Next-generation XPO1 inhibitor shows improved efficacy and in vivo tolerability in hematological malignancies.

The nuclear export receptor, Exportin 1 (XPO1), mediates transport of growth-regulatory proteins, including tumor suppressors, and is overactive in many cancers, including chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML) and aggressive lymphomas. Oral selective inhibitor of nuclear export (SINE) compounds that block XPO1 function were recently identified and hold promise as a new therapeutic paradigm in many neoplasms. One of these compounds, KPT-330 (selinexor), has made progress in Phase I/II clinical trials, but systemic toxicities limit its administration to twice-per-week and requiring supportive care. We designed a new generation SINE compound, KPT-8602, with a similar mechanism of XPO1 inhibition and potency but considerably improved tolerability. Efficacy of KPT-8602 was evaluated in preclinical animal models of hematological malignancies, including CLL and AML. KPT-8602 shows similar in vitro potency compared with KPT-330 but lower central nervous system penetration, which resulted in enhanced tolerability, even when dosed daily, and improved survival in CLL and AML murine models compared with KPT-330. KPT-8602 is a promising compound for further development in hematological malignancies and other cancers in which upregulation of XPO1 is seen. The wider therapeutic window of KPT-8602 may also allow increased on-target efficacy leading to even more efficacious combinations with other targeted anticancer therapies.

Antileukemic activity of nuclear export inhibitors that spare normal hematopoietic cells.

Drugs that target the chief mediator of nuclear export, chromosome region maintenance 1 protein (CRM1) have potential as therapeutics for leukemia, but existing CRM1 inhibitors show variable potencies and a broad range of cytotoxic effects. Here, we report the structural analysis and antileukemic activity of a new generation of small-molecule inhibitors of CRM1. Designated selective inhibitors of nuclear export (SINE), these compounds were developed using molecular modeling to screen a small virtual library of compounds against the nuclear export signal (NES) groove of CRM1. The 2.2-Å crystal structure of the CRM1-Ran-RanBP1 complex bound to KPT-251, a representative molecule of this class of inhibitors, shows that the drug occupies part of the groove in CRM1 that is usually occupied by the NES, but penetrates much deeper into the groove and blocks CRM1-directed protein export. SINE inhibitors exhibit potent antileukemic activity, inducing apoptosis at nanomolar concentrations in a panel of 14 human acute myeloid leukemia (AML) cell lines representing different molecular subtypes of the disease. When administered orally to immunodeficient mice engrafted with human AML cells, KPT-251 had potent antileukemic activity with negligible toxicity to normal hematopoietic cells. Thus, KPT-SINE CRM1 antagonists represent a novel class of drugs that warrant further testing in AML patients.

Karyopherins and nuclear import.

Proteins of the karyopherin alpha and karyopherin beta families play a central role in nucleocytoplasmic transport. Recently, crystal structures of karyopherin alpha and its complexes with nuclear localization signal peptides, a karyopherin beta2-Ran complex and complexes of full-length and fragments of karyopherin beta1 with import substrates, Ran and nucleoporins have been solved. These karyopherin structures provide valuable insights into understanding the molecular mechanism of nuclear import, especially substrate recognition, substrate release by GTPase and interactions with the nuclear pore complex.

Pictures in cell biology. Structures of nuclear-transport components.

The past three years have seen the solution of several nuclear transport component structures and recently of the structure of a regulator bound to part of a nuclear pore complex (NPC) protein. These structures have provided a wealth of valuable information about the proteins involved and suggested strategies for further investigation of their properties. We do not have space here to go into detail about this information, so instead we are illustrating the structures and providing primary references enabling interested readers to find further information. On this page, we are concentrating on the GTPase Ran and proteins that modulate its activity, and on the facing page are the other transport factors, some of which also interact directly with Ran. Notably absent at the moment are the nuclear pore complex component s, apart from one domain of RanBP2. Only when theses are characterized fully will we really be able to understand how transport substrates move across the nuclear envelope.

Structure of the nuclear transport complex karyopherin-beta2-Ran x GppNHp.

Transport factors in the karyopherin-beta (also called importin-beta) family mediate the movement of macromolecules in nuclear-cytoplasmic transport pathways. Karyopherin-beta2 (transportin) binds a cognate import substrate and targets it to the nuclear pore complex. In the nucleus, Ran x GTP binds karyopherin-beta2 and dissociates the substrate. Here we present the 3.0 A structure of the karyopherin-beta2-Ran x GppNHp complex where GppNHp is a non-hydrolysable GTP analogue. Karyopherin-beta2 contains eighteen HEAT repeats arranged into two continuous orthogonal arches. Ran is clamped in the amino-terminal arch and substrate-binding activity is mapped to the carboxy-terminal arch. A large loop in HEAT repeat 7 spans both arches. Interactions of the loop with Ran and the C-terminal arch implicate it in GTPase-mediated dissociation of the import-substrate. Ran x GppNHp in the complex shows extensive structural rearrangement, compared to Ran GDP, in regions contacting karyopherin-beta2. This provides a structural basis for the specificity of the karyopherin-beta family for the GTP-bound state of Ran, as well as a rationale for interactions of the karyopherin-Ran complex with the regulatory proteins ranGAP, ranGEF and ranBP1.

