The unstructured linker of Mlh1 contains a motif required for endonuclease function which is mutated in cancers.

Eukaryotic DNA mismatch repair (MMR) depends on recruitment of the Mlh1-Pms1 endonuclease (human MLH1-PMS2) to mispaired DNA. Both Mlh1 and Pms1 contain a long unstructured linker that connects the N- and carboxyl-terminal domains. Here, we demonstrated the Mlh1 linker contains a conserved motif (Saccharomyces cerevisiae residues 391-415) required for MMR. The Mlh1-R401A,D403A-Pms1 linker motif mutant protein was defective for MMR and endonuclease activity in vitro, even though the conserved motif could be >750 Å from the carboxyl-terminal endonuclease active site or the N-terminal adenosine triphosphate (ATP)-binding site. Peptides encoding this motif inhibited wild-type Mlh1-Pms1 endonuclease activity. The motif functioned in vivo at different sites within the Mlh1 linker and within the Pms1 linker. Motif mutations in human cancers caused a loss-of-function phenotype when modeled in S. cerevisiae. These results suggest that the Mlh1 motif promotes the PCNA-activated endonuclease activity of Mlh1-Pms1 via interactions with DNA, PCNA, RFC, or other domains of the Mlh1-Pms1 complex.

Mlh1 interacts with both Msh2 and Msh6 for recruitment during mismatch repair.

Eukaryotic DNA mismatch repair (MMR) initiates through mispair recognition by the MutS homologs Msh2-Msh6 and Msh2-Msh3 and subsequent recruitment of the MutL homologs Mlh1-Pms1 (human MLH1-PMS2). In bacteria, MutL is recruited by interactions with the connector domain of one MutS subunit and the ATPase and core domains of the other MutS subunit. Analysis of the S. cerevisiae and human homologs have only identified an interaction between the Msh2 connector domain and Mlh1. Here we investigated whether a conserved Msh6 ATPase/core domain-Mlh1 interaction and an Msh2-Msh6 interaction with Pms1 also act in MMR. Mutations in MLH1 affecting interactions with both the Msh2 and Msh6 interfaces caused MMR defects, whereas equivalent pms1 mutations did not cause MMR defects. Mutant Mlh1-Pms1 complexes containing Mlh1 amino acid substitutions were defective for recruitment to mispaired DNA by Msh2-Msh6, did not support MMR in reconstituted Mlh1-Pms1-dependent MMR reactions in vitro, but were proficient in Msh2-Msh6-independent Mlh1-Pms1 endonuclease activity. These results indicate that Mlh1, the common subunit of the Mlh1-Pms1, Mlh1-Mlh2, and Mlh1-Mlh3 complexes, but not Pms1, is recruited by Msh2-Msh6 through interactions with both of its subunits.

Rad27 and Exo1 function in different excision pathways for mismatch repair in Saccharomyces cerevisiae.

Eukaryotic DNA Mismatch Repair (MMR) involves redundant exonuclease 1 (Exo1)-dependent and Exo1-independent pathways, of which the Exo1-independent pathway(s) is not well understood. The exo1Δ440-702 mutation, which deletes the MutS Homolog 2 (Msh2) and MutL Homolog 1 (Mlh1) interacting peptides (SHIP and MIP boxes, respectively), eliminates the Exo1 MMR functions but is not lethal in combination with rad27Δ mutations. Analyzing the effect of different combinations of the exo1Δ440-702 mutation, a rad27Δ mutation and the pms1-A99V mutation, which inactivates an Exo1-independent MMR pathway, demonstrated that each of these mutations inactivates a different MMR pathway. Furthermore, it was possible to reconstitute a Rad27- and Msh2-Msh6-dependent MMR reaction in vitro using a mispaired DNA substrate and other MMR proteins. Our results demonstrate Rad27 defines an Exo1-independent eukaryotic MMR pathway that is redundant with at least two other MMR pathways.

Structural basis for the function and inhibition of dihydroorotate dehydrogenase from Schistosoma mansoni.

