Novel Transthyretin Amyloid Fibril Formation Inhibitors: Synthesis, Biological Evaluation, and X-Ray Structural Analysis

Transthyretin (TTR) is one of thirty non-homologous proteins whose misfolding, dissociation, aggregation, and deposition is linked to human amyloid diseases. Previous studies have identified that TTR amyloidogenesis can be inhibited through stabilization of the native tetramer state by small molecule binding to the thyroid hormone sites of TTR. We have evaluated a new series of β-aminoxypropionic acids (compounds 5–21), with a single aromatic moiety (aryl or fluorenyl) linked through a flexible oxime tether to a carboxylic acid. These compounds are structurally distinct from the native ligand thyroxine and typical halogenated biaryl NSAID-like inhibitors to avoid off-target hormonal or anti-inflammatory activity. Based on an in vitro fibril formation assay, five of these compounds showed significant inhibition of TTR amyloidogenesis, with two fluorenyl compounds displaying inhibitor efficacy comparable to the well-known TTR inhibitor diflunisal. Fluorenyl 15 is the most potent compound in this series and importantly does not show off-target anti-inflammatory activity. Crystal structures of the TTR∶inhibitor complexes, in agreement with molecular docking studies, revealed that the aromatic moiety, linked to the sp(2)-hybridized oxime carbon, specifically directed the ligand in either a forward or reverse binding mode. Compared to the aryl family members, the bulkier fluorenyl analogs achieved more extensive interactions with the binding pockets of TTR and demonstrated better inhibitory activity in the fibril formation assay. Preliminary optimization efforts are described that focused on replacement of the C-terminal acid in both the aryl and fluorenyl series (compounds 22–32). The compounds presented here constitute a new class of TTR inhibitors that may hold promise in treating amyloid diseases associated with TTR misfolding

Structural investigation of inhibitor designs targeting 3-dehydroquinate dehydratase from the shikimate pathway of <em>Mycobacterium tuberculosis</em>

International audienceThe shikimate pathway is essential in Mycobacterium tuberculosis and its absence in humans makes the enzymes of this pathway potential drug targets. In this report, we provide structural insights into ligand and inhibitor binding to 3-dehydroquinate dehydratase (dehydroquinase) from Mycobacterium tuberculosis (MtDHQase), the third enzyme of the shikimate pathway. The enzyme has been crystallized in complex with its reaction product, 3-dehydroshikimate, and with six different competitive inhibitors. The inhibitor 2,3-anhydroquinate mimics the flattened enol/enolate reaction intermediate and serves as an anchor molecule for four of the inhibitors investigated. MtDHQase also forms a complex with citrazinic acid, a planar analog of the reaction product. The structure of MtDHQase in complex with a 2,3-anhydroquinate moiety attached to a biaryl group shows that this group extends to an active site subpocket inducing significant structural re-arrangement. The flexible extensions of inhibitors designed to form π-stacking interactions with the catalytic Tyr24 residue have been investigated. The high resolution crystal structures of the MtDHQase complexes provide structural evidence for the role of the loop residues 19-24 in MtDHQase ligand binding and catalytic mechanism and provide rationale for the design and efficacy of inhibitors

Structural Insights into the Mechanism of the Allosteric Transitions of Mycobacterium tuberculosis cAMP Receptor Protein*

The cAMP receptor protein (CRP) from Mycobacterium tuberculosis is a cAMP-responsive global transcriptional regulator, responsible for the regulation of a multitude of diverse proteins. We have determined the crystal structures of the CRP·cAMP and CRP·N6-cAMP derivative-bound forms of the enzyme to 2.2- and 2.3 Å-resolution, respectively, to investigate cAMP-mediated conformational and structural changes. The allosteric switch from the open, inactive conformation to the closed, active conformation begins with a number of changes in the ligand-binding cavity upon cAMP binding. These subtle structural changes and numerous non-bonding interactions between cAMP, the N-domain residues, and the C-domain helices demonstrate that the N-domain hairpin loop acts as a structural mediator of the allosteric switch. Based on the CRP·N6-cAMP crystal structure, binding of N6-cAMP with a bulkier methylphenylethyl extension from the N6 atom stabilizes the cAMP-binding domain, N-domain hairpin, and C-terminal domain in a similar manner as that of the CRP·cAMP structure, maintaining structural integrity within the subunits. However, the bulkier N6 extension of N6-cAMP (in R conformation) is accommodated only in subunit A with minor changes, whereas in subunit B, the N6 extension is in the S conformation hindering the hinge region of the central helix. As a result, the entire N-domain and the C-domain of subunit B integrated by the cAMP portion of this ligand, together tilt away (∼7° tilt) from central helix C, positioning the helix-turn-helix motif in an unfavorable position for the DNA substrate, asymmetrically. Together, these crystal structures demonstrate the mechanism of action of the cAMP molecule and its role in integrating the active CRP structure