Structure-based drug discovery of carbonic anhydrase inhibitors

Figures & data

Scheme 1.  Catalytic and inhibition mechanisms (with zinc ion binders) of α-carbonic anhydrases (CAs) (hCA I amino acid numbering of the zinc ligands). A similar catalytic/inhibition mechanism is valid also for CAs from other classes (β-, γ- and ζ-CAs) but either the metal ion is coordinated by other amino acid residues or a Cd(II) ion is present instead of zinc at the active site.

Figure 1.  Clinically used/preclinical sulphonamide and sulphamate CAIs 1–15 and compounds developed in the last period (16–27).

Figure 2.  Human carbonic anhydrase (hCA) II in adduct with the sulphonamide incorporating an NO-donating moiety 18, as obtained by x-ray crystallography⁴⁹.

Figure 3.  Human carbonic anhydrase (hCA) II complexed with the thienylacetamido benzenesulphonamides 19 (X = H) and 20 (X = F), which illustrate that small changes at the aromatic ring lead to different conformations of the bound inhibitor within the enzyme active site⁵⁴.

Figure 4.  Human carbonic anhydrase (hCA) II complexed with the tosylureido benzenesulphonamide 21 (green) and triazinyl-substituted benzenesulphonamide 22 (blue)^56,62 (A); and with five ureido sulphonamides 23–27, compounds 23 (orange), 24 (pink), 25 (yellow), 26 (grey) and 27 (cyan) (B)⁵⁵.

Figure 5.  A. Structure of DTCs 28–30. B. Electronic density for the adduct of dithiocarbamate 29 bound within the active site of human carbonic anhydrase (hCA) II⁵⁹. The zinc ion is shown as the central sphere, and the amino acid residues involved in the binding are evidenced and numbered (hCA I numbering system)⁵⁹.

Table 1.  Inhibition constants of aromatic carboxylates 31–46 against isozymes human carbonic anhydrase (hCA) I and II (α-CA class) and β-CAs Can2 (from Cryptococcus neoformans) and Nce103 (from Candida albicans), for the CO₂ hydration reaction, at 20°C. The standard sulphonamide inhibitor acetazolamide (1) was also included as standard⁷¹.

No	R§	KI [μM]#			Nce103^b
		hCA Ia	hCA IIa	Can2b
31	H	730	30	1267	24.4
32	2,3,5,6-F4-	74	6	49.1	61.6
33	4-F-	7.39	3.34	0.13	1.09
34	4-Cl-	7.61	3.81	3.40	2.65
35	4-Br-	8.00	4.70	4.24	4.18
36	4-I-	8.18	5.85	5.46	5.17
37	4-O2N-	8.80	8.56	0.12	4.85
38	4-HO-	7.61	7.28	0.0095	0.11
39	4-MeO-	9.06	8.03	0.0099	0.15
40	4-H2N-	8.40	8.13	0.12	2.95
41	3-H2N-	8.79	8.63	3.06	3.40
42	4-NC-	6.92	6.79	0.14	4.47
43	4-AcHN-	4.04	7.28	0.32	0.34
44	4-ClCH2-	3.88	4.36	3.65	4.71
45	4-Me2N-	3.94	3.33	0.13	5.20
46	4-Cl2NSO2-	3.0.2588	1.96	0.12	4.97
1	-		0.012	0.010	0.13

^#Errors were in the range of 3%–5% of the reported values, from three different assays.

^§As sodium salt.

^aHuman cloned isoforms, assay at pH 7.5.

^bFungal enzymes, at pH 8.3.

Table 2.  Carbonic anhydrases (CAs) from pathogenic bacteria cloned and characterised thus far (data for Vibrio cholerae are based on the sequence of a putative CA) and their inhibition studies⁸. The genome of many other bacteria contains CAs, which have not yet been cloned and characterised.

Bacterium	Family	Name	Inhibition study
			In vitro	In vivo
Neisseria gonorrhoeae	α	-	Sulphonamides,Anions	sulphonamides
Neisseria sicca	α	-	Sulphonamides	Sulphonamides
Helicobacter pylori	α	hpαCA	Sulphonamides,Anions	sulphonamides
	β	hpβCA	Sulphonamides,Anions	sulphonamides
Escherichia coli	β	-	NI	NI
Haemophilus influenzae	β	HICA	Bicarbonate	NI
Mycobacterium tuberculosis	β	mtCA 1	Sulphonamides	NA
	β	mtCA 2	Sulphonamides	NA
	β	mtCA 3	Sulphonamides	NA
Brucella suis	β	bsCA 1	Sulphonamides	Sulphonamides
	β	bsCA 2	Sulphonamides	Sulphonamides
Streptococcus pneumoniae	β	PCA	Sulphonamides, Anions	NI
Salmonella enterica	β	stCA 1	Sulphonamides, Anions	NI
	β	stCA 2	Sulphonamides, Anions	NI
Vibrio cholerae	unknown	-	NI	NI

- means not named.

