Krista K. Bowman

Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity

The Ras gene is frequently mutated in cancer, and mutant Ras drives tumorigenesis. Although Ras is a central oncogene, small molecules that bind to Ras in a well-defined manner and exert inhibitory effects have not been uncovered to date. Through an NMR-based fragment screen, we identified a group of small molecules that all bind to a common site on Ras. High-resolution cocrystal structures delineated a unique ligand-binding pocket on the Ras protein that is adjacent to the switch I/II regions and can be expanded upon compound binding. Structure analysis predicts that compound-binding interferes with the Ras/SOS interactions. Indeed, selected compounds inhibit SOS-mediated nucleotide exchange and prevent Ras activation by blocking the formation of intermediates of the exchange reaction. The discovery of a small-molecule binding pocket on Ras with functional significance provides a new direction in the search of therapeutically effective inhibitors of the Ras oncoprotein.

Somatic mutations in p85α promote tumorigenesis through class IA PI3K activation

Members of the mammalian phosphoinositide-3-OH kinase (PI3K) family of proteins are critical regulators of various cellular process including cell survival, growth, proliferation, and motility. Oncogenic activating mutations in the p110alpha catalytic subunit of the heterodimeric p110/p85 PI3K enzyme are frequent in human cancers. Here we show the presence of frequent mutations in p85alpha in colon cancer, a majority of which occurs in the inter-Src homology-2 (iSH2) domain. These mutations uncouple and retain p85alphas p110-stabilizing activity, while abrogating its p110-inhibitory activity. The p85alpha mutants promote cell survival, AKT activation, anchorage-independent cell growth, and oncogenesis in a p110-dependent manner.

Oncogenic ERBB3 Mutations in Human Cancers

The human epidermal growth factor receptor (HER) family of tyrosine kinases is deregulated in multiple cancers either through amplification, overexpression, or mutation. ERBB3/HER3, the only member with an impaired kinase domain, although amplified or overexpressed in some cancers, has not been reported to carry oncogenic mutations. Here, we report the identification of ERBB3 somatic mutations in ~11% of colon and gastric cancers. We found that the ERBB3 mutants transformed colonic and breast epithelial cells in a ligand-independent manner. However, the mutant ERBB3 oncogenic activity was dependent on kinase-active ERBB2. Furthermore, we found that anti-ERBB antibodies and small molecule inhibitors effectively blocked mutant ERBB3-mediated oncogenic signaling and disease progression in vivo.

Structural studies of neuropilin/antibody complexes provide insights into semaphorin and VEGF binding

Neuropilins (Nrps) are co‐receptors for class 3 semaphorins and vascular endothelial growth factors and important for the development of the nervous system and the vasculature. The extracellular portion of Nrp is composed of two domains that are essential for semaphorin binding (a1a2), two domains necessary for VEGF binding (b1b2), and one domain critical for receptor dimerization (c). We report several crystal structures of Nrp1 and Nrp2 fragments alone and in complex with antibodies that selectively block either semaphorin or vascular endothelial growth factor (VEGF) binding. In these structures, Nrps adopt an unexpected domain arrangement in which the a2, b1, and b2 domains form a tightly packed core that is only loosely connected to the a1 domain. The locations of the antibody epitopes together with in vitro experiments indicate that VEGF and semaphorin do not directly compete for Nrp binding. Based upon our structural and functional data, we propose possible models for ligand binding to neuropilins.

Structural basis of Nav1.7 inhibition by an isoform-selective small-molecule antagonist.

