Martin Geiser

X-Ray Structure of the hRORα LBD at 1.63 Å: Structural and Functional Data that Cholesterol or a Cholesterol Derivative Is the Natural Ligand of RORα

Abstract The retinoic acid-related orphan receptor α (RORα) is an orphan member of the subfamily 1 of nuclear hormone receptors. No X-ray structure of RORα has been described so far, and no ligand has been identified. We describe the first crystal structure of the ligand binding domain (LBD) of RORα, at 1.63 A resolution. This structure revealed a ligand present in the ligand binding pocket (LBP), which was identified by X-ray crystallography as cholest-5-en-3β-ol (cholesterol). Moreover, RORα transcriptional activity could be modulated by changes in intracellular cholesterol level or mutation of residues involved in cholesterol binding. These findings suggest that RORα could play a key role in the regulation of cholesterol homeostasis and thus represents an important drug target in cholesterol-related diseases.

Structural Basis for the Exceptional in vivo Efficacy of Bisphosphonate Drugs

To understand the structural basis for bisphosphonate therapy of bone diseases, we solved the crystal structures of human farnesyl pyrophosphate synthase (FPPS) in its unliganded state, in complex with the nitrogen‐containing bisphosphonate (N‐BP) drugs zoledronate, pamidronate, alendronate, and ibandronate, and in the ternary complex with zoledronate and the substrate isopentenyl pyrophosphate (IPP). By revealing three structural snapshots of the enzyme catalytic cycle, each associated with a distinct conformational state, and details about the interactions with N‐BPs, these structures provide a novel understanding of the mechanism of FPPS catalysis and inhibition. In particular, the accumulating substrate, IPP, was found to bind to and stabilize the FPPS–N‐BP complexes rather than to compete with and displace the N‐BP inhibitor. Stabilization of the FPPS–N‐BP complex through IPP binding is supported by differential scanning calorimetry analyses of a set of representative N‐BPs. Among other factors such as high binding affinity for bone mineral, this particular mode of FPPS inhibition contributes to the exceptional in vivo efficacy of N‐BP drugs. Moreover, our data form the basis for structure‐guided design of optimized N‐BPs with improved pharmacological properties.

The hypervariable region in the genes coding for entomopathogenic crystal proteins of Bacillus thuringiensis: nucleotide sequence of the kurhd1 gene of subsp. kurstaki HD1

One of the genes for the entomophatogenic crystal protein of Bacillus thuringiensis (subsp. kurstaki strain HD1) has been cloned in Escherichia coli, and its nucleotide sequence determined completely. The gene is contained within a 4360-bp-long HpaI-PstI DNA restriction fragment and codes for a polypeptide of 1,155 amino acid residues. The protoxin protein has a predicted Mr of 130,625. The E. coli-derived protoxin gene product is biologically active against Heliothis virescens larvae in a biotest assay. Extensive computer comparisons with other published B. thuringiensis subsp. kurstaki strains HD1, HD73, and B. thuringiensis subsp. sotto gene sequences reveal hypervariable regions in the first half of the protoxin coding sequence. These regions are responsible for the biological activity of the protein product of the cloned gene, and may explain the different biological activities of these different protoxins.

Allosteric non-bisphosphonate FPPS inhibitors identified by fragment-based discovery.

Bisphosphonates are potent inhibitors of farnesyl pyrophosphate synthase (FPPS) and are highly efficacious in the treatment of bone diseases such as osteoporosis, Pagets disease and tumor-induced osteolysis. In addition, the potential for direct antitumor effects has been postulated on the basis of in vitro and in vivo studies and has recently been demonstrated clinically in early breast cancer patients treated with the potent bisphosphonate zoledronic acid. However, the high affinity of bisphosphonates for bone mineral seems suboptimal for the direct treatment of soft-tissue tumors. Here we report the discovery of the first potent non-bisphosphonate FPPS inhibitors. These new inhibitors bind to a previously unknown allosteric site on FPPS, which was identified by fragment-based approaches using NMR and X-ray crystallography. This allosteric and druggable pocket allows the development of a new generation of FPPS inhibitors that are optimized for direct antitumor effects in soft tissue.

Discovery of 3-(1H-indol-3-yl)-4-[2-(4-methylpiperazin-1-yl)quinazolin-4-yl]pyrrole-2,5-dione (AEB071), a potent and selective inhibitor of protein kinase C isotypes

A series of novel maleimide-based inhibitors of protein kinase C (PKC) were designed, synthesized, and evaluated. AEB071 (1) was found to be a potent, selective inhibitor of classical and novel PKC isotypes. 1 is a highly efficient immunomodulator, acting via inhibition of early T cell activation. The binding mode of maleimides to PKCs, proposed by molecular modeling, was confirmed by X-ray analysis of 1 bound in the active site of PKCalpha.

Crystal Structures of Human MdmX (HdmX) in Complex with p53 Peptide Analogues Reveal Surprising Conformational Changes

p53 tumor suppressor activity is negatively regulated through binding to the oncogenic proteins Hdm2 and HdmX. The p53 residues Leu26, Trp23, and Phe19 are crucial to mediate these interactions. Inhibiting p53 binding to both Hdm2 and HdmX should be a promising clinical approach to reactivate p53 in the cancer setting, but previous studies have suggested that the discovery of dual Hdm2/HdmX inhibitors will be difficult. We have determined the crystal structures at 1.3 Å of the N-terminal domain of HdmX bound to two p53 peptidomimetics without and with a 6-chlorine substituent on the indole (which binds in the same subpocket as Trp23 of p53). The latter compound is the most potent peptide-based antagonist of the p53-Hdm2 interaction yet to be described. The x-ray structures revealed surprising conformational changes of the binding cleft of HdmX, including an “open conformation” of Tyr99 and unexpected “cross-talk” between the Trp and Leu pockets. Notably, the 6-chloro p53 peptidomimetic bound with high affinity to both HdmX and Hdm2 (Kd values of 36 and 7 nm, respectively). Our results suggest that the development of potent dual inhibitors for HdmX and Hdm2 should be feasible. They also reveal possible conformational states of HdmX, which should lead to a better prediction of its interactions with potential biological partners.

