kinetic Characterization of Irreversible Covalent Inhibitors of KRASG12C by Intact Protein MS and Targeted MRM.

With recent advances and success in several drugs designed to treat acute and chronic diseases, targeted covalent inhibitors show a resurgence in drug discovery. As covalent inhibition is time-dependent, the preferred quantitative potency metric of irreversible inhibitors is the second-order rate constant kinact/Ki, rather than IC50. Here, we present the development of a mass spectrometry-based platform for rapid kinetic analysis of irreversible covalent inhibitors. Using a simple liquid handling robot for automated sample preparation and a solid-phase extraction-based RapidFire-MS system for rapid MS analysis, kinetic characterization of covalent inhibitors was performed in high throughput both by intact protein analysis and targeted multiple reaction monitoring (MRM). In addition, a bimolecular reaction model was applied to extract kinact/Ki in data fitting, providing tremendous flexibility in the experimental design to characterize covalent inhibitors with various properties. Using KRASG12C inhibitors as a test case, the platform was demonstrated to be effective for studying covalent inhibitors with a wide range of kinact/Ki values from single digit to 3 × 105 M-1 s-1.

RTK-Dependent Inducible Degradation of Mutant PI3Kα Drives GDC-0077 (Inavolisib) Efficacy.

PIK3CA is one of the most frequently mutated oncogenes; the p110a protein it encodes plays a central role in tumor cell proliferation. Small-molecule inhibitors targeting the PI3K p110a catalytic subunit have entered clinical trials, with early-phase GDC-0077 studies showing antitumor activity and a manageable safety profile in patients with PIK3CA-mutant breast cancer. However, preclinical studies have shown that PI3K pathway inhibition releases negative feedback and activates receptor tyrosine kinase signaling, reengaging the pathway and attenuating drug activity. Here we discover that GDC-0077 and taselisib more potently inhibit mutant PI3K pathway signaling and cell viability through unique HER2-dependent mutant p110a degradation. Both are more effective than other PI3K inhibitors at maintaining prolonged pathway suppression. This study establishes a new strategy for identifying inhibitors that specifically target mutant tumors by selective degradation of the mutant oncoprotein and provide a strong rationale for pursuing PI3Kα degraders in patients with HER2-positive breast cancer. SIGNIFICANCE: The PI3K inhibitors GDC-0077 and taselisib have a unique mechanism of action; both inhibitors lead to degradation of mutant p110a protein. The inhibitors that have the ability to trigger specific degradation of mutant p110a without significant change in wild-type p110a protein may result in improved therapeutic index in PIK3CA-mutant tumors.See related commentary by Vanhaesebroeck et al., p. 20.This article is highlighted in the In This Issue feature, p. 1.

Dynamic Organization of Myristoylated Src in the Live Cell Plasma Membrane.

The spatial organization of lipid-anchored proteins in the plasma membrane directly influences cell signaling, but measuring such organization in situ is experimentally challenging. The canonical oncogene, c-Src, is a lipid anchored protein that plays a key role in integrin-mediated signal transduction within focal adhesions and cell-cell junctions. Because of its activity in specific plasma membrane regions, structural motifs within the protein have been hypothesized to play an important role in its subcellular localization. This study used a combination of time-resolved fluorescence fluctuation spectroscopy and super-resolution microscopy to quantify the dynamic organization of c-Src in live cell membranes. Pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) showed that a small fraction of c-Src transiently sorts into membrane clusters that are several times larger than the monomers. Photoactivated localization microscopy (PALM) confirmed that c-Src partitions into clusters with low probability and showed that the characteristic size of the clusters is 10-80 nm. Finally, time-resolved fluorescence anisotropy measurements were used to quantify the rotational mobility of c-Src to determine how it interacts with its local environment. Taken together, these results build a quantitative description of the mobility and clustering behavior of the c-Src nonreceptor tyrosine kinase in the live cell plasma membrane.

Emerging concepts in the regulation of the EGF receptor and other receptor tyrosine kinases.

The activation of receptor tyrosine kinases in response to extracellular signals is a principal component of metazoan signaling. Structural analysis of the extracellular and intracellular domains of these receptors has shed substantial light on the mechanisms underlying their activation. A remaining challenge is to understand how these domains operate together in the context of the full-length receptors. With a focus on the epidermal growth factor (EGF) receptor, this review highlights recent advances towards this goal. Although receptor tyrosine kinases are divergent in terms of the details of how they operate, these studies reveal common mechanisms that ensure activation in the proper context. Understanding these mechanisms provides insights into the vulnerabilities of these receptors to disease-causing mutations.

