Hydroxypyridinethione Inhibitors of Human Insulin-Degrading Enzyme.

Insulin-degrading enzyme (IDE) is a human mononuclear Zn2+ -dependent metalloenzyme that is widely regarded as the primary peptidase responsible for insulin degradation. Despite its name, IDE is also critically involved in the hydrolysis of several other disparate peptide hormones, including glucagon, amylin, and the amyloid ß-protein. As such, the study of IDE inhibition is highly relevant to deciphering the role of IDE in conditions such as type-2 diabetes mellitus and Alzheimer disease. There have been few reported IDE inhibitors, and of these, inhibitors that directly target the active-site Zn2+ ion have yet to be fully explored. In an effort to discover new, zinc-targeting inhibitors of IDE, a library of ∼350 metal-binding pharmacophores was screened against IDE, resulting in the identification of 1-hydroxypyridine-2-thione (1,2-HOPTO) as an effective Zn2+ -binding scaffold. Screening a focused library of HOPTO compounds identified 3-sulfonamide derivatives of 1,2-HOPTO as inhibitors of IDE (Ki values of ∼50 µM). Further structure-activity relationship studies yielded several thiophene-sulfonamide HOPTO derivatives with good, broad-spectrum activity against IDE that have the potential to be useful pharmacological tools for future studies of IDE.

Identification of Adenosine Deaminase Inhibitors by Metal-binding Pharmacophore Screening.

Adenosine deaminase (ADA) is a human mononuclear Zn2+ metalloenzyme that converts adenosine to inosine. ADA is a validated drug target for cancer, but there has been little recent work on the development of new therapeutics against this enzyme. The lack of new advancements can be partially attributed to an absence of suitable assays for high-throughput screening (HTS) against ADA. To facilitate more rapid drug discovery efforts for this target, an in vitro assay was developed that utilizes the enzymatic conversion of a visibly emitting adenosine analogue to the corresponding fluorescent inosine analogue by ADA, which can be monitored via fluorescence intensity changes. Utilizing this assay, a library of ∼350 small molecules containing metal-binding pharmacophores (MBPs) was screened in an HTS format to identify new inhibitor scaffolds against ADA. This approach yielded a new metal-binding scaffold with a Ki value of 26±1 µM.

Targeting Metalloenzymes for Therapeutic Intervention.

Metalloenzymes are central to a wide range of essential biological activities, including nucleic acid modification, protein degradation, and many others. The role of metalloenzymes in these processes also makes them central for the progression of many diseases and, as such, makes metalloenzymes attractive targets for therapeutic intervention. Increasing awareness of the role metalloenzymes play in disease and their importance as a class of targets has amplified interest in the development of new strategies to develop inhibitors and ultimately useful drugs. In this Review, we provide a broad overview of several drug discovery efforts focused on metalloenzymes and attempt to map out the current landscape of high-value metalloenzyme targets.

Structure-Activity Relationships in Metal-Binding Pharmacophores for Influenza Endonuclease.

Metalloenzymes represent an important target space for drug discovery. A limitation to the early development of metalloenzyme inhibitors has been the lack of established structure-activity relationships (SARs) for molecules that bind the metal ion cofactor(s) of a metalloenzyme. Herein, we employed a bioinorganic perspective to develop an SAR for inhibition of the metalloenzyme influenza RNA polymerase PAN endonuclease. The identified trends highlight the importance of the electronics of the metal-binding pharmacophore (MBP), in addition to MBP sterics, for achieving improved inhibition and selectivity. By optimization of the MBPs for PAN endonuclease, a class of highly active and selective fragments was developed that displays IC50 values <50 nM. This SAR led to structurally distinct molecules that also displayed IC50 values of ∼10 nM, illustrating the utility of a metal-centric development campaign in generating highly active and selective metalloenzyme inhibitors.

Breathing new life into West Nile virus therapeutics; discovery and study of zafirlukast as an NS2B-NS3 protease inhibitor.

The West Nile virus (WNV) has spread throughout the world causing neuroinvasive diseases with no treatments available. The viral NS2B-NS3 protease is essential for WNV survival and replication in host cells and is a promising drug target. Through an enzymatic screen of the National Institute of Health clinical compound library, we report the discovery of zafirlukast, an FDA approved treatment for asthma, as an inhibitor for the WNV NS2B-NS3 protease. Zafirlukast was determined to inhibit the protease through a mixed mode mechanism with an IC50 value of 32 µM. A structure activity relationship study of zafirlukast revealed the cyclopentyl carbamate and N-aryl sulfonamide as structural elements crucial for NS2B-NS3 protease inhibition. Replacing the cyclopentyl with a phenyl improved inhibition, resulting in an IC50 of 22 µM. Experimental and computational docking analysis support the inhibition model of zafirlukast and analogs binding at an allosteric site on the NS3 protein, thereby disrupting the NS2B cofactor from binding, resulting in protease inhibition.

Isosteres of hydroxypyridinethione as drug-like pharmacophores for metalloenzyme inhibition.

Hydroxypyridinethiones (HOPTOs) are strong ligands for metal ions and potentially useful pharmacophores for inhibiting metalloenzymes relevant to human disease. However, HOPTOs have been sparingly used in drug discovery efforts due, in part, to concerns that this scaffold will act as a promiscuous, non-selective metalloenzyme inhibitor, as well as possess poor pharmacokinetics (PK), which may undermine drug candidates containing this functional group. To advance HOPTOs as a useful pharmacophore for metalloenzyme inhibitors, a library of 22 HOPTO isostere compounds has been synthesized and investigated. This library demonstrates that it is possible to maintain the core metal-binding pharmacophore (MBP) while generating diversity in structure, electronics, and PK properties. This HOPTO library has been screened against a set of four different metalloenzymes, demonstrating that while the same metal-binding donor atoms are maintained, there is a wide range of activity between metalloenzyme targets. Overall, this work shows that HOPTO isosteres are useful MBPs and valuable scaffolds for metalloenzyme inhibitors.

A FRET-based assay for the discovery of West Nile Virus NS2B-NS3 protease inhibitors.

The West Nile Virus (WNV) has been a worldwide epidemic since the early 1990s. Currently there are no therapeutic treatments for WNV infections. One particular avenue of treatment is inhibition of the NS2B-NS3 protease, an enzyme that is crucial for WNV replication. In our effort to increase the number of NS2B-NS3 protease inhibitors, we report a novel FRET-based high throughput assay for the discovery of WNV NS2B-NS3 protease inhibitors. For this assay, a FRET-based peptide substrate was synthesized and kinetically characterized with the NS2B-NS3 protease. The new substrate exhibits a K(m) of 3.35 ± 0.31 µM, a k(cat) of 0.0717 ± 0.0016 s(-1) and a k(cat)/K(m) of 21,400 ± 2000 M(-1) s(-1).