Efficient transformation of Bacillus thuringiensis and B. cereus via electroporation: transformation of acrystalliferous strains with a cloned delta-endotoxin gene.

SummaryElectroporation was used as a method to transform intact cells of Bacillus thuringiensis and B. cereus. With our optimized method a range of plasmid vectors could be transformed into strains of B. thuringiensis at frequencies of up to 107 transformants/μg DNA. This high frequency allows cloning experiments to be bone directly in B. thuringiensis. A bifunctional vector capable of replicating in Escherichia coli and in Bacillus spp. was constructed. The kurhd1 protoxin gene was cloned into this shuttle vector to produce plasmid pXI93, then transformed into B. thuringiensis HDl cryB and B. cereus 569K. The cloned protoxin gene was expressed in sporulating cultures of both strain HD1 cryB (pXI93) and 569K (pXI93), producing crystal protein active in biotests against larvae of Heliothis virescens. This demonstrates the usefulness of the electroporation method for the introduction of cloned toxin genes, in either their native or modified form, into a variety of host strains.

High-level expression in insect cells and purification of secreted monomeric single-chain Fv antibodies

We have constructed a recombinant baculovirus encoding an anti-(phenyl-oxazolone) single-chain Fv antibody (anti-phOx-scFv) fused to the baculovirus GP67 secretion signal sequence, 6 liters of Sf9 insect cells were infected with this virus at a multiplicity of infection of one and cultured in a bioreactor for 72 h. The dialyzed supernatant was subjected to cation exchange chromatography at pH 6.0 followed by size exclusion chromatography on a Sephadex G100 superfine matrix. This rapid protocol resulted in the isolation of monomeric scFv with a purity of greater than 98%. The final yield was 32 mg/l (10(9) cells/l). Partial amino-terminal sequencing revealed that the GP67 signal sequence was completely removed upon secretion. The dissociation constant of the scFv monomers is about 1 x 10(-4) M. By competitive ELISA scFv dimers yielded a half maximum inhibitory concentration of 3.4 x 10(-7 M which matches the earlier measured Kd for the anti-phOx-scFv (3.2-5.3 x 10-7 M. Marks et al. (1991) J. Mol. Biol. 222, 581-597: Marks et al. (1992) Bio/Technology 10, 779-783). This method is readily scaled up for the preparation of scFv antibodies in high yield and purity obviating any affinity chromatography and/or refolding steps by exploitation of insect cell expression as an efficient alternative to E. coli expression.

Evaluation of antibodies fused to minor coat protein III and major coat protein VIII of bacteriophage M 13

A gene coding for an anti-(2-phenyl-5-oxazolone) single-chain Fv antibody (Ab) fragment (anti-phOx scFv) was cloned in-frame into phagemid vectors upstream from genes encoding (i) the wild-type (wt) minor coat protein (cp) III of the filamentous bacteriophage M13 of Escherichia coli, (ii) a truncated version of cpIII (amino acid (aa) positions 198-406), (iii) the wt major cpVIII, or (iv) a hybrid of interleukin-1 beta (IL-1 beta; aa 10-152) and wt cpVIII. Recombinant (re-) phage obtained by phagemid rescue were examined for the efficiency of displaying these various anti-phOx scFv::cp hybrids with commercially available anti-M13 enzyme-linked immunosorbent assays (ELISA), by immunoblotting with an anti-c-myc Ab, and by selection experiments. We found that the highest ELISA signals were obtained with the cpIII constructs and also that more immunoreactive material was detected by blotting than with Ab::cpVIII fusions. Consequently, more scFv::cpIII than scFv::cpVIII phage could be recovered in micropanning experiments with the antigen phOx as target.

Crystal structure of human estrogen-related receptor alpha in complex with a synthetic inverse agonist reveals its novel molecular mechanism.

Inverse agonists of the constitutively active human estrogen-related receptorα (ERRα, NR3B1) are of potential interest for several disease indications (e.g. breast cancer, metabolic diseases, or osteoporosis). ERRα is constitutively active, because its ligand binding pocket (LBP) is practically filled with side chains (in particular with Phe328, which is replaced by Ala in ERRβ and ERRγ). We present here the crystal structure of the ligand binding domain of ERRα (containing the mutation C325S) in complex with the inverse agonist cyclohexylmethyl-(1-p-tolyl-1H-indol-3-ylmethyl)-amine (compound 1a), to a resolution of 2.3Å. The structure reveals the dramatic multiple conformational changes in the LBP, which create the necessary space for the ligand. As a consequence of the new side chain conformation of Phe328 (on helix H3), Phe510(H12) has to move away, and thus the activation helix H12 is displaced from its agonist position. This is a novel mechanism of H12 inactivation, different from ERRγ, estrogen receptor (ER) α, and ERβ. H12 binds (with a surprising binding mode) in the coactivator groove of its ligand binding domain, at a similar place as a coactivator peptide. This is in contrast to ERRγ but resembles the situation for ERα (raloxifene or 4-hydroxytamoxifen complexes). Our results explain the novel molecular mechanism of an inverse agonist for ERRα and provide the basis for rational drug design to obtain isotype-specific inverse agonists of this potential new drug target. Despite a practically filled LBP, the finding that a suitable ligand can induce an opening of the cavity also has broad implications for other orphan nuclear hormone receptors (e.g. the NGFI-B subfamily).