Probing chelation motifs in HIV integrase inhibitors.

A series of HIV integrase (HIV-1 IN) inhibitors were synthesized to evaluate the role of the metal-binding group (MBG) in this class of metalloenzyme inhibitors. A total of 21 different raltegravir-chelator derivative (RCD) compounds were prepared that differed only in the nature of the MBG. These IN strand-transfer inhibitors (INSTIs) were evaluated in vitro in cell-free enzyme activity assays, and the in vitro results were further validated in cell culture experiments. All of the active compounds showed selective inhibition of the strand-transfer reaction over 3'-processing, suggesting a common mode of action with raltegravir. The results of the in vitro activity suggest that the nature of the MBG donor atoms, the overall MBG structure, and the specific arrangement of the MBG donor atom triad are essential for obtaining maximal HIV-1 IN inhibition. At least two compounds (RCD-4, RCD-5) containing a hydroxypyrone MBG were found to display superior strand-transfer inhibition when compared to an abbreviated analogue of raltegravir (RCD-1). By isolating and examining the role of the MBG in a series of INSTIs, we have identified a scaffold (hydroxypyrones) that may provide access to a unique class of HIV-1 IN inhibitors, and may help overcome rising raltegravir resistance.

Development of a high-throughput screen and its use in the discovery of Streptococcus pneumoniae immunoglobulin A1 protease inhibitors.

Streptococcus pneumoniae relies on a number of virulence factors, including immunoglobulin A1 protease (IgA1P), a Zn(2+) metalloprotease produced on the extracellular surface of the bacteria, to promote pathogenic colonization. IgA1P exhibits a unique function, in that it catalyzes the proteolysis of human IgA1 at its hinge region to leave the bacterial cell surface masked by IgA1 Fab, enabling the bacteria to evade the host's immune system and adhere to host epithelial cells to promote colonization. Thus, S. pneumoniae IgA1P has emerged as a promising antibacterial target; however, the lack of an appropriate screening assay has limited the investigation of this metalloprotease virulence factor. Relying on electrostatics-mediated AuNP aggregation, we have designed a promising high-throughput colorimetric assay for IgA1P. By using this assay, we have uncovered inhibitors of the enzyme that should be useful in deciphering its role in pneumococcal colonization and virulence.

Antagonism of a zinc metalloprotease using a unique metal-chelating scaffold: tropolones as inhibitors of P. aeruginosa elastase.

Tropolone emerged from the screening of a chelator fragment library (CFL) as an inhibitor of the Zn(2+)-dependent virulence factor, Pseudomonas aeruginosa elastase (LasB). Based on this initial hit, a series of substituted tropolone-based LasB inhibitors was prepared, and a compound displaying potent activity in vitro and in a bacterial swarming assay was identified. Importantly, this inhibitor was found to be specific for LasB over other metalloenzymes, validating the usage of tropolone as a viable scaffold for identifying first-in-class LasB inhibitors.

3-Hydroxy-1-alkyl-2-methylpyridine-4(1H)-thiones: Inhibition of the Pseudomonas aeruginosa Virulence Factor LasB.

Bacterial resistance coupled to our current arsenal of antibiotics presents us with a growing threat to public health, thus warranting the exploration of alternative antibacterial strategies. In particular, the targeting of virulence factors has been regarded as a "second generation" antibiotic approach. In Pseudomonas aeruginosa, a Zn(2+) metalloprotease virulence factor, LasB or P. aeruginosa elastase, has been implicated in the development of P. aeruginosa-related keratitis, pneumonia and burn infection. Moreover, the enzyme also plays a critical role in swarming and biofilm formation, both of which are processes that have been linked to antibiotic resistance. To further validate the importance of LasB in P. aeruginosa infection, we describe our efforts toward the discovery of non-peptidic small molecule inhibitors of LasB. Using identified compounds, we have confirmed the role that LasB plays in P. aeruginosa swarming and demonstrate the potential for LasB-targeted small molecules in studying antimicrobial resistant P. aeruginosa phenotypes.

Identifying chelators for metalloprotein inhibitors using a fragment-based approach.

Fragment-based lead design (FBLD) has been used to identify new metal-binding groups for metalloenzyme inhibitors. When screened at 1 mM, a chelator fragment library (CFL-1.1) of 96 compounds produced hit rates ranging from 29% to 43% for five matrix metalloproteases (MMPs), 24% for anthrax lethal factor (LF), 49% for 5-lipoxygenase (5-LO), and 60% for tyrosinase (TY). The ligand efficiencies (LE) of the fragment hits are excellent, in the range of 0.4-0.8 kcal/mol. The MMP enzymes all generally elicit the same chelators as hits from CFL-1.1; however, the chelator fragments that inhibit structurally unrelated metalloenzymes (LF, 5-LO, TY) vary considerably. To develop more advanced hits, one hit from CFL-1.1, 8-hydroxyquinoline, was elaborated at four different positions around the ring system to generate new fragments. 8-Hydroxyquinoline fragments substituted at either the 5- or 7-positions gave potent hits against MMP-2, with IC(50) values in the low micromolar range. The 8-hydroxyquinoline represents a promising new chelator scaffold for the development of MMP inhibitors that was discovered by use of a metalloprotein-focused chelator fragment library.