Screening Of Novel Inhibitors Of HMG-CoA Reductase From Streptococcus Pneumoniae And Studies On The Interaction Between Inhibitors And HMGR

Streptococcus pneumoniae is one of the major causes of bacterial disease in human, such as meningitis, septicemia and pneumonia. About3700people die of the infection of Streptococcus pneumoniae everyday, and most of them are children under five years old whose Immunity is very weak. Due to the reason of environmental pollution, abusing of antibiotics, and so on, Streptococcus pneumoniae have new Drug resistance to the traditional drugs. So the developing of new inhibitors to resist streptococcus pneumonia has great significance.As the oldest known biomolecules, Isoprenoids serve numerous biochemical functions. So the rutes of IPP biosynthesis attract resaerchers’attention. Two pathways for the biosynthesis of IPP have been described, the mevalonate pathway and the recently discovered deoxyxylulose5-phosphate (DXP) pathway. Streptococcus pneumonia use the mevalonate pathway for the synthesis of IPP. And we choose the HMG-CoA reductase, which catalyzes the conversion of mevaonate to HMG-CoA, enabling the organism to use mevalonate as its sole source of carbon, as the target. This paper discusses this enzyme from the flowing aspects:(1) the reasonable three-dimensional (3-D) structure of Class II HMGR from S. pneumoniae (SP-HMGR-II) was built using the SWISSMODEL server, the X-ray crystallographic structural of HMGR-II from P. mevalonii was selected as template. By using the docking process, a serial of hit compounds, such as D7S and LH, were screening from SPECs database.(2) The activity assay shows that a series of hit compounds exhibit high inhibition activity, such as compound D7S21, with the IC50values of11.5μM.(3) In order to find the interaction mechanism between the active site of Class II HMGR and its inhibitor, the molecular docking results show that the compounds exhibit that the compounds can form strongly hydrogen bonds with the residues N212, H378and K263. But the K263and H378are located the catalytic region of the enzyme, and are probably participate the catalysis of HMGR. Besides, N212is partly responsible for the NADPH affinity. The occupying not only part of the HMG-CoA pocket but also part of the NADPH pocket maybe the main reason for its high inhibition activity. In order to confirm this conclusion, the site-directed mutagenesis and fluorescence quenching was performed.(4) We have obtained the mutants of these residues, such as K263A,N212A,N212D, H378A, Q382A,whereas some of these mutant proteins, just like K263A, N212A, H378A exhibit less enzymatic acticity to continue the enzymatic assays studies. The other two mutants have some catalytic activity, although the catalytic constant (Kcat) variant was-10-fold lower compared with the value of parental enzyme of HMGR. Besides, the inhibitory activity of compounds D7S14and D7S21against the N212D variant of SP-HMGR-II are about3-fold higher than parental enzyme, at the same time, the IC50value of D7S14and D7S21against Q382A variant of SP-HMGR-II are about2-fold higher than parental enzyme.(5) Because some of the mutants exhibit less enzymatic acticity, the fluorescence quenching was performed. Mutating of these residues significantly affect the binding of hit compound D7S21, the Kd values obtained from fluorescence quenching for D7S21binding to the variants are smaller than the parental SP-HMGR-II, especially for Q382A and K263A, whose Kd value are about10-fold smaller than the parental HMGR.(6) The results of site-directed mutagenesis and fluorescence quenching successfully supports our theoretical supposition, and suggests that the H378, N212, K263are essential in hit compounds binging and there is directly interaction between these residues and the present hit compounds.