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.

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 retinoic acid-related orphan receptor ␣ (ROR␣) is an orphan member of the subfamily 1 of nuclear hormone receptors. Our recent structural and functional studies have led to the hypothesis that cholesterol or a cholesterol derivative is the natural ligand of ROR␣. We have now solved the x-ray crystal structure of the ligand binding domain of ROR␣ in complex with cholesterol-3-O-sulfate following a ligand exchange experiment. In contrast to the 3-hydroxyl of cholesterol, the 3-O-sulfate group makes additional direct hydrogen bonds with three residues of the ROR␣ ligand binding domain, namely NH-Gln 289 , NH-Tyr 290 , and NH1-Arg 370 . When compared with the complex with cholesterol, seven well ordered water molecules have been displaced, and the ligand is slightly shifted toward the hydrophilic part of the ligand binding pocket, which is ideally suited for interactions with a sulfate group. These additional ligand-protein interactions result in an increased affinity of cholesterol sulfate when compared with cholesterol, as shown by mass spectrometry analysis done under native conditions and differential scanning calorimetry. Moreover, mutational studies show that the higher binding affinity of cholesterol sulfate translates into an increased transcriptional activity of ROR␣. Our findings suggest that cholesterol sulfate could play a crucial role in the regulation of ROR␣ in vivo.The group of retinoic acid-related orphan nuclear receptors (ROR) 1 is encoded by three different genes (␣, ␤, and ␥) (1). ROR␣ has been implicated in numerous age-related phenotypes such as atherosclerosis, cerebellar atrophy, immunodeficiency, and bone metabolism (2). ROR␣ was still considered an orphan receptor until we recently reported the first crystal structure of the ROR␣ LBD. It had revealed a ligand that was unexpectedly present, namely cholesterol (3). We also had shown that the transcriptional activity of ROR␣ could be modulated by changes in intracellular cholesterol level or mutation of residues involved in cholesterol binding. This has led to the hypothesis that ROR␣ could play a key role in the regulation of cholesterol homeostasis and thus represents an important drug target in cholesterol-related diseases. Despite the relatively high homology between ROR␣ LBD and ROR␤ LBD (63%), cholesterol seems not to be a ligand for the ROR␤ isoform, as reported recently by . This indicates a possible distinct function for ROR␤ and ROR␣. An inspection of the x-ray structure of the complex between ROR␣ LBD and cholesterol had shown that in the hydrophilic part of the LBP, there is space for a substituent attached to the hydroxy group of cholesterol, if water molecules are displaced (3). The presence of three arginines (Arg 292 , Arg 370 , and Arg 367 ) and of two free backbone amide nitrogens (NH-Gln 289 and NH-Tyr 290 ) strongly suggested a negatively charged substituent with at least two hydrogen-bond acceptor functionalities. Docking studies led to the prediction that cholesterol sulfate should have higher affinity than cholest...

GPR15 is an orphan G protein-coupled receptor (GPCR) that is found in lymphocytes. It functions as a co-receptor of simian immunodeficiency virus and HIV-2 and plays a role in the trafficking of T cells to the lamina propria in the colon and to the skin. We describe the purification from porcine colonic tissue extracts of an agonistic ligand for GPR15 and its functional characterization. In humans, this ligand, which we named GPR15L, is encoded by the gene and has some features similar to the CC family of chemokines. was found in some human and mouse epithelia exposed to the environment, such as the colon and skin. In humans, was also abundant in the cervix. In skin, was readily detected after immunologic challenge and in human disease, for example, in psoriatic lesions. Allotransplantation of skin from -deficient mice onto wild-type mice resulted in substantial graft protection, suggesting nonredundant roles for GPR15 and GPR15L in the generation of effector T cell responses. Together, these data identify a receptor-ligand pair that is required for immune homeostasis at epithelia and whose modulation may represent an alternative approach to treating conditions affecting the skin such as psoriasis.

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...