Brain Pharmacokinetics of Non‐Imidazole Biphenyl H3 Receptor Antagonists: a Liquid Chromatography/Electrospray‐Mass Spectrometry and ex vivo Binding Study in Rats

In the present article, we report on the kinetics of brain penetration in rats of the H3R antagonist 1,1′‐[1,1′‐biphenyl‐4,4′‐diylbis(methylene)]bis‐[piperidine] ( 1 ), which had shown a favorable in vitro pharmacological profile and in vivo potency in preventing scopolamine‐induced amnesia. Two different approaches were employed: high‐performance liquid chromatography/electrospray‐mass spectrometry (HPLC/ESI‐MS) and ex vivo binding against the labeled agonist [³H]‐(R)‐α‐methylhistamine ([³H]RAMHA). Starting from the structure of 1 , the rigid piperidine ring was replaced by a flexible dipropylamino group (see 2 ) or by a morpholino ring (see 3 ), endowed with lower basicity. The effect of replacement on rat plasma and brain disposition in the 24 h after administration was analyzed. High (μM ) and persistent concentrations of 1 were found in rat plasma, while plasma levels were significantly lower (range: 0–200 nM ) for the other two derivatives. This could be explained, among other factors, by the higher stability, observed for 1 , to liver metabolic cleavage. The applied chemical modulation had an important effect on in vivo brain disposition, as, despite the comparable physico‐chemical properties, 2 did not show the tendency to accumulate within the brain, as stated by its brain vs. plasma concentration ratios, if compared to 1 . These structure? property relationships should be taken into account in the pharmacokinetic optimization of new series of H3 receptor antagonists.     

Two-compartment tumor metabolism: Autophagy in the tumor microenvironment and oxidative mitochondrial metabolism (OXPHOS) in cancer cells

Previously, we proposed a new paradigm to explain the compartment-specific role of autophagy in tumor metabolism. In this model, autophagy and mitochondrial dysfunction in the tumor stroma promotes cellular catabolism, which results in the production of recycled nutrients. These chemical building blocks and high-energy “fuels” would then drive the anabolic growth of tumors, via autophagy resistance and oxidative mitochondrial metabolism in cancer cells. We have termed this new form of stromal-epithelial metabolic coupling: “two-compartment tumor metabolism.” Here, we stringently tested this energy-transfer hypothesis, by genetically creating (1) constitutively autophagic fibroblasts, with mitochondrial dysfunction or (2) autophagy-resistant cancer cells, with increased mitochondrial function. Autophagic fibroblasts were generated by stably overexpressing key target genes that lead to AMP-kinase activation, such as DRAM and LKB1. Autophagy-resistant cancer cells were derived by overexpressing GOLPH3, which functionally promotes mitochondrial biogenesis. As predicted, DRAM and LKB1 overexpressing fibroblasts were constitutively autophagic and effectively promoted tumor growth. We validated that autophagic fibroblasts showed mitochondrial dysfunction, with increased production of mitochondrial fuels (L-lactate and ketone body accumulation). Conversely, GOLPH3 overexpressing breast cancer cells were autophagy-resistant, and showed signs of increased mitochondrial biogenesis and function, which resulted in increased tumor growth. Thus, autophagy in the tumor stroma and oxidative mitochondrial metabolism (OXPHOS) in cancer cells can both dramatically promote tumor growth, independently of tumor angiogenesis. For the first time, our current studies also link the DNA damage response in the tumor microenvironment with “Warburg-like” cancer metabolism, as DRAM is a DNA damage/repair target gene.     