Detection and use of pseudo-translation in determination of protein structures.

Two types of pseudo-translation symmetry, pseudo-twofold translational symmetry and pseudo-body-centered symmetry, have been found in protein crystals of chorismate mutase and cyclophilin C. Statistics on diffraction intensity from these two crystals showed that the presence of pseudo-translations in atomic space yielded a distribution of systematically strong and weak reflections at low resolution. The diffraction pattern resulting from pseudo-translational symmetry was apparently similar to that from true crystallographic symmetry at 4 A resolution, but was distinct at high resolution. Pseudo-translation can be detected by comparing the average magnitudes of certain parity groups of reflections in three-dimensional hkl space. Based on the structures of chorismate mutase and cyclophilin C, the ratio of >1.2 for the average magnitudes of parity groups is sufficient to indicate the existence of pseudo-translation. Although pseudo-translation often makes structure determination more difficult, the additional information of pseudo-translation has been used successfully in the structure determination of chorismate mutase by multiple isomorphous replacement and of cyclophilin C by molecular replacement. Thus, examination of pseudo-translation is recommended at an early stage of structure determination.

High affinity binding of the pleckstrin homology domain of mSos1 to phosphatidylinositol (4,5)-bisphosphate.

mSos1 has been implicated in coupling mammalian tyrosine kinases to the Ras GTPase. Because activation of Ras induced by growth factor stimulation likely requires the localization of mSos1 to the plasma membrane, we have investigated the possibility that the PH domain of mSos1 might mediate an interaction of mSos1 with phospholipid membranes. A glutathione S-transferase fusion protein containing the pleckstrin homology (PH) domain of mSos1 bound specifically and tightly to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) with a Kd of 1.8 +/- 0.4 microM. This interaction was saturable and was competed away with the soluble head group of PI(4,5)P2, inositol 1,4, 5-triphosphate. Substitution of Arg452 within the PH domain with Ala had only a slight effect on binding to PI(4,5)P2, whereas substitution of Arg459 severely compromised the ability of the mSos1 PH domain to bind to PI(4,5)P2 containing vesicles. Purified full-length mSos1 and mSos1 complexed with Grb2 were also tested for binding to various phosphoinositol derivatives and demonstrated a specific interaction with PI(4,5)P2, although these interactions were weaker (Kd = approximately 53 and approximately 69 microM, respectively) than that of the PH domain alone. These findings suggest that the PH domain of mSos1 can interact in vitro with phospholipid vesicles containing PI(4,5)P2 and that this interaction is facilitated by the ionic interaction of Arg459 with the negatively charged head group of PI(4,5)P2. The association of the mSos1 PH domain with phospholipid may therefore play a role in regulating the function of this enzyme in vivo.

The Grb2-mSos1 complex binds phosphopeptides with higher affinity than Grb2.

Epidermal growth factor (EGF) stimulation leads to autophosphorylation of the epidermal growth factor receptor (EGFR) and tyrosine phosphorylation of Shc. The Grb2 SH2 domain binds to Tyr1068 of EGFR and Tyr317 of Shc while its SH3 domains bind to mSos1. Therefore, EGF treatment potentially results in the formation of several multimeric signaling complexes, including EGFR-Grb2-mSos1, EGFR-Shc-Grb2-mSos1, and Shc-Grb2-mSos1, linking the receptor to activation of the Ras GTPase. We have purified Grb2, mSos1, and the Grb2-mSos1 complex to high homogeneity, and used these isolated proteins to obtain binding affinities of mSos1 for Grb2 and of either Grb2 or Grb2-mSos1 for phosphotyrosine-containing peptides. mSos1 bound Grb2 with a KD of 0.4 microM; the stoichiometry of the Grb2-mSos1 complex was 1:1. An EGFR-derived phosphopeptide bound Grb2 with a KD of 0.7 microM, whereas the Shc-derived phosphopeptide bound Grb2 with a KD of 0.2 microM. Since Grb2 exists in a stable complex with mSos1, and both proteins can exist in a constitutive complex in unstimulated cells, we performed phosphopeptide binding studies on the Grb2-mSos1 complex to gain a better understanding of binding events in the intact cell. Grb2-mSos1 bound to both EGFR- and Shc-derived phosphopeptides with higher affinities (KD of 0.3 microM and 31 nM, respectively) than Grb2 alone. These findings suggest that the proximity of mSos1 to Grb2 in the complex can influence the interactions of the Grb2 SH2 domain with phosphopeptides and raise the possibility that in the Grb2-mSos1 complex the SH2 and SH3 domains of Grb2 are not independent of each other but may be indirectly linked by mSos1.