A channel involved in pain perception Voltage-gated sodium (Nav) channels propagate electrical signals in muscle cells and neurons. In humans, Nav1.7 plays a key role in pain perception. It is challenging to target a particular Nav isoform; however, arylsulfonamide antagonists selective for Nav1.7 have been reported recently. Ahuja et al. characterized the binding of these small molecules to human Nav channels. To further investigate the mechanism, they engineered a bacterial Nav channel to contain features of the Nav1.7 voltage-sensing domain that is targeted by the antagonist and determined the crystal structure of the chimera bound to an inhibitor. The structure gives insight into the mechanism of voltage sensing and will enable the design of more-selective Nav channel antagonists. Science, this issue p. 10.1126/science.aac5464 Structural studies give insight into how a human sodium channel involved in pain perception can be selectively inhibited. INTRODUCTION Voltage-gated sodium (Nav) channels open and close ion-selective pores in response to changes in membrane potential, and this gating underlies the generation of action potentials. Nav channels are large membrane proteins that contain four peripheral voltage-sensor domains (VSD1–4) that influence the functional state of the central ion-conducting pore. Mutations within the nine human Nav channel isoforms are associated with migraine (Nav1.1), epilepsy (Nav1.1–Nav1.3, Nav1.6), pain (Nav1.7–Nav1.9), cardiac (Nav1.5), and muscle paralysis (Nav1.4) syndromes. Accordingly, Nav channel blockers are used for the treatment of many neurological and cardiovascular disorders. These drugs bind within the central pore domain and generally lack isoform selectivity owing to the high sequence conservation found among Nav channels, limiting their therapeutic utility. In this study, we focused on a recently identified class of isoform-selective small-molecule antagonists that target a unique binding site on the fourth voltage-sensor domain, VSD4. Here we report the structural determination of such small-molecule aryl sulfonamide antagonists in complex with human Nav1.7 VSD4. Our studies demonstrate how this important new class of gating modifier engages VSD4 to inhibit Nav channel activity through a “voltage-sensor trapping” mechanism. RATIONALE For structural studies, we devised a novel protein-engineering strategy that overcomes the technical complexities of producing full-length human Nav channels. Exploiting the evolutionary relationship between human and bacterial Nav channels, we fused portions of Nav1.7 VSD4 onto the bacterial channel NavAb. Using ligand-binding assays and alanine-scanning mutagenesis, we demonstrated that the antagonist binding site present in the human Nav1.7 channel is preserved within this human VSD4-NavAb chimeric channel. This chimeric construct allowed purification, crystallization, and structure determination of potent aryl sulfonamide antagonists in complex with the human Nav1.7 VSD4 binding site. RESULTS Functional studies using patch-clamp electrophysiology revealed that aryl sulfonamide inhibitors bind with high affinity to an isoform-selective and extracellularly accessible site on VSD4. These inhibitors show a high level of state dependence, potently blocking human Nav1.7 only when VSD4 is in its activated conformation. Our crystallographic studies revealed that the anionic warhead from the aryl sulfonamide inhibitors directly engages the fourth gating charge residue (R4) on the voltage-sensing S4 helix, effectively trapping VSD4 in its activated state. Isoform selectivity is achieved by inhibitor interactions with nonconserved residues found on the S2 and S3 transmembrane helices. The drug receptor site is partially submerged within the membrane bilayer, and a peripherally bound phospholipid was observed to form a tripartite complex with the antagonist and channel. CONCLUSION A new crystallization strategy has enabled the structural determination of VSD4 from human Nav1.7 in complex with potent, state-dependent, isoform-selective small-molecule antagonists. Mechanistically, inhibitor binding traps VSD4 in an activated conformation, which stabilizes a nonconductive state of the channel, and likely prevents recovery from inactivation. Unique phospholipid interactions and an exposed inhibitor binding site expand the importance of the membrane bilayer in ion channel biology. We anticipate that these structures will enable drug design efforts aimed at other voltage-gated ion channels and may accelerate the development of new treatments for pain that selectively target Nav1.7. Drug binding sites in sodium channels. (Left) Top-view model of human Nav1.7. When open, sodium passes through the channel. Blocking drugs lacking isoform selectivity bind to a conserved site within the central pore. Isoform-selective inhibitors bind to a distinct site on VSD4. (Right) Strategy for Nav1.7 crystallography. Portions of Nav1.7 VSD4 were grafted onto a tetrameric channel (NavAb) and crystallized. (Inset) Side view of aryl sulfonamide binding site with the S4 helix and arginine gating charges highlighted pink. Voltage-gated sodium (Nav) channels propagate action potentials in excitable cells. Accordingly, Nav channels are therapeutic targets for many cardiovascular and neurological disorders. Selective inhibitors have been challenging to design because the nine mammalian Nav channel isoforms share high sequence identity and remain recalcitrant to high-resolution structural studies. Targeting the human Nav1.7 channel involved in pain perception, we present a protein-engineering strategy that has allowed us to determine crystal structures of a novel receptor site in complex with isoform-selective antagonists. GX-936 and related inhibitors bind to the activated state of voltage-sensor domain IV (VSD4), where their anionic aryl sulfonamide warhead engages the fourth arginine gating charge on the S4 helix. By opposing VSD4 deactivation, these compounds inhibit Nav1.7 through a voltage-sensor trapping mechanism, likely by stabilizing inactivated states of the channel. Residues from the S2 and S3 helices are key determinants of isoform selectivity, and bound phospholipids implicate the membrane as a modulator of channel function and pharmacology. Our results help to elucidate the molecular basis of voltage sensing and establish structural blueprints to design selective Nav channel antagonists.

Molecular basis of Tank-binding kinase 1 activation by transautophosphorylation.

