9 research outputs found
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Covalent inactivation of Mycobacterium thermoresistibile inosine-5'-monophosphate dehydrogenase (IMPDH).
Inosine-5'-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme involved in nucleotide biosynthesis. Because of its critical role in purine biosynthesis, IMPDH is a drug design target for immunosuppressive, anticancer, antiviral and antimicrobial chemotherapy. In this study, we use mass spectrometry and X-ray crystallography to show that the inhibitor 6-Cl-purine ribotide forms a covalent adduct with the Cys-341 residue of Mycobacterium thermoresistibile IMPDH
Lst4, the yeast Fnip1/2 orthologue, is a DENN-family protein.
The folliculin/Fnip complex has been demonstrated to play a crucial role in the mechanisms underlying Birt-Hogg-DubĆ© (BHD) syndrome, a rare inherited cancer syndrome. Lst4 has been previously proposed to be the Fnip1/2 orthologue in yeast and therefore a member of the DENN family. In order to confirm this, we solved the crystal structure of the N-terminal region of Lst4 from Kluyveromyces lactis and show it contains a longin domain, the first domain of the full DENN module. Furthermore, we demonstrate that Lst4 through its DENN domain interacts with Lst7, the yeast folliculin orthologue. Like its human counterpart, the Lst7/Lst4 complex relocates to the vacuolar membrane in response to nutrient starvation, most notably in carbon starvation. Finally, we express and purify the recombinant Lst7/Lst4 complex and show that it exists as a 1 : 1 heterodimer in solution. This work confirms the membership of Lst4 and the Fnip proteins in the DENN family, and provides a basis for using the Lst7/Lst4 complex to understand the molecular function of folliculin and its role in the pathogenesis of BHD syndrome.AP, BKB and RKN were supported by the Myrovlytis Trust. DBA was supported by a NHMRC CJ Martin Fellowship (APP1072476). LHW was supported by Medical Research Council (MRC) studentship, MR/J006580/1 and TPL by University College London. SD was supported by Fondation de France, La Ligue National contre le Cancer (ComitĆ© de Paris / Ile-de-France and ComitĆ© de lāOise); TLB and NZ thank the University of Cambridge and The Wellcome Trust for facilities and support.This is the final version of the article. It was first available from Royal Society Publishing via http://dx.doi.org/10.1098/rsob.15017
Fragment-Based Approach to Targeting Inosine-5'-monophosphate Dehydrogenase (IMPDH) from Mycobacterium tuberculosis.
Tuberculosis (TB) remains a major cause of mortality worldwide, and improved treatments are needed to combat emergence of drug resistance. Inosine 5'-monophosphate dehydrogenase (IMPDH), a crucial enzyme required for de novo synthesis of guanine nucleotides, is an attractive TB drug target. Herein, we describe the identification of potent IMPDH inhibitors using fragment-based screening and structure-based design techniques. Screening of a fragment library for Mycobacterium thermoresistible ( Mth) IMPDH ĪCBS inhibitors identified a low affinity phenylimidazole derivative. X-ray crystallography of the Mth IMPDH ĪCBS-IMP-inhibitor complex revealed that two molecules of the fragment were bound in the NAD binding pocket of IMPDH. Linking the two molecules of the fragment afforded compounds with more than 1000-fold improvement in IMPDH affinity over the initial fragment hit
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Fragment-Based Approach to Targeting Inosine-5'-monophosphate Dehydrogenase (IMPDH) from Mycobacterium tuberculosis.
