Fatty acid binding to the human transport proteins FABP3, FABP4, and FABP5 from a Ligand's perspective.

Fatty acid binding proteins (FABPs) are a family of amphiphilic transport proteins with high diversity in terms of their amino acid sequences and binding preferences. Beyond their main biological role as cytosolic fatty acid transporters, many aspects regarding their binding mechanism and functional specializations in human cells remain unclear. In this work, the binding properties and thermodynamics of FABP3, FABP4, and FABP5 were analyzed under various physical conditions. For this purpose, the FABPs were loaded with fatty acids bearing fluorescence or spin probes as model ligands, comparing their binding affinities via microscale thermophoresis (MST) and continuous-wave electron paramagnetic resonance (CW EPR) spectroscopy. The CW EPR spectra of non-covalently bound 5- and 16-DOXYL stearic acid (5/16-DSA) deliver in-depth information about the dynamics and chemical environments of ligands inside the binding pockets of the FABPs. EPR spectral simulations allow the construction of binding curves, revealing two different binding states ('intermediately' and 'strongly' bound). The proportion of bound 5/16-DSA depends strongly on the FABP concentration and the temperature but with remarkable differences between the three isoforms. Additionally, the more dynamic state ('intermediately bound') seems to dominate at body temperature with thermodynamic preference. The ligand binding studies were supplemented by aggregation studies via dynamic light scattering and bioinformatic analyses. Beyond the remarkably fine-tuned binding properties exhibited by each FABP, which were discernible with our EPR-centered approach, the results of this work attest to the power of simple spectroscopic experiments to provide new insights into the ligand binding mechanisms of proteins in general on a molecular level.

The prognostic impact of the serum heart-type fatty acid-binding protein level in patients with sepsis who were admitted to the non-surgical intensive-care unit.

Ongoing myocardial damage at the acme of the sepsis status has not been sufficiently evaluated. The clinical data of 160 sepsis patients who require intensive care and 127 outpatients with chronic heart failure (HF) were compared as a retrospective cohort study. Thereafter, the sepsis patients were divided into 3 groups according to the serum heart-type fatty acid-binding protein (H-FABP) quartiles [low H-FABP = Q1 (n = 39), middle H-FABP = Q2/Q3 (n = 81), and high H-FABP = Q4 group (n = 40)]. The H-FABP level was measured within 15 min of admission. The serum H-FABP levels in the sepsis patients [26.6 (9.3-79.0) ng/ml] were significantly higher than in the choric HF patients [6.6 (4.6-9.7) ng/ml]. A Kaplan-Meier curve showed that the survival rate of the high-H-FABP group was significantly lower than that of the middle- and low-H-FABP groups. The multivariate Cox regression analysis for the 365-day mortality showed that the high-H-FABP group (hazard ratio: 6.544, 95% confidence interval [CI] 2.026-21.140; p = 0.002) was an independent predictor of the 365-day mortality. The same trend in the prognostic impact was significantly (p = 0.015) observed in the cohort that had not been suffering from the cardiac disease before admission. The serum H-FABP level was an independent predictor of the 365-day mortality in the patients who were emergently hospitalized in the intensive-care unit due to sepsis. Ongoing myocardial damage was detected in the majority of patients with sepsis, suggesting that ongoing myocardial damage might be a candidate predictor of adverse outcomes in sepsis patients.

Identification of B Cell Epitopes of Blo t 13 Allergen and Cross-Reactivity with Human Adipocytes and Heart Fatty Acid Binding Proteins.

Cross-reactivity between allergens and human proteins could have a clinical impact in allergic diseases. Blo t 13 is an allergen from the mite Blomia tropicalis, which belongs to the fatty acid binding protein (FABP) family and has structural homology with human FABPs. This work aimed to map B cell epitopes on Blo t 13 and to identify epitopes involved in cross-reactivity with human heart FABP (FABP3) and adipocyte FABP (FABP4). Sera from 25 patients with house dust mite (HDM) allergy that were sensitized to Blo t 13 were used for testing the reactivity of immunoglobulin E (IgE) and IgG to FABP. The epitope mapping of Blo t 13 was performed using overlapping peptides, and cross-reactivity between Blo t 13 and human FABP was analyzed using human sera and anti-Blo t 13 monoclonal antibodies. IgE antibodies to all FABPs were detected in 14/25 serum samples, and IgG was detected in 25/25 serum samples. The cross-reactivity of Blo t 13 was 42% with FABP3 and 48% with FABP4. Two IgE-binding regions were identified in Blo t 13; one between residues 54 and 72 (the main cross-reacting region) and another between residues 111 to 129. Our results suggest that exposure to the Blo t 13 allergen could induce an auto-reactive response to endogenous FABP in allergic patients sensitized to Blo t 13.

Identification of Fatty Acid Binding Protein 5 Inhibitors Through Similarity-Based Screening.

Fatty acid binding protein 5 (FABP5) is a promising target for development of inhibitors to help control pain and inflammation. In this work, computer-based docking (DOCK6 program) was employed to screen ∼2 M commercially available compounds to FABP5 based on an X-ray structure complexed with the small molecule inhibitor SBFI-26 previously identified by our group (also through virtual screening). The goal was discovery of additional chemotypes. The screen resulted in the purchase of 78 candidates, which led to the identification of a new inhibitor scaffold (STK-0) with micromolar affinity and apparent selectivity for FABP5 over FABP3. A second similarity-based screen resulted in three additional hits (STK-15, STK-21, STK-22) from which preliminary SAR could be derived. Notably, STK-15 showed comparable activity to the SBFI-26 reference under the same assay conditions (1.40 vs 0.86 µM). Additional molecular dynamics simulations, free energy calculations, and structural analysis (starting from DOCK-generated poses) revealed that R enantiomers (dihydropyrrole scaffold) of STK-15 and STK-22 have a more optimal composition of functional groups to facilitate additional H-bonds with Arg109 of FABP5. This observation suggests enantiomerically pure compounds could show enhanced activity. Overall, our study highlights the utility of using similarity-based screening methods to discover new inhibitor chemotypes, and the identified FABP5 hits provide a strong starting point for future efforts geared to improve activity.