André Strauss

Targeting Bcr-Abl by combining allosteric with ATP-binding-site inhibitors

In an effort to find new pharmacological modalities to overcome resistance to ATP-binding-site inhibitors of Bcr–Abl, we recently reported the discovery of GNF-2, a selective allosteric Bcr–Abl inhibitor. Here, using solution NMR, X-ray crystallography, mutagenesis and hydrogen exchange mass spectrometry, we show that GNF-2 binds to the myristate-binding site of Abl, leading to changes in the structural dynamics of the ATP-binding site. GNF-5, an analogue of GNF-2 with improved pharmacokinetic properties, when used in combination with the ATP-competitive inhibitors imatinib or nilotinib, suppressed the emergence of resistance mutations in vitro, displayed additive inhibitory activity in biochemical and cellular assays against T315I mutant human Bcr–Abl and displayed in vivo efficacy against this recalcitrant mutant in a murine bone-marrow transplantation model. These results show that therapeutically relevant inhibition of Bcr–Abl activity can be achieved with inhibitors that bind to the myristate-binding site and that combining allosteric and ATP-competitive inhibitors can overcome resistance to either agent alone.

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.

Solution Conformations and Dynamics of ABL Kinase-Inhibitor Complexes Determined by NMR Substantiate the Different Binding Modes of Imatinib/Nilotinib and Dasatinib

Current structural understanding of kinases is largely based on x-ray crystallographic studies, whereas very little data exist on the conformations and dynamics that kinases adopt in the solution state. ABL kinase is an important drug target in the treatment of chronic myelogenous leukemia. Here, we present the first characterization of ABL kinase in complex with three clinical inhibitors (imatinib, nilotinib, and dasatinib) by modern solution NMR techniques. Structural and dynamical results were derived from complete backbone resonance assignments, experimental residual dipolar couplings, and 15N relaxation data. Residual dipolar coupling data on the imatinib and nilotinib complexes show that the activation loop adopts the inactive conformation, whereas the dasatinib complex preserves the active conformation, which does not support contrary predictions based upon molecular modeling. Nanosecond as well as microsecond dynamics can be detected for certain residues in the activation loop in the inactive and active conformation complexes.

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.

Amino–acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells useful for NMR studies

Culture conditions for successful amino–acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells are described. The method was applied to the selective labeling of the catalytic domain of c-Abl kinase with 15N-phenylalanine, 15N-glycine, 15N-tyrosine or 15N-valine. For the essential amino acids phenylalanine, tyrosine and valine high 15N-label incorporation rates of ≥90% and approximately the expected number of resonances in the HSQC spectra were observed, which was not the case for the non-essential amino acid glycine. The method should be applicable to amino-acid-type selective isotope labeling of other recombinant proteins which have not been amenable to NMR analysis.

Binding or bending: distinction of allosteric Abl kinase agonists from antagonists by an NMR-based conformational assay.

Allosteric inhibitors of Bcr-Abl have emerged as a novel therapeutic option for the treatment of CML. Using fragment-based screening, a search for novel Abl inhibitors that bind to the myristate pocket was carried out. Here we show that not all myristate ligands are functional inhibitors, but that the conformational state of C-terminal helix_I is a structural determinant for functional activity. We present an NMR-based conformational assay to monitor the conformation of this crucial helix_I and show that myristate ligands that bend helix_I are functional antagonists, whereas ligands that bind to the myristate pocket but do not induce this conformational change are kinase agonists. Activation of c-Abl by allosteric agonists has been confirmed in a biochemical assay.

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).

A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility

An easy to use and robust approach for amino acid type selective isotope labeling in insect cells is presented. It relies on inexpensive commercial media and can be implemented in laboratories without sophisticated infrastructure. In contrast to previous protocols, where either high protein amounts or high incorporation ratios were obtained, here we achieve both at the same time. By supplementing media with a well considered amount of yeast extract, similar protein amounts as with full media are obtained, without compromising on isotope incorporation. In single and dual amino acid labeling experiments incorporation ratios are consistently ≥90% for all amino acids tested. This enables NMR studies of eukaryotic proteins and their interactions even for proteins with low expression levels. We show applications with human kinases, where protein–ligand interactions are characterized by 2D [15N, 1H]- and [13C, 1H]-HSQC spectra.

Root induction on several Solanaceae species by Agrobacterium rhizogenes and the determination of root tropane alkaloid content.

Using Agrobacterium rhizogenes, roots were induced on explants of 24 different Solanaceae species and established as in vitro cultures. Some of the root clones produced tropane alkaloids at levels similar to roots of the corresponding intact plants and maintained these levels over several passages.