Tuberculosis (TB) remains a major cause of mortality worldwide, and improved treatments are needed to combat emergence of drug resistance. Inosine 5'-monophosphate dehydrogenase (IMPDH), a crucial enzyme required for de novo synthesis of guanine nucleotides, is an attractive TB drug target. Herein, we describe the identification of potent IMPDH inhibitors using fragment-based screening and structure-based design techniques. Screening of a fragment library for Mycobacterium thermoresistible ( Mth) IMPDH ĪCBS inhibitors identified a low affinity phenylimidazole derivative. X-ray crystallography of the Mth IMPDH ĪCBS-IMP-inhibitor complex revealed that two molecules of the fragment were bound in the NAD binding pocket of IMPDH. Linking the two molecules of the fragment afforded compounds with more than 1000-fold improvement in IMPDH affinity over the initial fragment hit
Synthesis and Structure\u2013Activity relationship of 1-(5-isoquinolinesulfonyl)piperazine analogues as inhibitors of Mycobacterium tuberculosis IMPDH
Tuberculosis (TB) is a major infectious disease associated increasingly with drug resistance. Thus, new anti-tubercular agents with novel mechanisms of action are urgently required for the treatment of drug-resistant TB. In prior work, we identified compound 1 (cyclohexyl(4-(isoquinolin-5-ylsulfonyl)piperazin-1-yl)methanone) and showed that its anti-tubercular activity is attributable to inhibition of inosine-5'-monophosphate dehydrogenase (IMPDH) in Mycobacterium tuberculosis. In the present study, we explored the structure-activity relationship around compound 1 by synthesizing and evaluating the inhibitory activity of analogues against M. tuberculosis IMPDH in biochemical and whole-cell assays. X-ray crystallography was performed to elucidate the mode of binding of selected analogues to IMPDH. We establish the importance of the cyclohexyl, piperazine and isoquinoline rings for activity, and report the identification of an analogue with IMPDH-selective activity against a mutant of M. tuberculosis that is highly resistant to compound 1. We also show that the nitrogen in urea analogues is required for anti-tubercular activity and identify benzylurea derivatives as promising inhibitors that warrant further investigation
Fragment-Based Approach to Targeting Inosine-5ā²-monophosphate Dehydrogenase (IMPDH) from <i>Mycobacterium tuberculosis</i>
Tuberculosis (TB)
remains a major cause of mortality worldwide,
and improved treatments are needed to combat emergence of drug resistance.
Inosine 5ā²-monophosphate dehydrogenase (IMPDH), a crucial enzyme
required for <i>de novo</i> synthesis of guanine nucleotides,
is an attractive TB drug target. Herein, we describe the identification
of potent IMPDH inhibitors using fragment-based screening and structure-based
design techniques. Screening of a fragment library for <i>Mycobacterium
thermoresistible</i> (<i>Mth</i>) IMPDH ĪCBS
inhibitors identified a low affinity phenylimidazole derivative. X-ray
crystallography of the <i>Mth</i> IMPDH ĪCBSāIMPāinhibitor
complex revealed that two molecules of the fragment were bound in
the NAD binding pocket of IMPDH. Linking the two molecules of the
fragment afforded compounds with more than 1000-fold improvement in
IMPDH affinity over the initial fragment hit
The Inosine Monophosphate Dehydrogenase, GuaB2, Is a Vulnerable New Bactericidal Drug Target for Tuberculosis
VCC234718, a molecule with growth
inhibitory activity against Mycobacterium tuberculosis (<i>Mtb</i>), was identified by phenotypic screening of
a 15344-compound library. Sequencing of a VCC234718-resistant mutant
identified a Y487C substitution in the inosine monophosphate dehydrogenase,
GuaB2, which was subsequently validated to be the primary molecular
target of VCC234718 in <i>Mtb</i>. VCC234718 inhibits <i>Mtb</i> GuaB2 with a <i>K</i><sub>i</sub> of 100 nM
and is uncompetitive with respect to IMP and NAD<sup>+</sup>. This
compound binds at the NAD<sup>+</sup> site, after IMP has bound, and
makes direct interactions with IMP; therefore, the inhibitor is by
definition uncompetitive. VCC234718 forms strong pi interactions with
the Y487 residue side chain from the adjacent protomer in the tetramer,
explaining the resistance-conferring mutation. In addition to sensitizing <i>Mtb</i> to VCC234718, depletion of GuaB2 was bactericidal in <i>Mtb</i> in vitro and in macrophages. When supplied at a high
concentration (ā„125 Ī¼M), guanine alleviated the toxicity
of VCC234718 treatment or GuaB2 depletion via purine salvage. However,
transcriptional silencing of <i>guaB2</i> prevented <i>Mtb</i> from establishing an infection in mice, confirming that <i>Mtb</i> has limited access to guanine in this animal model.
Together, these data provide compelling validation of GuaB2 as a new
tuberculosis drug target