In vitro inhibition of Mycobacterium tuberculosis β-carbonic anhydrase 3 with Mono- and dithiocarbamates and evaluation of their toxicity using zebrafish developing embryos

Figures & data

Figure 1. General structures of dithiocarbamates (DTC) and monothiocarbamates (MTC).

Figure 2. Chemical structures of MTCs 1-7 and DTCs 8-14.

Table 1. Inhibition data of Mtb β-CA3 and human CA isoforms hCA I and II for compounds 1–14, determined by the stopped-flow CO₂ hydrase assay³⁵, using acetazolamide (AAZ) as a standard drug.

	KI (nM)^a
Compounds	Mtb β-CA3	hCA I	hCA II
1	558.6	>2000^26	46.7^26
2	747.6	569^26	>2000^26
3	812.0	876^26	22.4^26
4	83.3	>2000^26	43.6^26
5	93.0	>2000^26	43.7^26
6	780.7	949^26	45.9^26
7	97.9	891^26	26.7^26
8	43.0	0.97^25	0.95^25
9	2.4	0.88^25	0.95^25
10	8.0	12.6^25	0.92^25
11	>2000	415^27	67.2^27
12	2.6	33.5^25	33.0^25
13	>2000	496^27	80.5^27
14	>2000	494^27	48.7^27
AAZ	104	250.0	12.0

^a Mean from 3 different assays, by a stopped flow technique (errors were in the range of ± 5–10% of the reported values).

Table 2. LC₅₀ of the CAIs 1–14.

Compounds	LC50 dose	In vivo studies (μM)^a
1	1 mM	400
2	1 mM	125
3	2 mM	500
4	125 μM	35
5	1 mM	400
6	2 mM	500
7	2 mM	500
8	2 μM	1
9	125 μM	18
10	2 mM	500
11	1 mM	250
12	600 μM	300^b
13	1 mM	500
14	25 μM	5

^a The concentrations do not induce any phenotypic defects in zebrafish larvae at 5 days of exposure and safe for inhibition studies of M. marinum in zebrafish².

^b The compound 12 (Fc14-584b) is screened for toxicity and for in vivo inhibition studies as reported earlier².

Figure 3. Images of zebrafish larvae treated with different inhibitors. The identification number of each compound is in parenthesis. The images of the zebrafish larvae after 5 days of exposure to inhibitors that are considered safe for in vivo use to inhibit M. marinum growth in larvae². The concentrations of the MTCs and DTCs shown here are the highest ones generally not inducing phenotypic defects at the end of 5 days of exposure. For compound 8, even the lowest concentration used caused the absence of swim bladder (arrow), suggesting that this compound is not suitable for further characterization².

Table 3. Effect of inhibitors on phenotypic parameters of the larvae 5 days of after exposure^a,b.

Compounds	Hatching	Oedema	Heartbeat	Yolk sac	Body shape	Swim bladder
1	100	0	0	0	6	0
2	100	5	4	5	5	0
3	100	0	0	0	2	0
4	100	0	3	0	0	0
5	100	2	0	3	1	0
6	100	0.7	0	0	0.7	0.7
7	100	0	0	1	1	0
8	80	10	100	0	8	22
9	0	NA	NA	NA	NA	NA
10	100	0	0	0	0	0
11	100	0	0	0	0	0
12^c	NA	NA	NA	NA	NA	NA^2
13	100	2	0	2	4	0
14	100	12	7	8	10	14

NA: not applicable.

^a The parameters were assessed at LC50 at 5 day of exposure to the compounds.

^b The values shown are in percent of larvae.

^c The compound was screened for toxicity in our previous studies.

Table 4. The compounds that inhibit Mtb β-CA3 efficiently and show minimal toxicity.

Compounds	KI (nM)^a	Safe concentration (μM)^b
1	558.6	400
3	812.0	500
5	93.0	400
7	97.9	500
10	8.0	500

^a Inhibition of β-CA3 in vitro.

^b The concentration that can be used for in vivo studies.

Khalifah RG. The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow kinetic studies on the native human isoenzymes B and C. J Biol Chem 1971;246:2561–73.

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Aspatwar A, Hammaren M, Koskinen S, et al. beta-CA-specific inhibitor dithiocarbamate Fc14-584B: a novel antimycobacterial agent with potential to treat drug-resistant tuberculosis. J Enzyme Inhib Med Chem 2017;32:832–40.

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