Rickettsia prowazekii methionine aminopeptidase as a promising target for the development of antibacterial agents

Abstract

Methionine aminopeptidase (MetAP) is a class of ubiquitous enzymes essential for the survival of numerous bacterial species. These enzymes are responsible for the cleavage of N-terminal formyl-methionine initiators from nascent proteins to initiate post-translational modifications that are often essential to proper protein function. Thus, inhibition of MetAP activity has been implicated as a novel antibacterial target. We tested this idea in the present study by targeting the MetAP enzyme in the obligate intracellular pathogen Rickettsia prowazekii. We first identified potent RpMetAP inhibitory species by employing an in vitro enzymatic activity assay. The molecular docking program AutoDock was then utilized to compare published crystal structures of inhibited MetAP species to docked poses of RpMetAP. Based on these in silico and in vitro screens, a subset of 23 compounds was tested for inhibition of R. prowazekii growth in a pulmonary vascular endothelial cell (EC) culture infection model system. All compounds were tested over concentration ranges that were determined to be non-toxic to the ECs and 10 of the 23 compounds displayed substantial inhibition of R. prowazekii growth. These data highlight the therapeutic potential for inhibiting RpMetAP as a novel antimicrobial strategy and set the stage for future studies in pre-clinical animal models of infection.

<p>Pre-Clinical Pharmacokinetics, Tissue Distribution and Physicochemical Studies of CLBQ14, a Novel Methionine Aminopeptidase Inhibitor for the Treatment of Infectious Diseases</p>

“…The metalloprotease, methionine amino peptidase (MetAP), has been reported as a novel therapeutic target for infectious diseases. [3][4][5][6][7][8][9][10][11] It has been validated as a target for various infectious diseases caused by methicillinresistant Staphylococcus aureus and Escherichia coli, 3 Mycobacterium tuberculosis (Mtb), [4][5][6][7] Cryptosporidium parvum, 8 Plasmodium falciparum, 9 Leishmania donovani, 10 Enterococcus faecalis, 11,12 Rickettsia prowazekii, 13 and Acinetobacter baumannii. 14 It has also been investigated as a potential therapeutic target for several other diseases including cancer and rheumatoid arthritis.…”

The identification of inhibitory compounds of Rickettsia prowazekii methionine aminopeptidase for antibacterial applications

“…Because R. prowazekii is an obligate intracellular pathogen, the parasite cannot survive for extended periods outside of a host. Consequently, screening campaigns targeting R. prowazekii must therefore be performed within host cells, 21 affording the bacteria an additional resistance mechanism; the host cells must first absorb the compounds, which must then be absorbed by the bacteria. For this reason, R. prowazekii is resistant to a wide number of commercially available antibiotics and few antibiotics are approved to treat this infection.…”

“…For this reason, R. prowazekii is resistant to a wide number of commercially available antibiotics and few antibiotics are approved to treat this infection. 19 Thus, Rp MetAP was chosen as the enzymatic target and was expressed and purified according to our previously published methods with slight modifications 21–25 (see Supplementary Content). Additionally, the choice of the metal cofactor to be utilized in the enzymatic activity assay is important, as inhibitory values are dependent upon cofactor identity.…”

“…Here, Mn(II) was used for our enzyme assays as it is the suggested native cofactor in human isoforms 11 and is the cofactor present in available crystal structures of Rp MetAP. 21…”

“…With the enzyme in hand, enzymatic activity was monitored via a fluorescence-based activity assay where methionine-amino-methylcoumarin (Met-AMC) was utilized as the substrate. 18,21 Enzymatic activity cleaves the peptide bond of the Met-AMC substrate, resulting in free AMC, which is fluorescent (excitation: 360 nm; emission: 460 nm) (Scheme 1). Because only the product of enzymatic activity is fluorescent, an increase in fluorescence over time is directly related to enzyme activity.…”