Conformational coupling across the plasma membrane in activation of the EGF receptor.

How the epidermal growth factor receptor (EGFR) activates is incompletely understood. The intracellular portion of the receptor is intrinsically active in solution, and to study its regulation, we measured autophosphorylation as a function of EGFR surface density in cells. Without EGF, intact EGFR escapes inhibition only at high surface densities. Although the transmembrane helix and the intracellular module together suffice for constitutive activity even at low densities, the intracellular module is inactivated when tethered on its own to the plasma membrane, and fluorescence cross-correlation shows that it fails to dimerize. NMR and functional data indicate that activation requires an N-terminal interaction between the transmembrane helices, which promotes an antiparallel interaction between juxtamembrane segments and release of inhibition by the membrane. We conclude that EGF binding removes steric constraints in the extracellular module, promoting activation through N-terminal association of the transmembrane helices.

Architecture and membrane interactions of the EGF receptor.

Dimerization-driven activation of the intracellular kinase domains of the epidermal growth factor receptor (EGFR) upon extracellular ligand binding is crucial to cellular pathways regulating proliferation, migration, and differentiation. Inactive EGFR can exist as both monomers and dimers, suggesting that the mechanism regulating EGFR activity may be subtle. The membrane itself may play a role but creates substantial difficulties for structural studies. Our molecular dynamics simulations of membrane-embedded EGFR suggest that, in ligand-bound dimers, the extracellular domains assume conformations favoring dimerization of the transmembrane helices near their N termini, dimerization of the juxtamembrane segments, and formation of asymmetric (active) kinase dimers. In ligand-free dimers, by holding apart the N termini of the transmembrane helices, the extracellular domains instead favor C-terminal dimerization of the transmembrane helices, juxtamembrane segment dissociation and membrane burial, and formation of symmetric (inactive) kinase dimers. Electrostatic interactions of EGFR's intracellular module with the membrane are critical in maintaining this coupling.

Regulation of the catalytic activity of the EGF receptor.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase involved in cell growth that is often misregulated in cancer. Several recent studies highlight the unique structural mechanisms involved in its regulation. Some elucidate the important role that the juxtamembrane segment and the transmembrane helix play in stabilizing the activating asymmetric kinase dimer, and suggest that its activation mechanism is likely to be conserved among the other human EGFR-related receptors. Other studies provide new explanations for two long observed, but poorly understood phenomena, the apparent heterogeneity in ligand binding and the formation of ligand-independent dimers. New insights into the allosteric mechanisms utilized by intracellular regulators of EGFR provide hope that allosteric sites could be used as targets for drug development.

Catalytic control in the EGF receptor and its connection to general kinase regulatory mechanisms.

In contrast to the active conformations of protein kinases, which are essentially the same for all kinases, inactive kinase conformations are structurally diverse. Some inactive conformations are, however, observed repeatedly in different kinases, perhaps reflecting an important role in catalysis. In this review, we analyze one of these recurring conformations, first identified in CDK and Src kinases, which turned out to be central to understanding of how kinase domain of the EGF receptor is activated. This mechanism, which involves the stabilization of the active conformation of an α helix, has features in common with mechanisms operative in several other kinases.

Mechanism for activation of the EGF receptor catalytic domain by the juxtamembrane segment.

Signaling by the epidermal growth factor receptor requires an allosteric interaction between the kinase domains of two receptors, whereby one activates the other. We show that the intracellular juxtamembrane segment of the receptor, known to potentiate kinase activity, is able to dimerize the kinase domains. The C-terminal half of the juxtamembrane segment latches the activated kinase domain to the activator, and the N-terminal half of this segment further potentiates dimerization, most likely by forming an antiparallel helical dimer that engages the transmembrane helices of the activated receptor. Our data are consistent with a mechanism in which the extracellular domains block the intrinsic ability of the transmembrane and cytoplasmic domains to dimerize and activate, with ligand binding releasing this block. The formation of the activating juxtamembrane latch is prevented by the C-terminal tails in a structure of an inactive kinase domain dimer, suggesting how alternative dimers can prevent ligand-independent activation.

Autoinhibition regulates the motility of the C. elegans intraflagellar transport motor OSM-3.

