Novel Human Aminopeptidase N Inhibitors: Discovery and Optimization of Subsite Binding Interactions

Abstract

Aminopeptidase N (APN/CD13) is a zinc-dependent M1 aminopeptidase that contributes to cancer progression by promoting angiogenesis, metastasis, and tumor invasion. We have previously identified hydroxamic acid-containing analogues that are potent inhibitors of the APN homologue from the malarial parasite Plasmodium falciparum M1 aminopeptidase (PfA-M1). Herein, we describe the rationale that underpins the repurposing of PfA-M1 inhibitors as novel APN inhibitors. A series of novel hydroxamic acid analogues were developed using a structure-based design approach and evaluated their inhibition activities against APN. N-(2-(Hydroxyamino)-2-oxo-1-(3′,4′,5′-trifluoro-[1,1′-biphenyl]-4-yl)ethyl)-4-(methylsulfonamido)benzamide (6ad) proved to be an extremely potent inhibitor of APN activity in vitro, selective against other zinc-dependent enzymes such as matrix metalloproteases, and possessed limited cytotoxicity against Ad293 cells and favorable physicochemical and metabolic stability properties. The combined results indicate that compound 6ad may be a useful lead for the development of anticancer agents.

Phosphonic Acid Analogs of Fluorophenylalanines as Inhibitors of Human and Porcine Aminopeptidases N: Validation of the Importance of the Substitution of the Aromatic Ring

“…Critical relevance of its activity for cancer progression caused intensive studies to be undertaken to develop new drugs directed towards this enzyme, including enzyme inhibitors and APN-targeted carrier constructs [15,16]. As a consequence, a variety of APN inhibitors have been developed [17][18][19][20][21][22][23].…”

A Structure−Activity Relationship Study of Novel Hydroxamic Acid Inhibitors around the S1 Subsite of Human Aminopeptidase N

“…A set of target compounds were designed to investigate the binding interactions at the S1 pocket of APN as well as to achieve favourable aqueous solubility (Figure 2). According to the previous molecular modelling studies, the 3,4,5‐trifluorophenyl ring formed various hydrophobic interactions, creating a stable binding site for the inhibitors within the S1 subsite of APN [26] . Therefore, the complete removal of the trifluorophenyl group would allow us to investigate if the biphenyl system is essential for obtaining potency against APN.…”

“…The molecular sizes of pyridine and pyrimidine are comparable to that of benzene. The designed analogues would retain the biphenyl core scaffold so that the compounds could engage in π‐stacking hydrophobic interactions with the phenylalanine residues of domain IV as described in earlier studies [26] . Introducing additional polar atoms into the molecule increases the hydrogen bonding capacity, which may also enhance binding interactions with APN.…”

“…We previously synthesised a series of hydroxamic acid analogues to optimise the binding interactions around and beyond the S1′ subsite of APN. We discovered that the sulfonamide 3 showed the most potent inhibition against APN ( K i (app) =4.8 nM), which was a 500‐fold improved activity compared to bestatin ( K i (app) =2.4 μM) [26] . Despite its potent activity and promising pharmacokinetic properties, compound 3 possessed sub‐optimal aqueous solubility (12.5–25 μg/mL in pH 2.0 and 6.0) [26] which could potentially lead to a limited absorption and low efficacy in the cellular environment.…”

IRAP Inhibitors: M1-Aminopeptidase Family Inspiration

“…For the former, the replacement of peptide bonds with ureido equivalents in the hydroxamic acid series led to some potent APN inhibitors such as 15. In the latter case, another series of hydroxamate based inhibitors exemplified by 16 (Ki 4.5 nM) were described by Lee J. et al (2019). These molecules have no obvious peptide character and may offer improved opportunity for achieving selectivity.…”