Pharmacological, Physiochemical, and Drug-Relevant Biological Properties of Short Chain Fatty Acid Hexosamine Analogues Used in Metabolic Glycoengineering

Abstract

In this study, we catalog structure activity relationships (SAR) of several short chain fatty acid (SCFA)-modified hexosamine analogues used in metabolic glycoengineering (MGE) by comparing in silico and experimental measurements of physiochemical properties important in drug design. We then describe the impact of these compounds on selected biological parameters that influence the pharmacological properties and safety of drug candidates by monitoring P-glycoprotein (Pgp) efflux, inhibition of cytochrome P450 3A4 (CYP3A4), hERG channel inhibition, and cardiomyocyte cytotoxicity. These parameters are influenced by length of the SCFAs (e.g., acetate vs n-butyrate), which are added to MGE analogues to increase the efficiency of cellular uptake, the regioisomeric arrangement of the SCFAs on the core sugar, the structure of the core sugar itself, and by the type of N-acyl modification (e.g., N-acetyl vs N-azido). By cataloging the influence of these SAR on pharmacological properties of MGE analogues, this study outlines design considerations for tuning the pharmacological, physiochemical, and the toxicological parameters of this emerging class of small molecule drug candidates.

Keywords: P450; Pgp efflux; carbohydrate drug design; hERG; metabolic oligosaccharide engineering.

Figure 1.. Overview of SCFA hexosamine analog “druggability.”

(A) The hexosamine scaffold has over 100,000 theoretical permutations , in which the R₁, R₃, R₄, and R₆ naturally-occurring or synthetic ester-linked SCFA groups can be mix-and-matched to improve the efficiency of analog uptake into cells and tune biological activity while the R’ group is either the natural acetate or non-natural “NAz” moiety in this study (overall, several dozen R’-modified analogs have been reported ^,). (B) The removal of the ester-linked protecting groups by non-specific intracellular esterases “activates” the core sugar for incorporation into glycosylation pathways ^,. In the current work, we expand the evaluation of these compounds to include (C) physicochemical properties relevant to pharmacokinetics and (D) biological endpoints related to ADME.

Figure 2.. In silico prediction of ADME properties.

(A) Representative physiochemical profiles of SCFA-hexosamine analogs were calculated using SwissADME. The shaded red area indicates the regions optimal physiochemical parameters for drug-likeness for oral administration. The calculated parameters for the six physicochemical properties (lipophilicity, size, polarity, solubility, flexibility and saturation) for each type of analog shown are outlined in red. (B) BOILED-Egg plot showing calculated log P (WLOGP) versus total polar surface area (TPSA) illustrating the predicted abilities of MGE analogs to penetrate the BBB (yellow) or undergo passive human intestinal absorption (HIA) through the gastrointestinal tract (white area). The blue dots indicate that all analogs evaluated were expected to be efflulated from the central nervous system via P-glycoprotein transporters (Pgp).

Figure 3.. HPLC analysis.

Retention times were determined for each analog using RP-HPLC; selected example sets of retention times differences between analogs are shown in panels (A-I) as discussed in more detail in the main text.

Figure 4.. k-Cluster analysis of RT data.

(A) Retention times were cataloged for each analog using RP-HPLC, representative data is shown. (B) Ordered pairs of retention times for all possible combinations of analogs were generated and the difference in retention times between each analog was calculated (ΔRT). (C) Using the differences between retention times for all pairs of analogs that were calculated (ΔRT), a k-cluster analysis was performed in order to group analog pairs that have similar changes in retention time after a specific change(s) in chemical structure. Each colored group represents a set of chemical transformations that produces a similar net change in retention time for each ordered pair of analogs. See the Supporting Information (Table S2) for a specific list of chemical transformations in each cluster.

Figure 5.. Chromatographic hydrophobicity index (CHI) analyses

(A) CHI values for MGE analogs were determined using set of molecules for calibrating RP-HPLC. (B) The calculated CHI scores for the analogs shown are listed, with each color representing a similar group of chemical transformations for the given hexosamine cores. (C) Experimental CHI scores correlate poorly with in silico modeling predictions using Swiss-ADME for consensus log P values. (D) The analogs tested for their CHI were ranked according to their experimentally determined values with the in silico lipophilicity rankings listed in parentheses. No analogs were calculated to have the same CHI score.

Figure 6.. Aqueous solubility map.

Colored squares indicate the type of hexosamine core following the conventions provided by Glycopedia (http://www.glycopedia.eu/IMG/pdf/the_symbolic_representation_of_monosaccharides_2014.pdf). The area of each square is proportional to the experimentally measured quantitative value for water solubility (exact values are provided in the Supporting Information (Table S4)). Each node of the map is connected to another node that is differentiated by the listed modification(s) and A, B, C(i) and C(ii) denote relationships discussed in the main text.

Figure 7.. Pgp efflux and CYP3A4 inhibition.

The indicated analogs were screened for (A) Pgp efflux (B) and CYP3A4 inhibition. Heat maps were generated to depict the combinatorial effects of the core hexosamine, SCFA modification, and composition of the N-acyl group. Gray areas denote analogs that do not exist or were not tested.

Figure 8.. HERG channel activity inhibition.

(A) E-1041, a positive control and (B-H) MGE analogs were screened for HERG channel inhibition. (I) Non-linear regression was used to fit the IC₅₀ curves to the inhibition data and IC₅₀ values were approximated (see Supplementary Table S7).

Figure 9.

Cardiomyocytes were incubated for 12, 24, and 48 h with (A) 3,4,6-O-Bu₃GlcNAc and (B) 3,4,6-O-Bu₃ManNAc at 50 μM each. Significantly higher cell death was apparent beginning 12 to 24 h after commencing incubation with each analog. (C). MTT assays conducted after 24 and 48 h confirm the dramatic decline in cell viability upon treatment with these “3,4,6” tri-butanylated hexosamine analogs.