The monofunctional chorismate mutase from Bacillus subtilis. Structure determination of chorismate mutase and its complexes with a transition state analog and prephenate, and implications for the mechanism of the enzymatic reaction.

Structures have been determined for chorismate mutase from Bacillus subtilis and of complexes of this enzyme with product and an endo-oxabicyclic transition state analog using multiple isomorphous replacement plus partial structure phase combination and non-crystallographic averaging. In addition to 522 water molecules, the model includes 1380 of the 1524 amino acid residues of the four trimers (each containing 3 x 127 amino acid residues) in the asymmetric unit. Refinement to 1.9 A resolution yields 0.194 for R and r.m.s. deviations from ideal values of 0.014 A for bond lengths and 2.92 degrees for bond angles. The trimer resembles a beta-barrel structure in which a core beta-sheet is surrounded by helices. The structures of the two complexes locate the active sites which are at the interfaces of adjacent pairs of monomers in the trimer. These structures have been refined at 2.2 A to a crystallographic R value of 0.18 and show r.m.s. deviations from ideal values of 0.013 A for bond lengths and 2.84 degrees or 3.05 degrees for bond angles, respectively. The final models have 1398 amino acid residues, nine prephenate molecules and 503 water molecules in the product complex, and 1403 amino acid residues, 12 inhibitor molecules and 530 water molecules in the transition state complex. The active sites of all three of these structures are very similar and provide a structural basis for the biochemical studies that indicate a pericyclic mechanism for conversion of chorismate to prephenate. The absence of reactive catalytic residues on the enzyme, the selective binding of the single reactive conformation of chorismate, the stabilization of the polar transition state, and the possible role of the C-terminal region in "capping" the active site are factors which relate these structures to the million-fold rate enhancement of this reaction.

Crystal structures of the monofunctional chorismate mutase from Bacillus subtilis and its complex with a transition state analog.

We have solved the structure of a chorismate mutase (chorismate pyruvatemutase, EC 5.4.99.5), the 1.9-A crystal structure of the monofunctional enzyme from Bacillus subtilis. The structure determination process was an unusual one, involving 12 monomers of the enzyme in the asymmetric unit. This structure was solved by the multiple isomorphous replacement method with partial structure phase combination and molecular averaging. The final model, which includes 1380 residues and 522 water molecules in an asymmetric unit, has been refined at 1.9 A and the current crystallographic R value is 0.201. The B. subtilis chorismate mutase is a homotrimer, with beta-sheets from each monomer packing to form the core of a pseudo-alpha beta-barrel with helices on the outside of the trimer. In addition, the active sites have been located by using data from a complex with an endo-oxabicyclic inhibitor that mimics the transition state of the reaction. The structure of this complex has been refined to 2.2 A with a current R value of 0.182 for a model that includes 1388 residues, 12 inhibitor molecules, and 530 water molecules in the asymmetric unit. In each trimer, three equivalent active sites are located at the interfaces of two adjacent subunits.

Escherichia coli aspartate carbamoyltransferase: the probing of crystal structure analysis via site-specific mutagenesis.

Crystal structures are known for aspartate carbamoyltransferase (ATCase) in the T and R states, with and without the allosteric activator adenosine triphosphate (ATP) or inhibitor cytidine triphosphate (CTP). Visual inspection of X-ray crystal structures does not provide all of the information necessary for the determination of structure--function relationships in protein molecules. This problem is compounded because the crystalline states of the molecule may introduce effects due to crystal packing, restricted flexibility and less than optimum enzymatic conditions. Therefore, alternative techniques are required to test mechanisms conjectured from three-dimensional crystal structures of proteins. The technique of site-specific mutagenesis allows the researcher to test structure--function models based on three-dimensional structures and to obtain further insight into characteristics of the enzyme. Site-specific mutagenesis has been used to probe residues believed to be critical in the structure and function of ATCase. Selection of residues to be mutated has depended extensively on three-dimensional crystal structures of the enzyme. To date, 48 site-specific mutations at 37 different amino acid sites have been published. Although a total of 118 mutants at 58 different sites has been communicated to our laboratory, only published mutants will be considered in this review. In this paper, we compile for the first time, review, and analyze the site-specific mutants of ATCase. Site-specific mutagenesis of proteins has become a powerful technique in modern-day molecular biology, especially in studying a molecule as large as aspartate carbamoyltransferase. In this review, the role of site-specific mutagenesis of ATCase is discussed and improvements in the analysis are suggested.