{"id":1056,"date":"2019-10-22T16:20:40","date_gmt":"2019-10-22T16:20:40","guid":{"rendered":"http:\/\/web.devl.snet.biobio.tamu.edu\/saclab\/?page_id=1056"},"modified":"2020-10-28T19:14:40","modified_gmt":"2020-10-28T19:14:40","slug":"central-carbon-metabolism","status":"publish","type":"page","link":"https:\/\/saclab.biobio.tamu.edu\/index.php\/research\/tuberculosis\/central-carbon-metabolism\/","title":{"rendered":"Central carbon metabolism"},"content":{"rendered":"

[vc_row][vc_column][vc_custom_heading text=”CENTRAL CARBON METABOLISM” font_container=”tag:h2|font_size:30px|text_align:left|color:%23000000|line_height:40px” google_fonts=”font_family:Roboto%3A100%2C100italic%2C300%2C300italic%2Cregular%2Citalic%2C500%2C500italic%2C700%2C700italic%2C900%2C900italic|font_style:700%20bold%20regular%3A700%3Anormal”][\/vc_column][\/vc_row][vc_row][vc_column][vc_tour interval=”0″ type=”rd_vtab_1″ pos=”rd_vtab_left”][vc_tab title=”malate synthase” tab_id=”8ca68d4e-cbfb-0″][vc_custom_heading text=”malate synthase” font_container=”tag:h2|font_size:20px|text_align:left|line_height:10px” google_fonts=”font_family:Roboto%3A100%2C100italic%2C300%2C300italic%2Cregular%2Citalic%2C500%2C500italic%2C700%2C700italic%2C900%2C900italic|font_style:700%20bold%20regular%3A700%3Anormal”][vc_column_text]The glyoxylate shunt plays an important role in fatty acid metabolism and has been shown to be critical to survival of several pathogens involved in chronic infections. For\u00a0Mycobacterium tuberculosis<\/em>\u00a0(Mtb<\/em>), a strain with a defective glyoxylate shunt was previously shown to be unable to establish infection in a mouse model. We report the development of phenyl-diketo acid (PDKA) inhibitors of malate synthase (GlcB), one of two glyoxylate shunt enzymes, using structure-based methods. PDKA inhibitors were active against\u00a0Mtb<\/em>\u00a0grown on acetate, and overexpression of GlcB ameliorated this inhibition. Crystal structures of complexes of GlcB with PDKA inhibitors guided optimization of potency. A selected PDKA compound demonstrated efficacy in a mouse model of tuberculosis. The discovery of these PDKA derivatives provides chemical validation of GlcB as an attractive target for tuberculosis therapeutics.[\/vc_column_text][vc_single_image image=”1407″ img_size=”800*600″ onclick=”custom_link” img_link_target=”_blank” link=”https:\/\/www.sciencedirect.com\/science\/article\/pii\/S107455211200381X?via%3Dihub#fig3″][vc_column_text]<\/p>\n

Figure :<\/span>\u00a0Comparing Binding of PDKA Analogs to GlcB<\/strong><\/p>\n

(A and B) Binding of GlcB to inhibitor\u00a04<\/strong>\u00a0(A) and inhibitor\u00a011<\/strong>\u00a0(B) colored by element, with Cprotein<\/sub>\u00a0in gray and Cligand<\/sub>\u00a0in black. Hydrogen bonds are indicated by solid blue lines, and distances from key positions on the phenyl ring to protein residues are marked as dashed lines.<\/p>\n

(C) Crystal structure overlay of GlcB complexed with PDKA (in magenta);\u00a01<\/strong>,\u00a04<\/strong>,\u00a07<\/strong>, and\u00a011<\/strong>, represented by ball-and-stick, and CoA, represented by a stick model (colored by element), with the Mg atom in green, illustrate the relative positions of the ligands occupying the active site channel (presented by protein surface calculated in CHIMERA (Pettersen et\u00a0al., 2004<\/a>). The CoA model and protein surface were made from chain A of the 2GQ3 model.<\/p>\n

[\/vc_column_text][vc_column_text]<\/p>\n

Fragment screening and high throughput screening are complementary approaches that combine with structural biology to explore the binding capabilities of an active site. We have used a fragment-based approach on malate synthase (GlcB) from\u00a0Mycobacterium tuberculosis<\/em>\u00a0and discovered several novel binding chemotypes. In addition, the crystal structures of GlcB in complex with these fragments indicated conformational changes in the active site that represent the enzyme conformations during catalysis. Additional structures of the complex with malate and of the apo form of GlcB supported that hypothesis. Comparative analysis of GlcB structures in complex with 18 fragments allowed us to characterize the preferred chemotypes and their binding modes. The fragment structures showed a hydrogen bond to the backbone carbonyl of Met-631. We successfully incorporated an indole group from a fragment into an existing phenyl-diketo acid series. The resulting indole-containing inhibitor was 100-fold more potent than the parent phenyl-diketo acid with an IC50<\/sub>\u00a0value of 20 nm<\/span>.<\/p>\n

