Optical Modulation of Antibiotic Resistance by Photoswitchable Cystobactamids

Abstract

The rise of antibiotic resistance causes a serious health care problem, and its counterfeit demands novel, innovative concepts. The combination of photopharmacology, enabling a light-controlled reversible modulation of drug activity, with antibiotic drug design has led to first photoswitchable antibiotic compounds derived from established scaffolds. In this study, we converted cystobactamids, gyrase-inhibiting natural products with an oligoaryl scaffold and highly potent antibacterial activities, into photoswitchable agents by inserting azobenzene in the N-terminal part and/or an acylhydrazone moiety near the C-terminus, yielding twenty analogs that contain mono- as well as double-switches. Antibiotic and gyrase inhibition properties could be modulated 3.4-fold and 5-fold by light, respectively. Notably, the sensitivity of photoswitchable cystobactamids towards two known resistance factors, the peptidase AlbD and the scavenger protein AlbA, was light-dependent. While irradiation of an analog with an N-terminal azobenzene with 365 nm light led to less degradation by AlbD, the AlbA-mediated inactivation was induced. This provides a proof-of-principle that resistance towards photoswitchable antibiotics can be optically controlled.

Keywords: antibiotics; antimicrobial resistance; natural products; oligoarylamides; photopharmacology.

Figure 1

Chemical structures of natural and synthetic cystobactamids and of albicidin.

Figure 2

Cystobactamids with N‐terminal photoswitches. A) Compound structures. Photoswitchable functional groups are highlighted in red, and the final synthetic step connecting the shown N‐terminal A–B rings to the Eastern part is indicated in blue; B) E→Z isomerization of 6 a; C–E) Characterization of 6 a; C) UV spectra of 6 a (0.5 mM in DMSO) before irradiation (in red) and after irradiation with 365 nm light (in green); D) LC/UV‐chromatographic traces before irradiation (above) and after irradiation with 365 nm light (below); E) calculated kinetic parameters: rate constant k, activation energy E_A , preexponential factor A, half‐life t_1/2.

Scheme 1

Final steps for the synthesis of C‐terminal hydrazobactamids and overview of synthesized analogs. Reagents, conditions, isolated yields: a) DCM, TFA, TiPS, rt, 2.5 h, quant.; b) HATU, DiPEA, DMF, rt, 15 min, then tert‐butyl carbazate, rt, 3 h, 91 %; c) Pd(PPh3)4, PhSiH3, THF rt, 2.5 h, 60 %; d) DCM, TFA, rt, 2 h, quant.; e) aldehyde in THF, rt, 15 min, 10–81 %.

Figure 3

Characterization of hydrazobactamid 10 c. A) Chemical structure and photo‐inducible E→Z isomerization of acylhydrazone moiety (highlighted in blue and violet); B) UV spectra of 10 c (0.5 mM in DMSO) in its thermally adapted state before irradiation (in black) and after irradiation with 365 nm light (in violet) and with 450 nm light (in blue); C) Photoswitching cycles, recorded by the UV absorption at 350 nm after alternating irradiation with 365 nm light (light blue periods) and 450 nm (dark blue periods).

Scheme 2

A) Synthesis of red‐shifted azobenzenes; B) Synthetic route to doubly photoswitchable cystobactamids. Photoswitchable moieties are highlighted in blue and red. Reagents, conditions, isolated yields: a) LTMP, THF, −78 °C 30 min, then 12 in THF −85 °C, 2 h, 49–51 %; b) DCM, TFA, rt, 2 h; quant; c) TFAA, TEA, DCM, rt, 4 h, 66 %; d) DCM, TFA, TiPS, rt, 2.5 h; e) HATU, DiPEA, DMF, rt, 30 min then tert‐butyl carbazate, rt, 3 h; f) NH₃ (7 N) in MeOH, THF, 45 °C, 10 h, 48 % 3 steps; g) 14 a–c, HATU, DiPEA, DMF, rt, 30 min then 16 rt, 1 h; h) Pd(PPh₃)₄, PhSiH₃, THF rt, 2.5 h, 20–60 % 2 steps; i) aldehyde in THF, rt, 15 min., 19–58 %.

Figure 4

Characterization of doubly switchable cystobactamids. A) UV spectra of 20 a (0.5 mM in DMSO) in its thermally adapted state before irradiation (in black) and after irradiation with light of five different wavelengths; B) Repeated photoswitching of 20 a, recorded by the UV absorption at 350 nm after alternating irradiation with 365 nm light (dark blue periods) and 450 nm (light blue periods). C) Arrhenius plot of the logarithmic rate constants of thermal isomerization to the ground state versus reciprocal temperature for four cystobactamids. D) Kinetic data for 10 a, 19 a, 20 b and 20 a. Activation energy E_A and half‐life t _1/2 were calculated based on reaction speeds in a temperature range from 30–50 °C (Supporting Information Figures S10–S21, Table S3). Data for azobenzene were taken from Ref. [21].

Figure 5

Light‐dependent inactivation of photoswitchable cystobactamids by AlbD. A) Enzymatic reaction. The photoswitchable moiety and the cleavage site are marked in red. B) LC/UV‐chromatographic traces of 6 a after 30 minutes incubation, in presence (+) or absence (−) of AlbD. Before incubation, 6 a was either irradiated (365 nm) or not (dark).

Figure 6

Light‐dependent inactivation of photoswitchable cystobactamids by AlbA. Top left: Petri plate layout. Other panels: Images of the Petri plates from the agar diffusion assay. Albicidin or cystobactamids were pre‐irradiated with 365 nm light (+UV) or not (‐UV), deposited on an agar plate inoculated with E. coli BW25113 in the presence (+) or absence (−) of AlbA (1 equivalent). Growth inhibition was visible as an inhibition zone, whose diameter was measured and listed in Table 4.