MrgX2 is a promiscuous receptor for basic peptides causing mast cell pseudo-allergic and anaphylactoid reactions.

Activation of MrgX2, an orphan G protein-coupled receptor expressed on mast cells, leads to degranulation and histamine release. Human MrgX2 binds promiscuously to structurally diverse peptides and small molecules that tend to have basic properties (basic secretagogues), resulting in acute histamine-like adverse drug reactions of injected therapeutic agents. We set out to identify MrgX2 orthologues from other mammalian species used in nonclinical stages of drug development. Previously, the only known orthologue of human MrgX2 was from mouse, encoded by Mrgprb2. MrgX2 genes of rat, dog (beagle), minipig, pig, and Rhesus and cynomolgus monkey were identified by bioinformatic approaches and verified by their ability to mediate calcium mobilization in transfected cells in response to the classical MrgX2 agonist, compound 48/80. The peptide GSK3212448 is an inhibitor of the PRC2 epigenetic regulator that caused profound anaphylactoid reactions upon intravenous infusion to rat. We showed GSK3212448 to be a potent MrgX2 agonist particularly at rat MrgX2. We screened sets of drug-like molecules and peptides to confirm the highly promiscuous nature of MrgX2. Approximately 20% of drug-like molecules activated MrgX2 (pEC50 ranging from 4.5 to 6), with the principle determinant being basicity. All peptides tested of net charge +3 or greater exhibited agonist activity, including the cell penetrating peptides polyarginine (acetyl-Arg9-amide) and TAT (49-60), a fragment of HIV-1 TAT protein. Finally, we showed that the glycopeptide antibiotic vancomycin, which is associated with clinical pseudo-allergic reactions known as red man syndrome, is an agonist of MrgX2.

A Qualified Success: Discovery of a New Series of ATAD2 Bromodomain Inhibitors with a Novel Binding Mode Using High-Throughput Screening and Hit Qualification.

The bromodomain of ATAD2 has proved to be one of the least-tractable proteins within this target class. Here, we describe the discovery of a new class of inhibitors by high-throughput screening and show how the difficulties encountered in establishing a screening triage capable of finding progressible hits were overcome by data-driven optimization. Despite the prevalence of nonspecific hits and an exceptionally low progressible hit rate (0.001%), our optimized hit qualification strategy employing orthogonal biophysical methods enabled us to identify a single active series. The compounds have a novel ATAD2 binding mode with noncanonical features including the displacement of all conserved water molecules within the active site and a halogen-bonding interaction. In addition to reporting this new series and preliminary structure-activity relationship, we demonstrate the value of diversity screening to complement the knowledge-based approach used in our previous ATAD2 work. We also exemplify tactics that can increase the chance of success when seeking new chemical starting points for novel and less-tractable targets.

Fragment-Based Discovery of Low-Micromolar ATAD2 Bromodomain Inhibitors.

Overexpression of ATAD2 (ATPase family, AAA domain containing 2) has been linked to disease severity and progression in a wide range of cancers, and is implicated in the regulation of several drivers of cancer growth. Little is known of the dependence of these effects upon the ATAD2 bromodomain, which has been categorized as among the least tractable of its class. The absence of any potent, selective inhibitors limits clear understanding of the therapeutic potential of the bromodomain. Here, we describe the discovery of a hit from a fragment-based targeted array. Optimization of this produced the first known micromolar inhibitors of the ATAD2 bromodomain.

Crystal structure of human cytochrome P450 2D6.

Cytochrome P450 2D6 is a heme-containing enzyme that is responsible for the metabolism of at least 20% of known drugs. Substrates of 2D6 typically contain a basic nitrogen and a planar aromatic ring. The crystal structure of human 2D6 has been solved and refined to 3.0A resolution. The structure shows the characteristic P450 fold as seen in other members of the family, with the lengths and orientations of the individual secondary structural elements being very similar to those seen in 2C9. There are, however, several important differences, the most notable involving the F helix, the F-G loop, the B'helix, beta sheet 4, and part of beta sheet 1, all of which are situated on the distal face of the protein. The 2D6 structure has a well defined active site cavity above the heme group, containing many important residues that have been implicated in substrate recognition and binding, including Asp-301, Glu-216, Phe-483, and Phe-120. The crystal structure helps to explain how Asp-301, Glu-216, and Phe-483 can act as substrate binding residues and suggests that the role of Phe-120 is to control the orientation of the aromatic ring found in most substrates with respect to the heme. The structure has been compared with published homology models and has been used to explain much of the reported site-directed mutagenesis data and help understand the metabolism of several compounds.

Potential of real-time measurement of GFP-fusion proteins.

Building on the basic design concepts of Randers-Eichhorn [Biotechnol. Bioeng. 55 (1997) 921], an on-line, real-time robust, steam sterilisable optical sensor for monitoring green fluorescent protein (GFP) has been developed. A general cloning vector for fusion expression proteins was constructed, allowing expression of both GFP and the target protein as a fusion. Cultivations were carried out at the 20l scale with the signal from the sensor being relayed directly to the control system of the bioreactors. The production of GFP was then measured on-line, the signal was interfaced directly with other controlling parameters, thereby allowing the microbial process to be controlled directly based on recombinant protein expression. A positive expression correlation between on-line and off-line data was obtained. Protein accretion measured off-line was quantified using both LC-MS and plate reader assays. The potential of such a sensor for many aspects of process development is considerable and we have developed a working system which allows the optimisation of production conditions, for example, linking pH control directly to the fusion protein. Results are also presented that illustrate GFP does not alter the cultivation characteristics of the target protein when compared to the native construct. Whether GFP expressed as a fusion influences the solubility of the target protein is also discussed.

CCR2: characterization of the antagonist binding site from a combined receptor modeling/mutagenesis approach.

We describe here a classical molecular modeling exercise that was carried out to provide a basis for the design of novel antagonist ligands of the CCR2 receptor. Using a theoretical model of the CCR2 receptor, docking studies were carried out to define plausible binding modes for the various known antagonist ligands, including our own series of indole piperidine compounds. On the basis of these results, a number of site-directed mutations (SDM) were designed that were intended to verify the proposed docking models. From these it was clear that further refinements would be necessary in the model. This was aided by the publication of a crystal structure of bovine rhodopsin, and a new receptor model was built by homology to this structure. This latest model enabled us to define ligand-docking hypotheses that were in complete agreement with the results of the SDM experiments.