Lauric acid and myristic acid from Allium sativum inhibit the growth of Mycobacterium tuberculosis H37Ra: in silico analysis reveals possible binding to protein kinase B

Figures & data

Table 1. Chromatographic fractions of petroleum ether extract of A. sativum Linn (Alliaceae).

Pooled fractions	Yield (mg)	Yield (%)	Rf value	Antimycobacterial activity
P1	4.3	0.8	0.99	–
P2	24.5	4.9	0.99	–
P3	32.2	6.4	0.99, 0.84	–
P4	26.4	5.2	0.84	–
P5	29.8	5.9	0.72	–
P6	36.8	7.3	0.62	–
P7	34.0	6.8	0.46,	–
P8	46.5	9.3	0.46, 0.21	–
P9	96.6	19.3	0.21, 0.1	+++
P10	58.9	11.7	0.01	++
P11	64.5	12.9	0.01	+

Of the eleven fractions, only P₉ exhibited significant antimycobacterial activity (mentioned in bold). Presence of activity is shown as ‘+’ whereas absence of activity is indicated as ‘–’. The retention factor values mentioned is for the TLC plates developed with petroleum ether: chloroform (4:1).

+ = 15–20 ± 0.5 of zone size (mm), ++ = 21–25 ± 0.5 (mm), +++ = 26–30 ± 0.5 (mm).

Figure 1. EI-MS for bioactive P₉ fraction was obtained in the positive ionization mode.

Table 2. Purification process of the petroleum ether extract of A. sativum with information on yield, specific inhibition activity and purification fold.

Purification process	Yield (mg)	Specific inhibition activity by plate assay	Purification fold (F3)
Solvent extraction	1000	0.03	1
Column chromatography	97	0.27	9
Semi-preparative HPLC	31	1.3	43

Specific inhibition activity (SIA) was determined by the ratio of zone of clearance to the amount of compound loaded in the well.

Table 3. The EI-Mass, ¹H-NMR and antimycobacterial activity by plate assay and MABA of the subfractions F₃, F₄ and F₅.

	EI-Mass spectrometry (m/z)			Antimycobacterial activity
Subfractions	For the subfraction	NIST database	Peak integral values of the resonances observed in the ^1H NMR spectrum (0.75–2.5 ppm)	Zone of clearance* (mm) in agar well diffusion assay	MIC (μg/mL) by MABA
F3	200.03	200.31	2.00, 2.04, 16.40, 2.90	28.0 ± 1.4	22.3 ± 0
F4	228.04	228.37	2.00, 2.04, 20.22, 3.22	20.0 ± 1.4	66.7 ± 2.8
F5	256.07	256.42	2.00, 2.39, 23.52, 3.10	12.0 ± 2.8	–

* The zone of clearance was measured with the 100 μg of the phytochemical.

Figure 2. (a) IR spectrum, (b) EI-MS, (c) COSY (expanded spectrum), (d) HSQC (expanded spectrum) in CDCl₃ for the bioactive subfraction (F₃).

Figure 3. Superposition of the modeled cytosolic domain of PknB on the loop-deficient structure available at the PDB (PDB ID: 1MRU). The modeled loop region, present in between the N-terminal and C-terminal lobe is indicated.

Table 4. The protein IDs, gene name and PDB codes for 12 membrane proteins are shown.

Protein IDs	Gene name	Protein description	PDB IDs
Rv1926c	mpt63	Immunogenic protein	1LMI
Rv2878c	mpt53	Soluble secreted antigen MPT53 precursor	1LU4
Rv0014c	PknB	Transmembrane serine/threonine protein kinase B	1MRU, 3ORM, 3ORI, 3F61, 3F69, 1O6Y
Rv0931c	pknD	Transmembrane serine/threonine protein kinase D	1RWI, 1RWL
Rv3804c	fbpA	Secreted antigen 85-A (Mycolyltransferase)	1SFR
Rv0129c	fbpC	Secreted antigen 85-C	1VA5, 1DQY, 1DQZ, 3HRH
Rv0983	pepD	Probable serine protease	1Y8T, 2Z9I
Rv0902c	prrB	Two component sensor histidine kinase	1YS3, 1YSR
Rv2945c	lppX	Probable conserved lipoprotein	2BYO
Rv0018c	pstP	Possible PstP PPP	2CM1, 1TXO
Rv2537c	aroD	3-Dehydroquinate dehydrastase	2DHQ, 1H05, 1H0S, 1H0R
Rv0985c	mscL	Possible large-conductance ion mechanosensitive	2OAR

The row having italic font contains the possible proteins responsible for signal transduction.

Table 5. Results of lauric acid docking in PknB modeled structure (modeled 1MRU) and PstP (pdb code 2CM1) using Autodock 4.0 software.

	PknB-LA		PstP-LA
Cluster rank	1	2	1	2	3	4	5
Lowest binding energy (kcal/mol)	−5.14	−3.79	−2.95	−2.89	−2.82	−2.79	−2.75
Mean binding energy (SD)	−4.32 (0.41)	−3.38 (0.36)	−2.72	−2.45	−2.36	−2.25	−2.15
Configurations in cluster	56	15	13	13	10	11	13

RMS-tolerance of 4 Å was considered for the clustering procedure, producing two and five distinct conformational clusters, out of 100 runs each. The first row indicates the number of the cluster, the lowest and mean binding energy values of each cluster are shown in the second and third rows, respectively. All energies are expressed in kilocalories/mole. The last row indicates the population of each cluster.

A threshold was set to >5 numbers of configurations in a cluster were chosen and displayed in order to avoid complexity.

Table 6. Binding site residues traced from the ligand with the distance of 4 Å using Swissprot deep viewer.

Binding site residues		L20	G21	G23	S26	V28	A41	K43	E62	V75	M95	E96	Y97	V98	M148	M158	D159
Frequency of occurrence (%)	Cluster rank 1	100	44	–	–	100	58	100	50	–	89	61	11	80	100	100	100
	Cluster rank 2	96	20	–	–	100	100	100	27	47	73	83	–	83	100	100	100

Three clusters show conserved residues of V28, K43, M148, M158 and D159 (highlighted in bold) of which only K43 and D159 are involved in electrostatic interaction to LA. All the values are rounded off by ±0.5%.

Figure 4. Proposed interaction of the mycobacterium PknB with LA. Intermolecular electrostatic interaction involving the amino acid residues, Lys43 and Asp159, is highlighted.

Figure 5. Variation in the RMSD values (y-axis) of all the backbone atoms as a function of simulation time (in ps). The data were acquired with a time interval of 5 ps.