Metabolic reprogramming of cancer-associated fibroblasts by TGF-β drives tumor growth: Connecting TGF-β signaling with “Warburg-like” cancer metabolism and L-lactate production

We have previously shown that a loss of stromal Cav-1 is a biomarker of poor prognosis in breast cancers. Mechanistically, a loss of Cav-1 induces the metabolic reprogramming of stromal cells, with increased autophagy/mitophagy, mitochondrial dysfunction and aerobic glycolysis. As a consequence, Cav-1-low CAFs generate nutrients (such as L-lactate) and chemical building blocks that fuel mitochondrial metabolism and the anabolic growth of adjacent breast cancer cells. It is also known that a loss of Cav-1 is associated with hyperactive TGF-β signaling. However, it remains unknown whether hyperactivation of the TGF-β signaling pathway contributes to the metabolic reprogramming of Cav-1-low CAFs. To address these issues, we overexpressed TGF-β ligands and the TGF-β receptor I (TGFβ-RI) in stromal fibroblasts and breast cancer cells. Here, we show that the role of TGF-β in tumorigenesis is compartment-specific, and that TGF-β promotes tumorigenesis by shifting cancer-associated fibroblasts toward catabolic metabolism. Importantly, the tumor-promoting effects of TGF-β are independent of the cell type generating TGF-β. Thus, stromal-derived TGF-β activates signaling in stromal cells in an autocrine fashion, leading to fibroblast activation, as judged by increased expression of myofibroblast markers, and metabolic reprogramming, with a shift toward catabolic metabolism and oxidative stress. We also show that TGF-β-activated fibroblasts promote the mitochondrial activity of adjacent cancer cells, and in a xenograft model, enhancing the growth of breast cancer cells, independently of angiogenesis. Conversely, activation of the TGF-β pathway in cancer cells does not influence tumor growth, but cancer cell-derived-TGF-β ligands affect stromal cells in a paracrine fashion, leading to fibroblast activation and enhanced tumor growth. In conclusion, ligand-dependent or cell-autonomous activation of the TGF-β pathway in stromal cells induces their metabolic reprogramming, with increased oxidative stress, autophagy/mitophagy and glycolysis, and downregulation of Cav-1. These metabolic alterations can spread among neighboring fibroblasts and greatly sustain the growth of breast cancer cells. Our data provide novel insights into the role of the TGF-β pathway in breast tumorigenesis, and establish a clear causative link between the tumor-promoting effects of TGF-β signaling and the metabolic reprogramming of the tumor microenvironment.     

Molecular Mechanism of Allosteric Communication in Hsp70 Revealed by Molecular Dynamics Simulations

Investigating ligand-regulated allosteric coupling between protein domains is fundamental to understand cell-life regulation. The Hsp70 family of chaperones represents an example of proteins in which ATP binding and hydrolysis at the Nucleotide Binding Domain (NBD) modulate substrate recognition at the Substrate Binding Domain (SBD). Herein, a comparative analysis of an allosteric (Hsp70-DnaK) and a non-allosteric structural homolog (Hsp110-Sse1) of the Hsp70 family is carried out through molecular dynamics simulations, starting from different conformations and ligand-states. Analysis of ligand-dependent modulation of internal fluctuations and local deformation patterns highlights the structural and dynamical changes occurring at residue level upon ATP-ADP exchange, which are connected to the conformational transition between closed and open structures. By identifying the dynamically responsive protein regions and specific cross-domain hydrogen-bonding patterns that differentiate Hsp70 from Hsp110 as a function of the nucleotide, we propose a molecular mechanism for the allosteric signal propagation of the ATP-encoded conformational signal.     

New retinoid derivatives as back-ups of Adarotene

Adarotene belongs to the so-called class of atypical retinoids. The presence of the phenolic hydroxyl group on Adarotene structure allows a rapid O-glucuronidation as a major mechanism of elimination of the drug, favoring a fast excretion of its glucuronide metabolite in the urines. A series of ether, carbamate and ester derivatives was synthesized. All of them were studied and evaluated for their stability at different pH. The cytotoxic activity in vitro on NCI-H460 non-small cell lung carcinoma and A2780 ovarian tumor cell lines was also tested. A potential back-up of Adarotene has been selected to be evaluated in tumor models.