# Mechanistic Studies of the Calcium-Dependent Antibiotics via Cofactor Engineering

## TL;DR

This study explores how modifying calcium-dependent antibiotics can change their cofactor requirements, offering new insights into their activation mechanisms.

## Contribution

The study introduces a new method to convert calcium-dependent antibiotics into boron-dependent ones through amino acid substitution.

## Key findings

- Electron withdrawing substituents on phenylboronic acid enhance the antibacterial activity of the boron-dependent antibiotic B1.
- CDA4b requires both calcium and phenylboronic acid for full activation and is less potent with only one cofactor.
- Modifications in friulimicin and daptomycin analogs did not yield active boron-dependent antibiotics.

## Abstract

The defining feature of calcium-dependent antibiotics
(CDAs) is
that they require the presence of calcium cation (Ca­(II)) as a cofactor
to exert antibacterial activity. We recently showed that substituting
two key aspartic acids (Asp) with serine (Ser) in laspartomycin C
(LspC) converts it from a CDA into a boron-dependent antibiotic (BDA).
This synthetic analog (termed B1) no longer depends on
Ca­(II) and requires only 10 μM of phenylboronic acid (PBA) to
become fully active. Such a calcium-to-boron dependence conversion
provides a new entry point to study the mechanistic details of the
cofactor dependence of CDAs, a rare phenomenon among bioactive small
molecules. Herein, we show that electron withdrawing substituents
on PBA enhance the antibacterial activity of B1. The
friulimicin and daptomycin synthetic analogs with the same Asp-to-Ser
substitution were inactive, whereas the CDA4b synthetic analog exhibited
dual cofactor dependence. CDA4b was fully activated when both Ca­(II)
and PBA were present and was 4-fold less potent in the presence of
only one or the other. These findings suggest that not only do CDAs
often have distinct cellular targets, the way they are activated by
Ca­(II) are also different. Such mechanistic diversity underscores
the strong potential of CDAs in drug development.