Crystal Structure of Streptococcus pneumoniae N -Acetylglucosamine-1-phosphate Uridyltransferase Bound to Acetyl-coenzyme A Reveals a Novel Active Site Architecture

International audienceThe bifunctional bacterial enzymeN-acetyl-glucosamine-1-phosphate uridyltransferase (GlmU) catalyzes the two-step formation of UDP-GlcNAc, a fundamental precursor in bacterial cell wall biosynthesis. With the emergence of new resistance mechanisms against β-lactam and glycopeptide antibiotics, the biosynthetic pathway of UDP-GlcNAc represents an attractive target for drug design of new antibacterial agents. The crystal structures of Streptococcus pneumoniae GlmU in unbound form, in complex with acetyl-coenzyme A (AcCoA) and in complex with both AcCoA and the end product UDP-GlcNAc, have been determined and refined to 2.3, 2.5, and 1.75 Å, respectively. TheS. pneumoniae GlmU molecule is organized in two separate domains connected via a long α-helical linker and associates as a trimer, with the 50-Å-long left-handed β-helix (LβH) C-terminal domains packed against each other in a parallel fashion and the C-terminal region extended far away from the LβH core and exchanged with the β-helix from a neighboring subunit in the trimer. AcCoA binding induces the formation of a long and narrow tunnel, enclosed between two adjacent LβH domains and the interchanged C-terminal region of the third subunit, giving rise to an original active site architecture at the junction of three subunits

Crystal structures of two human pyrophosphorylase isoforms in complexes with UDPGlc(Gal)NAc: role of the alternatively spliced insert in the enzyme oligomeric assembly and active site architecture

The recently published human genome with its relatively modest number of genes has highlighted the importance of post-transcriptional and post-translational modifications, such as alternative splicing or glycosylation, in generating the complexities of human biology. The human UDP-N-acetylglucosamine (UDPGlcNAc) pyrophosphorylases AGX1 and AGX2, which differ in sequence by an alternatively spliced 17 residue peptide, are key enzymes synthesizing UDPGlcNAc, an essential precursor for protein glycosylation. To better understand the catalytic mechanism of these enzymes and the role of the alternatively spliced segment, we have solved the crystal structures of AGX1 and AGX2 in complexes with UDPGlcNAc (at 1.9 and 2.4 Å resolution, respectively) and UDPGalNAc (at 2.2 and 2.3 Å resolution, respectively). Comparison with known structures classifies AGX1 and AGX2 as two new members of the SpsA-GnT I Core superfamily and, together with mutagenesis analysis, helps identify residues critical for catalysis. Most importantly, our combined structural and biochemical data provide evidence for a change in the oligomeric assembly accompanied by a significant modification of the active site architecture, a result suggesting that the two isoforms generated by alternative splicing may have distinct catalytic properties

Discovery of (2<i>S</i>)‑8-[(3<i>R</i>)‑3-Methylmorpholin-4-yl]-1-(3-methyl-2-oxobutyl)-2-(trifluoromethyl)-3,4-dihydro‑2<i>H</i>‑pyrimido[1,2‑<i>a</i>]pyrimidin-6-one: A Novel Potent and Selective Inhibitor of Vps34 for the Treatment of Solid Tumors

Vps34 (the human class III phosphoinositide 3-kinase) is a lipid kinase involved in vesicle trafficking and autophagy and therefore constitutes an interesting target for cancer treatment. Because of the lack of specific Vps34 kinase inhibitors, we aimed to identify such compounds to further validate the role of this lipid kinase in cancer maintenance and progression. Herein, we report the discovery of a series of tetrahydropyrimido­pyrimidinone derivatives. Starting with hit compound <b>1a</b>, medicinal chemistry optimization led to compound <b>31</b>. This molecule displays potent activity, an exquisite selectivity for Vps34 with excellent properties. The X-ray crystal structure of compound <b>31</b> in human Vps34 illustrates how the unique molecular features of the morpholine synthon bestows selectivity against class I PI3Ks. This molecule exhibits suitable in vivo mouse PK parameters and induces a sustained inhibition of Vps34 upon acute administration. Compound <b>31</b> constitutes an optimized Vps34 inhibitor that could be used to investigate human cancer biology