We report the X-ray crystal structure of a phosphodiesterase (PDE) that includes both catalytic and regulatory domains. PDE2A (215-900) crystallized as a dimer in which each subunit had an extended organization of regulatory GAF-A and GAF-B and catalytic domains connected by long ␣-helices. The subunits cross at the GAF-B/ catalytic domain linker, and each side of the dimer contains in series the GAF-A and GAF-B of one subunit and the catalytic domain of the other subunit. A dimer interface extends over the entire length of the molecule. The substrate binding pocket of each catalytic domain is occluded by the H-loop. We deduced from comparisons with structures of isolated, ligand-bound catalytic subunits that the H-loop swings out to allow substrate access. However, in dimeric PDE2A (215-900), the H-loops of the two catalytic subunits pack against each other at the dimer interface, necessitating movement of the catalytic subunits to allow for H-loop movement. Comparison of the unliganded GAF-B of PDE2A (215-900) with previous structures of isolated, cGMP-bound GAF domains indicates that cGMP binding induces a significant shift in the GAF-B/catalytic domain linker. We propose that cGMP binding to GAF-B causes movement, through this linker region, of the catalytic domains, such that the H-loops no longer pack at the dimer interface and are, instead, free to swing out to allow substrate access. This increase in substrate access is proposed as the basis for PDE2A activation by cGMP and may be a general mechanism for regulation of all PDEs.cGMP activation ͉ GAF domains ͉ PDE-2A T he cyclic nucleotides cAMP and cGMP are ubiquitous intracellular signaling molecules that mediate a vast array of biological processes throughout the body. The means by which these two molecules participate in diverse functions, in different cell types and within single cells, involves tight regulation of the spatial and temporal residence of their concentrations. The phosphodiesterases (PDEs) are a superfamily of enzymes that metabolically inactivate cAMP and cGMP (1) to play key roles in both these aspects of regulation. The PDEs are modular enzymes characterized by a relatively conserved C-terminal catalytic domain and a more variable N-terminal domain involved in regulation of activity, subcellular localization, and interactions with other proteins. There are 11 PDE gene families, with different families encoded by one to four genes, and further diversity derived from alternative splicing. The PDE families differ broadly in specificity and affinity for cAMP and cGMP. Much has been learned about the molecular bases for these differences from studies of X-ray crystal structures of the catalytic domains of

Monoacylglycerol lipase (MAGL) is the main enzyme responsible for degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the CNS. MAGL catalyzes the conversion of 2-AG to arachidonic acid (AA), a precursor to the proinflammatory eicosannoids such as prostaglandins. Herein we describe highly efficient MAGL inhibitors, identified through a parallel medicinal chemistry approach that highlighted the improved efficiency of azetidine and piperidine-derived carbamates. The discovery and optimization of 3-substituted azetidine carbamate irreversible inhibitors of MAGL were aided by the generation of inhibitor-bound MAGL crystal structures. Compound 6, a highly efficient and selective MAGL inhibitor against recombinant enzyme and in a cellular context, was tested in vivo and shown to elevate central 2-AG levels at a 10 mg/kg dose.

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The COVID-19 pandemic continues to be a public health threat with emerging variants of SARS-CoV-2. Nirmatrelvir (PF-07321332) is a reversible, covalent inhibitor targeting the main protease (Mpro) of SARS-CoV-2 and the active protease inhibitor in PAXLOVID (nirmatrelvir tablets and ritonavir tablets). One of the predominant SARS-CoV-2 variants emerging is the B.1.1.529 Omicron harboring a mutation at amino acid position 132 in the Mpro changing a proline to a histidine (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the Omicron Mpro (P132H) demonstrate that it is catalytically comparable to wildtype and that nirmatrelvir has similar potency against both wildtype and Omicron (P132H) Mpro with Ki of 0.933nM (wildtype) and 0.635nM (P132H) each, respectively. This observation is reinforced by our structural determination of nirmatrelvir bound to the omicron Mpro at 1.63Å resolution. These in vitro data suggest that PAXLOVID has the potential to maintain plasma concentrations of nirmatrelvir many-fold times higher than the amount required to stop the SARS-CoV-2 variant Omicron from replicating in cells.

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins.