Structure of lipoprotein lipase in complex with GPIHBP1.

Lipoprotein lipase (LPL) plays a central role in triglyceride (TG) metabolism. By catalyzing the hydrolysis of TGs present in TG-rich lipoproteins (TRLs), LPL facilitates TG utilization and regulates circulating TG and TRL concentrations. Until very recently, structural information for LPL was limited to homology models, presumably due to the propensity of LPL to unfold and aggregate. By coexpressing LPL with a soluble variant of its accessory protein glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) and with its chaperone protein lipase maturation factor 1 (LMF1), we obtained a stable and homogenous LPL/GPIHBP1 complex that was suitable for structure determination. We report here X-ray crystal structures of human LPL in complex with human GPIHBP1 at 2.5-3.0 Å resolution, including a structure with a novel inhibitor bound to LPL. Binding of the inhibitor resulted in ordering of the LPL lid and lipid-binding regions and thus enabled determination of the first crystal structure of LPL that includes these important regions of the protein. It was assumed for many years that LPL was only active as a homodimer. The structures and additional biochemical data reported here are consistent with a new report that LPL, in complex with GPIHBP1, can be active as a monomeric 1:1 complex. The crystal structures illuminate the structural basis for LPL-mediated TRL lipolysis as well as LPL stabilization and transport by GPIHBP1.

Ligandability Assessment of IL-1ß by Integrated Hit Identification Approaches.

Human interleukin-1ß (IL-1ß) is a pro-inflammatory cytokine that plays a critical role in the regulation of the immune response and the development of various inflammatory diseases. In this publication, we disclose our efforts toward the discovery of IL-1ß binders that interfere with IL-1ß signaling. To this end, several technologies were used in parallel, including fragment-based screening (FBS), DNA-encoded library (DEL) technology, peptide discovery platform (PDP), and virtual screening. The utilization of distinct technologies resulted in the identification of new chemical entities exploiting three different sites on IL-1ß, all of them also inhibiting the interaction with the IL-1R1 receptor. Moreover, we identified lysine 103 of IL-1ß as a target residue suitable for the development of covalent, low-molecular-weight IL-1ß antagonists.

Discovery of a selective and biologically active low-molecular weight antagonist of human interleukin-1ß.

Human interleukin-1ß (hIL-1ß) is a pro-inflammatory cytokine involved in many diseases. While hIL-1ß directed antibodies have shown clinical benefit, an orally available low-molecular weight antagonist is still elusive, limiting the applications of hIL-1ß-directed therapies. Here we describe the discovery of a low-molecular weight hIL-1ß antagonist that blocks the interaction with the IL-1R1 receptor. Starting from a low affinity fragment-based screening hit 1, structure-based optimization resulted in a compound (S)-2 that binds and antagonizes hIL-1ß with single-digit micromolar activity in biophysical, biochemical, and cellular assays. X-ray analysis reveals an allosteric mode of action that involves a hitherto unknown binding site in hIL-1ß encompassing two loops involved in hIL-1R1/hIL-1ß interactions. We show that residues of this binding site are part of a conformationally excited state of the mature cytokine. The compound antagonizes hIL-1ß function in cells, including primary human fibroblasts, demonstrating the relevance of this discovery for future development of hIL-1ß directed therapeutics.

2-Substituted 4-(thio)chromenone 6-O-sulfamates: potent inhibitors of human steroid sulfatase.

Steroid sulfatase (STS) has emerged as a highly attractive target for the therapy of a number of disorders. Starting with the known inhibitor estrone sulfamate (1) as lead compound and with the finding that steroid sulfamates containing a nonaromatic A-ring are inactive, chromen-4-one sulfamates were designed, prepared, and tested for their ability to block human STS. This new class of nonsteroidal inhibitors shows high potency when the sulfamate group and the side chain are situated in diagonally opposite positions (i.e., 2,6- and 3,7-substitution pattern). The highest activity is achieved with fully branched, bulky aliphatic side chains and with thiochromen-4-one as the core element. 2-(1-Adamantyl)-4H-thiochromen-4-on-6-O-sulfamate (6c) is the most potent STS inhibitor discovered so far, and it is about 170-fold superior to 1. As with 1, all chromenone sulfamates are irreversible inhibitors of STS with a biphasic time course of inactivation.

N-Acyl arylsulfonamides as novel, reversible inhibitors of human steroid sulfatase.

Steroid sulfatase (STS) is an attractive target for a range of oestrogen- and androgen-dependent diseases. In search of novel chemotypes of STS inhibitors, we had previously identified nortropinyl-arylsulfonylureas 1; however, while these compounds were good inhibitors of purified STS (lowest K(i)=76 nM), they showed only weak inhibition of STS activity in cells (lowest IC(50) around 2 microM). Extended structure-activity relationship studies involving modification of the phenylacetyl side chain and replacement of the nortropine element by simpler scaffolds led to the discovery of N-acyl arylsulfonamides, more specifically N-(Boc-piperidine-4-carbonyl)-benzenesulfonamides, as STS inhibitors, some of which exhibit improved cellular potency (best IC(50)=270 nM).

Nortropinyl-arylsulfonylureas as novel, reversible inhibitors of human steroid sulfatase.

Steroid sulfatase (STS) has emerged as an attractive target for a range of estrogen- and androgen-dependent diseases. Searching for novel chemotypes as STS inhibitors, we identified nortropinyl-arylsulfonylurea 3 as a hit from high-throughput screening. A series of analogues was prepared in order to explore the essential structural elements for STS inhibition, and first structure-activity relationships were established. Mechanistic investigations revealed that the compounds are reversible, competitive inhibitors of STS.