Tank-binding kinase (TBK)1 plays a central role in innate immunity: it serves as an integrator of multiple signals induced by receptor-mediated pathogen detection and as a modulator of IFN levels. Efforts to better understand the biology of this key immunological factor have intensified recently as growing evidence implicates aberrant TBK1 activity in a variety of autoimmune diseases and cancers. Nevertheless, key molecular details of TBK1 regulation and substrate selection remain unanswered. Here, structures of phosphorylated and unphosphorylated human TBK1 kinase and ubiquitin-like domains, combined with biochemical studies, indicate a molecular mechanism of activation via transautophosphorylation. These TBK1 structures are consistent with the tripartite architecture observed recently for the related kinase IKKβ, but domain contributions toward target recognition appear to differ for the two enzymes. In particular, both TBK1 autoactivation and substrate specificity are likely driven by signal-dependent colocalization events.

Disruption of PH–kinase domain interactions leads to oncogenic activation of AKT in human cancers

The protein kinase v-akt murine thymoma viral oncogene homolog (AKT), a key regulator of cell survival and proliferation, is frequently hyperactivated in human cancers. Intramolecular pleckstrin homology (PH) domain–kinase domain (KD) interactions are important in maintaining AKT in an inactive state. AKT activation proceeds after a conformational change that dislodges the PH from the KD. To understand these autoinhibitory interactions, we generated mutations at the PH–KD interface and found that most of them lead to constitutive activation of AKT. Such mutations are likely another mechanism by which activation may occur in human cancers and other diseases. In support of this likelihood, we found somatic mutations in AKT1 at the PH–KD interface that have not been previously described in human cancers. Furthermore, we show that the AKT1 somatic mutants are constitutively active, leading to oncogenic signaling. Additionally, our studies show that the AKT1 mutants are not effectively inhibited by allosteric AKT inhibitors, consistent with the requirement for an intact PH–KD interface for allosteric inhibition. These results have important implications for therapeutic intervention in patients with AKT mutations at the PH–KD interface.

The crystal structure of the catalytic domain of the NF-κB inducing kinase reveals a narrow but flexible active site.

The NF-κB inducing kinase (NIK) regulates the non-canonical NF-κB pathway downstream of important clinical targets including BAFF, RANKL, and LTβ. Despite numerous genetic studies associating dysregulation of this pathway with autoimmune diseases and hematological cancers, detailed molecular characterization of this central signaling node has been lacking. We undertook a systematic cloning and expression effort to generate soluble, well-behaved proteins encompassing the kinase domains of human and murine NIK. Structures of the apo NIK kinase domain from both species reveal an active-like conformation in the absence of phosphorylation. ATP consumption and peptide phosphorylation assays confirm that phosphorylation of NIK does not increase enzymatic activity. Structures of murine NIK bound to inhibitors possessing two different chemotypes reveal conformational flexibility in the gatekeeper residue controlling access to a hydrophobic pocket. Finally, a single amino acid difference affects the ability of some inhibitors to bind murine and human NIK with the same affinity.

Crystal structure of human cathepsin V.

Cathepsin V is a lysosomal cysteine protease that is expressed in the thymus, testis and corneal epithelium. We have determined the 1.6 A resolution crystal structure of human cathepsin V associated with an irreversible vinyl sulfone inhibitor. The fold of this enzyme is similar to the fold adopted by other members of the papain superfamily of cysteine proteases. This study provides a framework for understanding the structural basis for cathepsin Vs activity and will aid in the design of inhibitors of this enzyme. A comparison of cathepsin Vs active site with the active sites of related proteases revealed a number of differences, especially in the S2 and S3 subsites, that could be exploited in identifying specific cathepsin V inhibitors or in identifying inhibitors of other cysteine proteases that would be selective against cathepsin V.

Identification of amides derived from 1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid as potent inhibitors of human nicotinamide phosphoribosyltransferase (NAMPT).

Potent, 1H-pyrazolo[3,4-b]pyridine-containing inhibitors of the human nicotinamide phosphoribosyltransferase (NAMPT) enzyme were identified using structure-based design techniques. Many of these compounds exhibited nanomolar antiproliferation activities against human tumor lines in in vitro cell culture experiments, and a representative example (compound 26) demonstrated encouraging in vivo efficacy in a mouse xenograft tumor model derived from the A2780 cell line. This molecule also exhibited reduced rat retinal exposures relative to a previously studied imidazo-pyridine-containing NAMPT inhibitor. Somewhat surprisingly, compound 26 was only weakly active in vitro against mouse and monkey tumor cell lines even though it was a potent inhibitor of NAMPT enzymes derived from these species. The compound also exhibited only minimal effects on in vivo NAD levels in mice, and these changes were considerably less profound than those produced by an imidazo-pyridine-containing NAMPT inhibitor. The crystal structures of compound 26 and the corresponding PRPP-derived ribose adduct in complex with NAMPT were also obtained.