OSM-3 is a Kinesin-2 family member from Caenorhabditis elegans that is involved in intraflagellar transport (IFT), a process essential for the construction and maintenance of sensory cilia. In this study, using a single-molecule fluorescence assay, we show that bacterially expressed OSM-3 in solution does not move processively (multiple steps along a microtubule without dissociation) and displays low microtubule-stimulated adenosine triphosphatase (ATPase) activity. However, a point mutation (G444E) in a predicted hinge region of OSM-3's coiled-coil stalk as well as a deletion of that hinge activate ATPase activity and induce robust processive movement. These hinge mutations also cause a conformational change in OSM-3, causing it to adopt a more extended conformation. The motility of wild-type OSM-3 also can be activated by attaching the motor to beads in an optical trap, a situation that may mimic attachment to IFT cargo. Our results suggest that OSM-3 motility is repressed by an intramolecular interaction that involves folding about a central hinge and that IFT cargo binding relieves this autoinhibition in vivo. Interestingly, the G444E allele in C. elegans produces similar ciliary defects to an osm-3-null mutation, suggesting that autoinhibition is important for OSM-3's biological function.

Mechanism of transport of IFT particles in C. elegans cilia by the concerted action of kinesin-II and OSM-3 motors.

The assembly and function of cilia on Caenorhabditis elegans neurons depends on the action of two kinesin-2 motors, heterotrimeric kinesin-II and homodimeric OSM-3-kinesin, which cooperate to move the same intraflagellar transport (IFT) particles along microtubule (MT) doublets. Using competitive in vitro MT gliding assays, we show that purified kinesin-II and OSM-3 cooperate to generate movement similar to that seen along the cilium in the absence of any additional regulatory factors. Quantitative modeling suggests that this could reflect an alternating action mechanism, in which the motors take turns to move along MTs, or a mechanical competition, in which the motors function in a concerted fashion to move along MTs with the slow motor exerting drag on the fast motor and vice versa. In vivo transport assays performed in Bardet-Biedl syndrome (BBS) protein and IFT motor mutants favor a mechanical competition model for motor coordination in which the IFT motors exert a BBS protein-dependent tension on IFT particles, which controls the IFT pathway that builds the cilium foundation.

A lever-arm rotation drives motility of the minus-end-directed kinesin Ncd.

Kinesins are microtubule-based motor proteins that power intracellular transport. Most kinesin motors, exemplified by Kinesin-1, move towards the microtubule plus end, and the structural changes that govern this directional preference have been described. By contrast, the nature and timing of the structural changes underlying the minus-end-directed motility of Kinesin-14 motors (such as Drosophila Ncd) are less well understood. Using cryo-electron microscopy, here we demonstrate that a coiled-coil mechanical element of microtubule-bound Ncd rotates approximately 70 degrees towards the minus end upon ATP binding. Extending or shortening this coiled coil increases or decreases velocity, respectively, without affecting ATPase activity. An unusual Ncd mutant that lacks directional preference shows unstable nucleotide-dependent conformations of its coiled coil, underscoring the role of this mechanical element in motility. These results show that the force-producing conformational change in Ncd occurs on ATP binding, as in other kinesins, but involves the swing of a lever-arm mechanical element similar to that described for myosins.

Anisotropic dynamics of the JE-2147-HIV protease complex: drug resistance and thermodynamic binding mode examined in a 1.09 A structure.

The structure of HIV protease (HIV Pr) bound to JE-2147 (also named AG1776 or KNI-764) is determined here to 1.09 A resolution. This highest-resolution structure for HIV Pr allows refinement of anisotropic displacement parameters (ADPs) for all atoms. Clustering based on the directional information in ADPs defines two sets of subdomains such that within each set, subdomains undergo similar anisotropic motion. These sets are (a) the core of monomer A grouped with both substrate-binding flaps and (b) the core of monomer B coupled to both catalytic aspartates (25A/B). The four-stranded beta-sheet (1-4 A/B and 95-99 A/B) that forms a significant part of the dimer interface exhibits large anisotropic amplitudes that differ from those of the other sets of subdomains. JE-2147 is shown here to be a picomolar inhibitor (K(i) = 41 +/- 18 pM). The structure is used to interpret the mechanism of association of JE-2147, a second-generation inhibitor for which binding is enthalpically driven, with respect to first-generation inhibitors for which binding is predominantly entropically driven [Velazquez-Campoy, A., et al. (2001) Arch. Biochem. Biophys. 390, 169-175]. Relative to the entropically driven inhibitor complexes, the JE-2147-HIV Pr complex exhibits an approximately 0.5 A movement of the substrate flaps in toward the substrate, suggesting a more compatible enthalpically driven association. Domains of the protease identified by clustering of ADPs also suggest a model of enthalpy-entropy compensation for all HIV Pr inhibitors in which dynamic coupling of the flaps is offset by an increased level of motion of the beta-sheet domain of the dimer interface (1-4 A/B and 95-99 A/B).