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FIGURE:<\/a><\/strong><\/span><\/em><\/div>\n
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A<\/em>, a close-up of the active site with a superposition of GlcB crystal structures complexed with PDKA (white<\/em>) and fragment\u00a011<\/strong>\u00a0(cyan<\/em>) displaying protein surface. The\u00a0black arrows<\/em>\u00a0indicate changes upon binding of the Group 2 fragments: the widening of the cavity behind the active site Mg2+<\/sup>, the narrowing mouth of the main tunnel caused by the movement of the CoA binding loop (details are shown in\u00a0B<\/em>), and an additional portal to the solvent with the active site lid opening (details are shown in\u00a0C<\/em>). Non-carbon atoms are colored as follows: magnesium,\u00a0chartreuse<\/em>; oxygen,\u00a0red<\/em>; nitrogen,\u00a0blue<\/em>; sulfur,\u00a0yellow<\/em>. Images are rendered in Chimera.<\/p>\n<\/div>\n

[\/vc_column_text][vc_single_image image=”1405″ img_size=”745*364″][vc_column_text]<\/p>\n

FIGURE :<\/a><\/strong><\/span><\/em><\/div>\n
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Positions of malate in model 1N8W and in the crystal structures from GlcB preincubated with substrates at various times.<\/strong>\u00a0A<\/em>, overlay of different positions of malate in model 1N8W (light green<\/em>;\u00a0B<\/em>), malate-bound structure after 40-min preincubation (light blue<\/em>;\u00a0C<\/em>), apoenzyme resulting from preincubation at 60 min (gold<\/em>;\u00a0D<\/em>), and G459A mutant complex with malate after preincubation with substrates (pink<\/em>;\u00a0E<\/em>). Non-carbon atoms are colored as follows: magnesium,\u00a0chartreuse<\/em>; oxygen,\u00a0red<\/em>; nitrogen,\u00a0blue<\/em>; sulfur,\u00a0yellow<\/em>. Images are rendered in Chimera.<\/p>\n<\/div>\n

[\/vc_column_text][vc_column_text]Human infection by\u00a0Mycobacterium tuberculosis<\/i>\u00a0(Mtb) continues to be a global epidemic. Computer-aided drug design (CADD) methods are used to accelerate traditional drug discovery efforts. One noncovalent interaction that is being increasingly identified in biological systems but is neglected in CADD is the anion-\u03c0 interaction. The study reported herein supports the conclusion that anion-\u03c0 interactions play a central role in directing the binding of phenyl-diketo acid (PDKA) inhibitors to malate synthase (GlcB), an enzyme required for\u00a0Mycobacterium tuberculosis<\/i>\u00a0virulence. Using density functional theory methods (M06-2X\/6-31+G(d)), a GlcB active site template was developed for a predictive model through a comparative analysis of PDKA-bound GlcB crystal structures. The active site model includes the PDKA molecule and the protein determinants of the electrostatic, hydrogen-bonding, and anion-\u03c0 interactions involved in binding. The predictive model accurately determines the Asp 633-PDKA structural position upon binding and precisely predicts the relative binding enthalpies of a series of 2-ortho halide-PDKAs to GlcB. A screening model was also developed to efficiently assess the propensity of each PDKA analog to participate in an anion-\u03c0 interaction; this method is in good agreement with both the predictive model and the experimental binding enthalpies for the 2-ortho halide-PDKAs. With the screening and predictive models in hand, we have developed an efficient method for computationally screening and evaluating the binding enthalpy of variously substituted PDKA molecules. This study serves to illustrate the contribution of this overlooked interaction to binding affinity and demonstrates the importance of integrating anion-\u03c0 interactions into structure-based CADD.[\/vc_column_text][vc_single_image image=”1406″ img_size=”500*290″ onclick=”custom_link” img_link_target=”_blank” link=”https:\/\/pubs.acs.org\/doi\/10.1021\/acs.jcim.8b00417″][\/vc_tab][vc_tab title=”PrpR” tab_id=”1572885622847-5-4″][vc_custom_heading text=”PrpR” font_container=”tag:h2|font_size:20px|text_align:left|line_height:10px” google_fonts=”font_family:Roboto%3A100%2C100italic%2C300%2C300italic%2Cregular%2Citalic%2C500%2C500italic%2C700%2C700italic%2C900%2C900italic|font_style:700%20bold%20regular%3A700%3Anormal”][vc_column_text]The pathogenicity of\u00a0Mycobacterium tuberculosis<\/em>\u00a0depends upon its ability to catabolize host cholesterol. Upregulation of the methylcitrate cycle (MCC) is required to assimilate and detoxify propionyl-CoA, a cholesterol degradation product. The transcription of key genes\u00a0prpC<\/em>\u00a0and\u00a0prpD<\/em> in MCC is activated by MtPrpR, a member of a family of prokaryotic transcription factors whose structures and modes of action have not been clearly defined. We show that MtPrpR has a novel overall structure and directly binds to CoA or short-chain acyl-CoA derivatives to form a homotetramer that covers the binding cavity and locks CoA tightly inside the protein. The regulation of this process involves a [4Fe4S] cluster located close to the CoA-binding cavity on a neighboring chain. Mutations in the [4Fe4S] cluster binding residues rendered MtPrpR incapable of regulating MCC gene transcription. The structure of MtPrpR without the [4Fe4S] cluster-binding region shows a conformational change that prohibits CoA binding. The stability of this cluster means it is unlikely a redox sensor but may function by sensing ambient iron levels. These results provide mechanistic insights into this family of critical transcription factors who share similar structures and regulate gene transcription using a combination of acyl-CoAs and [4Fe4S] clusters.[\/vc_column_text]