Lucy
Lin
a,
Lewis D.
Turner
a,
Peter
Šilhár‡
a,
Sabine
Pellett
b,
Eric A.
Johnson
b and
Kim D.
Janda
*a
aDepartment of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. E-mail: kdjanda@scripps.edu
bDepartment of Bacteriology, University of Wisconsin, 1550 Linden Drive, Madison, Wisconsin 53706, USA
First published on 12th November 2020
Botulinum neurotoxin serotype A (BoNT/A) is an important therapeutic target owing to its extremely potent nature, but also has potential use as a biowarfare agent. Currently, no therapeutic exists to reverse the long-lasting paralysis caused by BoNT/A. Herein, we describe the identification of 3-hydroxy-1,2-dimethylpyridine-4(1H)-thione (3,4-HOPTO) as a metal binding warhead for the inhibition of BoNT/A1. An initial screen of 96 metal binding fragments identified three derivatives containing the 3,4-HOPTO scaffold to inhibit the BoNT/A1 light chain (LC) at >95% at 1 mM. Additional screening of a 3,4-HOPTO sub-library identified structure–activity relationships (SARs) between N-substituted 3,4-HOPTO derivatives and the BoNT/A1 LC. Subsequent synthesis was conducted to improve on inhibitory potency – achieving low μM biochemical IC50 values. Representative compounds were evaluated in a cellular-based assay and showed promising μM activity.
Currently, the standard treatment for botulism is an equine-derived antitoxin. The antitoxin must be administered within 72 hours of intoxication to maximize its effect, as it cannot enter the neuronal compartment to neutralize internalized BoNT/A, where the toxin remains active for several months.6–8 Additional limitations to this therapeutic strategy include adverse reactions to horse serum and high costs. Furthermore, even with the aid of antibody treatment, patients may spend weeks to months in the hospital, often requiring mechanical nutrition and ventilation;4 cost of treatment ranges from $55000–350
000 per patient.9,10 The high medical costs and burden on the healthcare system of several patients requiring mechanical ventilation has become painfully apparent in the current COVID-19 pandemic. Thus, there is an unmet need for a treatment strategy that is capable of arresting the activity of BoNT/A after motor neurons have been intoxicated.
BoNT/A is a 150 kD metalloprotease composed of a heavy chain (HC, 100 kD), which mediates specific neuronal cell association, endocytosis, and toxin translocation into cells, and a light chain (LC, 50 kD) which possesses the catalytic domain, the source of BoNT/A's proteolytic activity.11–13 Once translocated into neurons, the LC is free to cleave its substrate, synaptosomal nerve-associated protein-25 (SNAP-25). SNAP-25 is a SNAP receptor (SNARE) protein required for vesicle fusion to the cellular membrane.14 In the absence of fully intact SNAP-25, motor-neurons are unable to release acetylcholine, resulting in the flaccid muscle paralysis characteristic of botulism.14 Post-intoxication treatments for botulism must disrupt the action of the BoNT/A LC inside the neuronal cells. Thus, an ideal strategy to address this issue would be the development of a cell-permeable small molecule inhibitor.8 However, despite significant effort, no small molecule inhibitors of BoNT/A have seen clinical success.
Recently, our group reported the use of a bifunctional strategy which leverages the potency of a metal binding group (MBG) active site inhibitor tethered to a cysteine-reactive warhead to covalently inhibit enzymatic activity of the BoNT/A1 LC.15 While the proof-of concept inhibitors were successful, the potential off-target effects resulting from both the highly reactive warhead and the hydroxamic acid moiety remain a concern.16–18 Thus, there is a continued need for the discovery of MBGs that are capable of inhibiting the BoNT/A LC enzymatic activity, which can then be adapted for use in the bifunctional approach. Herein, we describe the identification and development of 3-hydroxy-1,2-dimethylpyridine-4(1H)-thione (3,4-HOPTO) as an active site inhibitor of BoNT/A1.
An additional screen of a 3,4-HOPTO-based library (eCFL-1)19 containing 18 compounds substituted on the nitrogen was carried out (Table 1). In general, phenyl derivatives that were directly attached to the nitrogen (7–14) were either inactive or had poor potency, with the exception of compound 7. The mono-para-substituted lipophilic ethoxy group appears to be better placed within the active site when compared to smaller (8 and 9) and larger (10 and 11) para-substituted compounds, suggesting a ‘medium’-sized substituent is most desired when there is a constrained vector for the phenyl group. Bulkier di-substituted compounds (12–14) were not well tolerated, outlining potential steric and orientation constraints of the active site in this region. An increase in flexibility introduced by the insertion of a one- or two-carbon linker between the phenyl ring and the nitrogen atom saw an increase in potency (15–24), supporting this notion. This is best demonstrated through the increase in potency between compounds 8 and 15. We hypothesize that the additional flexibility and length afforded by the linker may facilitate more favourable hydrophobic binding interactions, most notably π-stacking with aromatic residues in the BoNT/A1 LC active site. Moreover, there is little difference in inhibitory potency for compounds bearing the same substituent in different substitution patterns (i.e.16 and 17, 22 and 23), implying more steric freedom. The most influential factor determining changes in potency appears to be steric bulk, demonstrated by the negligible difference in potencies between compounds 18 and 19 where the pyridyl nitrogen has minimal effects on binding. Compounds 20–23 contain an ethylene linker and exhibit similar potencies to their methylene counterparts and are equipotent with compound 24 which possesses a large naphthalene group. This apparent tolerance for steric bulk in this region is consistent with previous work, which established the plasticity of the BoNT/A1 LC.27,28 An X-ray crystal structure (PDB ID: 4HEV) of an adamantane-based hydroxamate inhibitor in complex with the BoNT/A1 LC shows the bulky adamantane group occupying a hydrophobic pocket.27 In direct contrast to this, an X-ray crystal structure (PDB ID: 2IMB) of L-arginine hydroxamate bound in the LC shows the active site to be significantly more polar.29 The variability in the active site conformations means that larger, more bulky hydrophobic inhibitors can be tolerated, while maintaining the same configuration of the zinc ion and catalytic residues.
