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.
Here, we describe a method that offers a unique way to engineer plasmids with precision but without digestion using restriction enzymes for the insertion of DNA. The method allows the insertion of PCR fragments in between any two nucleotides within a target plasmid. The only requirement is that the amplified fragments must be embedded between DNA sequences homologous to the site in which the integration is planned. This method is an adaptation of the QuikChange Site-Directed Mutagenesis protocol. It is simpler than the existing cloning strategies and is suitable for multiparallel constructions of new plasmids. We have demonstrated its utility by constructing plasmids in which we have successfully integrated PCR fragments up to 1117 bp.
Bisphosphonates are potent inhibitors of farnesyl pyrophosphate synthase (FPPS) and are highly efficacious in the treatment of bone diseases such as osteoporosis, Paget's 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.
The crystal structure of the ligand binding domain (LBD) of the estrogen-related receptor ␣ (ERR␣, NR3B1) complexed with a coactivator peptide from peroxisome proliferator-activated receptor coactivator-1␣ (PGC-1␣) reveals a transcriptionally active conformation in the absence of a ligand. This is the first x-ray structure of ERR␣ LBD, solved to a resolution of 2.5 Å, and the first structure of a PGC-1␣ complex. The putative ligand binding pocket (LBP) of ERR␣ is almost completely occupied by side chains, in particular with the bulky side chain of Phe 328 (corresponding to Ala 272 in ERR␥ and Ala 350 in estrogen receptor ␣). Therefore, a ligand of a size equivalent to more than ϳ4 carbon atoms could only bind in the LBP, if ERR␣ would undergo a major conformational change (in particular the ligand would displace H12 from its agonist position). The x-ray structure thus provides strong evidence for ligand-independent transcriptional activation by ERR␣. The interactions of PGC-1␣ with ERR␣ also reveal for the first time the atomic details of how a coactivator peptide containing an inverted LXXLL motif (namely a LLXYL motif) binds to a LBD. In addition, we show that a PGC-1␣ peptide containing this nuclear box motif from the L3 site binds ERR␣ LBD with a higher affinity than a peptide containing a steroid receptor coactivator-1 motif and that the affinity is further enhanced when all three leucine-rich regions of PGC-1␣ are present.Nuclear hormone receptors (NRs) 1 are transcription factors that control essential developmental and physiological pathways (1). Although the transcriptional activity of NRs is often regulated by specific ligands, several members of the superfamily have no known natural ligands and are therefore referred to as orphan NRs (2). Estrogen-related receptor ␣ (ERR␣; NR3B1) was the first orphan NR to be identified on the basis of its similarity with estrogen receptor ␣ (ER␣; NR3A1) (3). ERR␣ and its relatives ERR (NR3B2) and ERR␥ (NR3B3) form a small family of orphan NRs that are evolutionarily related to the estrogen receptors ER␣ and ER. ERRs preferentially bind to DNA sites composed of a single half-site preceded by three nucleotides with the consensus sequence TNAAGGTCA, referred to as an ERR response element. It has been shown that ERR␣ also efficiently binds to estrogen response elements and that these receptors share common target genes (4). This observation was further supported by studies demonstrating cross-talk between the ER and ERR pathways (reviewed in Ref. 5). The most striking feature observed in the phenotype of mice lacking ERR␣ is their resistance to high fat diet-induced obesity and the impaired activity of enzymes implicated in lipid metabolism. This finding led to the hypothesis that ERR␣ could be implicated in obesity or metabolic diseases (6). A function of ERR␣ on bone metabolism has also been suggested (7,8). Finally, recent publications show that ERR␣ and ERR␥ are associated with biomarkers of breast cancer and further emphasize the importance of ER-ERR cross-talk (9...
The retinoic acid-related orphan receptor alpha (RORalpha) is an orphan member of the subfamily 1 of nuclear hormone receptors. No X-ray structure of RORalpha has been described so far, and no ligand has been identified. We describe the first crystal structure of the ligand binding domain (LBD) of RORalpha, 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-3beta-ol (cholesterol). Moreover, RORalpha transcriptional activity could be modulated by changes in intracellular cholesterol level or mutation of residues involved in cholesterol binding. These findings suggest that RORalpha could play a key role in the regulation of cholesterol homeostasis and thus represents an important drug target in cholesterol-related diseases.
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