Increased amounts of amino acids are essential for cancer cells to support their sustained growth and survival. Therefore, inhibitors of amino acid transporters, such as l-type amino acid transporter 1 (LAT1) have been developed. In this study, a previously reported LAT1-inhibitor (KMH-233) was studied for its hemocompatibility and toxicity towards human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (AoSMCs). Furthermore, the cytotoxic effects against human breast adenocarcinoma cells (MCF-7) and its ability to affect mammalian (or mechanistic) target of rapamycin (mTOR) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling were evaluated. Moreover, the effects of this inhibitor to modulate LAT1 function on the cell surface and the brain amino acid homeostasis were evaluated after intraperitoneal (i.p.) administration of LAT1-inhibitor (23 µmol/kg) in mice. The results showed that LAT1-inhibitor (KMH-233) is hemocompatible at concentrations below 25 µM and it does not affect coagulation in plasma. However, it can reduce the total protein amount of mTOR and NF-κB, resulting in increased apoptosis in LAT1-expressing cancer cells. Most importantly, the inhibitor did not affect mouse brain levels of l-Leu, l-Tyr or l-Trp or modulate the function of LAT1 on the MCF-7 cell surface. Therefore, this inhibitor can be considered as a safe but effective anti-cancer agent. However, due to the compensative mechanism of cancer cells for their increased amino acid demand, this compound is most effective inducing apoptosis when used in combinations with other chemotherapeutics, such as protease inhibitor, bestatin, as demonstrated in this study.

Ferulic acid (FA) is a natural phenolic antioxidant, which can exert also several other beneficial effects to combat neuroinflammation and neurodegenerative diseases, such as Alzheimer’s disease. One of these properties is the inhibition of several enzymes and factors, such as β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1), cyclooxygenases (COXs), lipoxygenases (LOXs), mammalian (or mechanistic) target for rapamycin (mTOR), and transcription factor NF-κB. We have previously synthesized three L-type amino acid transporter 1- (LAT1-) utilizing FA-derivatives with the aim to develop brain-targeted prodrugs of FA. In the present study, the cellular uptake and bioavailability of these FA-derivatives were evaluated in mouse primary astrocytic cell cultures together with their inhibitory effects towards BACE1, COX/LOX, mTOR, NF-κB, acetylcholinesterase (AChE), and oxidative stress. According to the results, all three FA-derivatives were taken up 200–600 times more effectively at 10 μM concentration into the astrocytes than FA, with one derivative having a high intracellular bioavailability (Kp,uu), particularly at low concentrations. Moreover, all of the derivatives were able to inhibit BACE1, COX/LOX, AChE, and oxidative stress measured as decreased cellular lipid peroxidation. Furthermore, one of the derivatives modified the total mTOR amount. Therefore, these derivatives have the potential to act as multifunctional compounds preventing β-amyloid accumulation as well as combating inflammation and reducing oxidative stress in the brain. Thus, this study shows that converting a parent drug into a transporter-utilizing derivative not only may increase its brain and cellular uptake, and bioavailability but can also broaden the spectrum of pharmacological effects elicited by the derivative.

Inherited retinal degenerations (IRDs) are a leading cause of blindness among the population of young people in the developed world. Approximately half of IRDs initially manifest as gradual loss of night vision and visual fields, characteristic of retinitis pigmentosa (RP). Due to challenges in genetic testing, and the large heterogeneity of mutations underlying RP, targeted gene therapies are an impractical largescale solution in the foreseeable future. For this reason, identifying key pathophysiological pathways in IRDs that could be targets for mutation-agnostic and disease-modifying therapies (DMTs) is warranted. In this study, we investigated the retinal proteome of three distinct IRD mouse models, in comparison to sex- and age-matched wild-type mice. Specifically, we used the Pde6β^Rd10 (rd10) and Rho^P23H/WT (P23H) mouse models of autosomal recessive and autosomal dominant RP, respectively, as well as the Rpe65^-/- mouse model of Leber's congenital amaurosis type 2 (LCA2). The mice were housed at two distinct institutions and analyzed using LC-MS in three separate facilities/instruments following data-dependent and data-independent acquisition modes. This cross-institutional and multi-methodological approach signifies the reliability and reproducibility of the results. The large-scale profiling of the retinal proteome, coupled with in vivo electroretinography recordings, provided us with a reliable basis for comparing the disease phenotypes and severity. Despite evident inflammation, cellular stress, and downscaled phototransduction observed consistently across all three models, the underlying pathologies of RP and LCA2 displayed many differences, sharing only four general KEGG pathways. The opposite is true for the two RP models in which we identify remarkable convergence in proteomic phenotype even though the mechanism of primary rod death in rd10 and P23H mice is different. Our data highlights the cAMP and cGMP second-messenger signaling pathways as potential targets for therapeutic intervention. The proteomic data is curated and made publicly available, facilitating the discovery of universal therapeutic targets for RP.

In order to efficiently combat neuroinflammation, it is essential to deliver the anti-inflammatory drugs to their target sites in the brain. Pro-drugs utilizing the L-type amino acid transporter 1 (LAT1) can be transported across the blood-brain barrier (BBB) and the cellular barriers of the brain’s parenchymal cells. In this study, we evaluated, for the first time, the efficacy of LAT1-utilizing prodrugs of ketoprofen (KPF) on cyclooxygenase (COX) enzymes in vitro and prostaglandin E2 production in vivo by using an enzymatic assay and liquid chromatography- tandem mass spectrometry method, respectively. Aliphatic amino acid-conjugated pro-drugs inhibited the peroxidase activity of COX in vitro in their intact form (85% inhibition, IC50 ≈ 1.1 µM and 79%, IC50 ≈ 2.3 µM), which was comparable to KPF (90%, IC50 ≈ 0.9). Thus, these compounds acted more as KPF derivatives rather than pro-drugs. In turn, aromatic amino acid-conjugated pro-drugs behaved differently. The ester pro-drug inhibited the COX peroxidase activity in vitro (90%, IC50 ≈ 0.6 µM) due to its bioconversion to KPF, whereas the amide pro-drug was inactive toward COX enzymes in vitro. However, the amide pro-drug released KPF in the mouse brain in sufficient and effective amounts measured as reduced PGE2 levels.