MD-Based Assessment of Covalent Inhibitors in Noncovalent Association Complexes: Learning from Cathepsin K as a Test Case.

A sufficiently stable noncovalent association complex between a covalent inhibitor and its protein target is regarded as a prerequisite for the formation of a covalent complex. As this transient form can hardly be assessed experimentally, computational modeling is required to probe the suitability of a given ligand at this particular stage. To investigate which criteria should be fulfilled by suitable candidates in a molecular dynamics (MD) assessment, a systematic study was conducted with 20 complexes of cathepsin K, a papain-like cysteine protease of pharmaceutical relevance. The covalent inhibitors in these complexes were converted to their pre-reaction states, and the resulting noncovalent complexes were subjected to MD simulations. The critical distance between the electrophilic and nucleophilic reaction partners was monitored as a potential parameter to assess the suitability for covalent bond formation. Across various warhead types, a distance between 3.6 and 4.0 Å, comparable to the sum of the generalized Born radii of carbon and sulfur, was found to be stably maintained under appropriate conditions. The protonation state of the catalytic dyad and the resulting solvation pattern dramatically affected the noncovalent binding mode and the distance of the warhead to the active site. For several complexes, fluctuations in the orientation of the warhead were observed due to torsional rotations in adjacent bonds. This observation helped to explain the gradual transitions from noncovalent to covalent complexes observed in the crystal structures of three closely related nitrile-based inhibitors. According to comparative simulations conducted for a set of 13 cathepsin S complexes, the overall findings of the study appear to be transferable to related cysteine proteases as targets of covalent inhibitors.

Glycolytic flux control by drugging phosphoglycolate phosphatase.

Targeting the intrinsic metabolism of immune or tumor cells is a therapeutic strategy in autoimmunity, chronic inflammation or cancer. Metabolite repair enzymes may represent an alternative target class for selective metabolic inhibition, but pharmacological tools to test this concept are needed. Here, we demonstrate that phosphoglycolate phosphatase (PGP), a prototypical metabolite repair enzyme in glycolysis, is a pharmacologically actionable target. Using a combination of small molecule screening, protein crystallography, molecular dynamics simulations and NMR metabolomics, we discover and analyze a compound (CP1) that inhibits PGP with high selectivity and submicromolar potency. CP1 locks the phosphatase in a catalytically inactive conformation, dampens glycolytic flux, and phenocopies effects of cellular PGP-deficiency. This study provides key insights into effective and precise PGP targeting, at the same time validating an allosteric approach to control glycolysis that could advance discoveries of innovative therapeutic candidates.

Melatonin- and Ferulic Acid-Based HDAC6 Selective Inhibitors Exhibit Pronounced Immunomodulatory Effects In Vitro and Neuroprotective Effects in a Pharmacological Alzheimer's Disease Mouse Model.

The structures of melatonin and ferulic acid were merged into tertiary amide-based histone deacetylase 6 (HDAC6) inhibitors to develop multi-target-directed inhibitors for neurodegenerative diseases to incorporate antioxidant effects without losing affinity and selectivity at HDAC6. Structure-activity relationships led to compound 10b as a hybrid molecule showing pronounced and selective inhibition of HDAC6 (IC50 = 30.7 nM, > 25-fold selectivity over other subtypes). This compound shows comparable DPPH radical scavenging ability to ferulic acid, comparable ORAC value to melatonin and comparable Cu2+ chelating ability to EDTA. It also lacks neurotoxicity on HT-22 cells, exhibits a pronounced immunomodulatory effect, and is active in vivo showing significantly higher efficacy in an AD mouse model to prevent both Aß25-35-induced spatial working and long-term memory dysfunction at lower dose (0.3 mg/kg) compared to positive control HDAC6 inhibitor ACY1215 and an equimolar mixture of the three entities ACY1215, melatonin and ferulic acid, suggesting potentially disease-modifying properties.

Tacrine-xanomeline and tacrine-iperoxo hybrid ligands: Synthesis and biological evaluation at acetylcholinesterase and M1 muscarinic acetylcholine receptors.

We synthesized a set of new hybrid derivatives (7-C8, 7-C10, 7-C12 and 8-C8, 8-C10, 8-C12), in which a polymethylene spacer chain of variable length connected the pharmacophoric moiety of xanomeline, an M1/M4-preferring orthosteric muscarinic agonist, with that of tacrine, a well-known acetylcholinesterase (AChE) inhibitor able to allosterically modulate muscarinic acetylcholine receptors (mAChRs). When tested in vitro in a colorimetric assay for their ability to inhibit AChE, the new compounds showed higher or similar potency compared to that of tacrine. Docking analyses were performed on the most potent inhibitors in the series (8-C8, 8-C10, 8-C12) to rationalize their experimental inhibitory power against AChE. Next, we evaluated the signaling cascade at M1 mAChRs by exploring the interaction of Gαq-PLC-ß3 proteins through split luciferase assays and the myo-Inositol 1 phosphate (IP1) accumulation in cells. The results were compared with those obtained on the known derivatives 6-C7 and 6-C10, two quite potent AChE inhibitors in which tacrine is linked to iperoxo, an exceptionally potent muscarinic orthosteric activator. Interestingly, we found that 6-C7 and 6-C10 behaved as partial agonists of the M1 mAChR, at variance with hybrids 7-Cn and 8-Cn containing xanomeline as the orthosteric molecular fragment, which were all unable to activate the receptor subtype response.