![]() | ||
Scheme 1 General synthesis of 3,4-HOPTO derivatives 27–44. Full synthetic details provided in ESI.† |
Compound | R group | IC50a (μM) | Compound | R group | IC50a (μM) |
---|---|---|---|---|---|
a IC50 values are reported as mean ± SD (n = 3). | |||||
27 |
![]() |
5.0 ± 1.0 | 35 |
![]() |
1.9 ± 0.2 |
28 |
![]() |
11.3 ± 3.4 | 36 |
![]() |
1.7 ± 0.4 |
29 |
![]() |
2.2 ± 0.8 | 37 |
![]() |
2.8 ± 1.1 |
30 |
![]() |
31.9 ± 1.7 | 38 |
![]() |
4.9 ± 2.6 |
31 |
![]() |
>50 | 39 |
![]() |
>50 |
32 |
![]() |
3.0 ± 0.5 | 40 |
![]() |
1.8 ± 0.1 |
33 |
![]() |
>50 | 41 |
![]() |
1.4 ± 0.1 |
34 |
![]() |
1.6 ± 0.2 | 42 |
![]() |
1.3 ± 0.3 |
21 |
![]() |
3.2 ± 0.7 | 43 |
![]() |
1.8 ± 0.4 |
44 |
![]() |
1.4 ± 0.5 |
Due to the favourable binding of para-substituted lipophilic phenyl derivatives (7 and 21), we decided to investigate the SAR in this region further (27–32). Lipophilic substituents (27–29) performed better than less lipophilic substituents (30 and 31). Interestingly, chloro derivative 28 was significantly less potent than both methyl (27) and bromo derivatives (29), suggesting a stronger role for electrostatic interactions than observed in earlier data. Compounds 30 and 31 exhibit significantly lower potencies than their lipophilic counterparts, which reflects previous results (9) (Table 1). Yet, 32 possesses a nitro group and shows comparable potency to compounds with lipophilic substituents. This strengthens the hypothesis that there may be an electrostatic component to binding in this region of the active site. The addition of a second –Cl to the ring (33) results in a complete loss of potency. However, extension of the methylene linker to an ethylene linker results in inhibitory activity being regained, with other ethylene-based compounds (21, 35–37) exhibiting equipotency. The additional flexibility and length likely allows the aromatic rings to engage more favourably with hydrophobic aromatic residues (Ile161, Phe194, and Phe369) within the P1 pocket of the active site.29 Past success with adamantane derivatives of BoNT/A1 LC active site inhibitors led us to synthesize 38 and 39. In this instance, the ethylene linker (39) resulted in a complete loss in activity while the methylene derivative (38) remained relatively potent. We thus concluded that the length and flexibility of this appendage has an optimum range, and that the pi-interactions of the aryl groups are likely important. Compounds 40–44 possess linear bi-aryl systems, all of which show equipotent single-digit μM activity and overall flat SAR.
![]() | ||
Fig. 3 IC50 data of 40 when tested against various MMPs. IC50 values are reported as mean ± SD (n = 3) and can be found in Table S2.† |
Cpd no. | R group | BoNT/A LC IC50a (μM) | hIPSC IC50a (μM) | Cpd no. | R group | BoNT/A LC IC50a (μM) | hIPSC IC50a (μM) |
---|---|---|---|---|---|---|---|
a IC50 values are reported as mean ± SD (n = 3). b SD not calculated due to no variation in fit value. | |||||||
32 |
![]() |
3.1 ± 0.5 | 100 ± 9 | 42 |
![]() |
1.3 ± 0.3 | 31 ± 3 |
34 |
![]() |
1.6 ± 0.2 | ∼200 | 43 |
![]() |
1.8 ± 0.4 | 39 ± 0b |
40 |
![]() |
1.8 ± 0.1 | 13 ± 2 | 44 |
![]() |
1.4 ± 0.5 | 51 ± 4 |
41 |
![]() |
1.4 ± 0.1 | 33 ± 6 |
Unsurprisingly, an overall decrease in potency was observed between enzyme- and cell-based IC50 values. In particular, poor cell-permeation due to the charged nature of the nitro group of 32 is likely a crucial factor in its poor inhibitory potency, although the lack of SNAP-25 protection afforded by 34 is unexplained. As seen in the enzyme IC50 data, there is a limited SAR trend between compounds 34 and 40–44. Interestingly, compound 40 exhibits the best protection against SNAP-25 cleavage, between 2- to 3-fold better than the other biphenyl derivatives 41–44 and only suffered a 7-fold reduction in cell potency compared to the enzyme assay. Despite possessing similar enzyme-potencies, 40 and 32, have drastically different cell potencies. It is therefore likely that any putative cell permeability issues are not related to the 3,4-HOPTO scaffold itself, and that potency in the cell assay may be tuned by improving physicochemical properties.
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/d0md00320d |
‡ Present address: 217 Kameničany, 01854 Slovakia. |
This journal is © The Royal Society of Chemistry 2021 |