Extended Table 4 |. SAR of amino acid differences between macolacin and colistin.

Note: Macolacin differs from colistin by three amino acids: Ser3, Ile7, Leu10 (orange). Macolacin analogs synthesized with only 2 amino acid changes (orange) compared to colistin were tested for antibacterial activity (MIC μg/mL) against pathogens with or without either mcr-1 or phoP/Q (n=2). The fold increase in MIC upon introduction of either mcr-1 or phoP/Q is shown in bold. In polymyxin family structures, the side chain of a D-configured amino acid at position 3 and an L-configured amino acid at position 10 are very close in three-dimensional space and likely interact together with lipid A to counter common amine containing modifications (i.e., PEtN or L-Ara4N). As the binding of colistin to lipid A is largely driven by electrostatic interactions, it is not surprising that compensating for appending a primary amine onto a lipid A phosphate involves replacing a positively charged Dab with a neutral residue (Ser).

Substitution	Polymyxin	Colistin	Macolacin	Macolacin-3Dab	Macolacin-7Leu	Macolacin-10Thr
Amino acid 3	Dab	Dab	Ser	Dab	Ser	Ser
Amino acid 7	Leu	Leu	lie	lie	Leu	lie
Amino acid 10	Thr	Thr	Let	Leu	Leu	Thr
Pathogen			MIC (μg/mL)
K. pneumoniae 10031	1	0.5	1	1	1	0.5–1
K. pneumoniae 0497 (mcr-1)	32	16	2	8	2	8
K. pneumoniae 13883	1	1	1	2	2	1
K. pneumoniae 13883 (mcr-1)	128	64	4	16	4	64
Fold increase in MIC	128	64	4	8	2	64
E. cloacae ATCC13047 (Δ phoP/Q)	1	1	1	2	2	1
E. cloacae ATCC13047 (Δ phoP/Q+phoP/Q)	32	32	2	8	1	16
Fold increase in MIC	32	32	2	4	0.5	16