NA, no activity in vivo (presumably due to penetration problems); NI, not investigated.

Table 3.  Inhibition data of the human (h) α-carbonic anhydrase (CA) isoforms hCA I and II and mycobacterial β-CA isoforms mtCA 1 and 3 with dithiocarbamates 47–73 by a stopped-flow, CO₂ hydrase assay⁶⁸.

R^1R^2N-CSS^−M^+ 47–73
Cmpnd	R1	R2	KI (nM)*				M
			hCA I	hCA II	mtCA 1	mtCA 3
47	H	Ph	4.8	4.5	5.6	2.5	Et3NH
48	H	O[(CH2CH2)]2N	4.8	3.6	6.1	2.4	K
49	H	MeN[(CH2CH2)]2N	33.5	33.0	4.7	2.6	K
50	H	2-butyl	21.1	29.4	6.0	3.6	K
51	H	O[(CH2CH2)]2N(CH2)2 31.8	36.3	7.1	2.8	K
52**	H	N[(CH2CH2)N]3	31.9	13.5	4.2	4.0	K
53	H	PhCH2	4.1	0.7	7.1	87.3	Na
54	H	4-PyridylCH2	3.5	16.6	5.4	5.7	Et3NH
55	H	[(CH2)5N]CH2CH2	4.5	20.3	9.1	8.8	K
56	H	2-thiazolyl	3.9	4.6	89.4	9.5	Et3NH
57	H	KOOCCH2	13.1	325	7.8	8.3	K
58	H	imidazol-1-yl-(CH2)3	8.6	24.7	5.3	8.7	A
59	Me	Me	699	6910	893	659	Na
60	Et	Et	790	3100	615	431	Na
61	(CH2)5		0.96	27.5	90.5	4.1	Na
62	n-Pr	n-Pr	1838	55.5	74.8	80.0	Na
63	n-Bu	n-Bu	43.1	50.9	81.7	72.8	Na
64	iso-Bu	iso-Bu	0.97	0.95	86.2	43.0	Na
65	n-Hex	n-Hex	48.0	51.3	95.4	51.7	Na
66	Et	n-Bu	157	27.8	91.6	63.5	Na
67	HOCH2CH2	HOCH2CH2	9.2	4.0	7.5	6.0	Na
68	Me	Ph	39.6	21.5	25.2	46.8	Na
69	Me	PhCH2	69.9	25.4	72.0	62.5	Na
70		O[(CH2CH2)]2	0.88	0.95	0.94	0.91	Na
71	NaS2CN[(CH2CH2)]2		12.6	0.92	7.7	8.0	Na
72	(NC)(Ph)C(CH2CH2)2		48.4	40.8	93.0	61.2	N
73^#	(S)-[CH2CH2CH2CH(COONa)]		2.5	17.3	7.1	6.4	Na
1	(acetazolamide	250	12	481	104	-

*Errors in the range of ±10% of the reported values, by a CO₂ hydrase assay method⁶⁸.

A, imidazol-1-yl-(CH₂)₃NH₃⁺.

**Tris-dithiocarbamate.

^#(S)-proline dithiocarbamate.

Table 4.  Inhibition data of mycobacterial β-carbonic anhydrase (CA) isoforms mtCA 1–3 with carboxylic acids 74–83 and sulphanilamide 84 by a stopped-flow, CO₂ hydrase assay⁸³.

Cmpnd	R	KI (μM)*
		mtCA 1	mtCA 2	mtCA 3
74	Br	3.91	2.66	0.97
75	I	4.94	2.14	0.14
76	CN	6.20	2.12	0.19
77	AcNH	4.76	3.67	0.48
78	NO2	5.87	8.10	0.52
79	-	4.89	5.27	0.11
80	2-OH	2.25	4.17	0.12
81	4-OH	3.03	1.56	0.60
82	OH	5.92	0.59	0.57
83	OMe	7.13	3.50	0.36
84	Sulphanilamide	9.84	29.6	7.11

*Errors in the range of ±10% of the reported values, by a CO₂ hydrase assay method (from three different measurements)³⁰.

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