Schistosomiasis is a serious public health problem, prevalent in tropical and subtropical areas, especially in poor communities without access to safe drinking water and adequate sanitation. Transmission has been reported in 78 countries, and its control depends on a single drug, praziquantel, which has been used over the past 30 years. Our work is focused on exploiting target-based drug discovery strategies to develop new therapeutics to treat schistosomiasis. In particular, we are interested in evaluating the enzyme dihydroorotate dehydrogenase (DHODH) as a drug target. DHODH is a flavoenzyme that catalyzes the stereospecific oxidation of (S)-dihydroorotate (DHO) to orotate during the fourth and only redox step of the de novo pyrimidine nucleotide biosynthetic pathway. Previously, we identified atovaquone, used in the treatment of malaria, and its analogues, as potent and selective inhibitors against Schistosoma mansoni DHODH (SmDHODH). In the present article, we report the first crystal structure of SmDHODH in complex with the atovaquone analogue inhibitor 2-((4-fluorophenyl)amino)-3-hydroxynaphthalene-1,4-dione (QLA). We discuss three major findings: (a) the open conformation of the active site loop and the unveiling of a novel transient druggable pocket for class 2 DHODHs; (b) the presence of a protuberant domain, only present in Schistosoma spp DHODHs, that was found to control and modulate the dynamics of the inhibitor binding site; (c) a detailed description of an unexpected binding mode for the atovaquone analogue to SmDHODH. Our findings contribute to the understanding of the catalytic mechanism performed by class 2 DHODHs and provide the molecular basis for structure-guided design of SmDHODH inhibitors. DATABASE: The structural data are available in Protein Data Bank (PDB) database under the accession code number 6UY4.

Ligand-based design, synthesis and biochemical evaluation of potent and selective inhibitors of Schistosoma mansoni dihydroorotate dehydrogenase.

Schistosomiasis ranks second only to malaria as the most common parasitic disease worldwide. 700 million people are at risk and 240 million are already infected. Praziquantel is the anthelmintic of choice but decreasing efficacy has already been documented. In this work, we exploited the inhibition of Schistosoma mansoni dihydroorotate dehydrogenase (SmDHODH) as a strategy to develop new therapeutics to fight schistosomiasis. A series of quinones (atovaquone derivatives and precursors) was evaluated regarding potency and selectivity against both SmDHODH and human DHODH. The best compound identified is 17 (2-hydroxy-3-isopentylnaphthalene-1,4-dione) with IC50 = 23 ±â€¯4 nM and selectivity index of 30.83. Some of the new compounds are useful pharmacological tools and represent new lead structures for further optimization.

Structural basis for the design of selective inhibitors for Schistosoma mansoni dihydroorotate dehydrogenase.

Trematode worms of the genus Schistosoma are the causing agents of schistosomiasis, a parasitic disease responsible for a considerable economic and healthy burden worldwide. In the present work, the characterization of the enzyme dihydroorotate dehydrogenase from Schistosoma mansoni (SmDHODH) is presented. Our studies demonstrated that SmDHODH is a member of class 2 DHODHs and catalyzes the oxidation of dihydroorotate into orotate using quinone as an electron acceptor by employing a ping-pong mechanism of catalysis. SmDHODH homology model showed the presence of all structural features reported for class 2 DHODH enzymes and reveal the presence of an additional protuberant domain predicted to fold as a flexible loop and absent in the other known class 2 DHODHs. Molecular dynamics simulations showed that the ligand-free forms of SmDHODH and HsDHODH undergo different rearrangements in solution. Well-known class 2 DHODH inhibitors were tested against SmDHODH and HsDHODH and the results suggest that the variable nature of the quinone-binding tunnel between human and parasite enzymes, as well as the differences in structural plasticity involving rearrangements of the N-terminal α-helical domain can be exploited for the design of SmDHODH selective inhibitors, as a strategy to validate DHODH as a drug target against schistosomiasis.

Pradimicin-IRD from Amycolatopsis sp. IRD-009 and its antimicrobial and cytotoxic activities.

A new polycyclic antibiotic, pradimicin-IRD, was isolated from actinobacteria Amycolatopsis sp. IRD-009 recovered from soil of Brazilian rainforest undergoing restoration area. This molecule is the major compound produced in solid culture media. The new compound was detected by a focused method of precursor ion (high-performance liquid chromatography coupled to tandem mass spectrometer) developed previously to identify unusual aminoglycosyl sugar moieties. The compound was isolated and its structure was, therefore, elucidated by high-resolution mass spectrometry, and 1D and 2D nuclear magnetic resonance experiments. Pradimicin-IRD displayed potential antimicrobial activity against Streptococcus agalactiae (MIC 3.1 µg/mL), Pseudomonas aeruginosa (MIC 3.1 µg/mL) and Staphylococcus aureus (MIC 3.1 µg/mL), and also cytotoxicity against tumour and non-tumour cell lines with IC50 values ranging from 0.8 µM in HCT-116 colon carcinoma cells to 2.7 µM in MM 200 melanoma cells. Particularly, these biological properties are described for the first time for this chemical class.

Natural products as inhibitors of Leishmania major dihydroorotate dehydrogenase.

The flavoenzyme dihydroorotate dehydrogenase (DHODH) catalyzes the fourth reaction of the de novo pyrimidine biosynthetic pathway, which exerts vital functions in the cells, especially within DNA and RNA biosynthesis. Thus, this enzyme stands out as a new key molecular target for parasites causing Neglected Diseases (NDs). Focused on contributing to the development of new therapeutic alternatives for NDs, in this study, for the first time, a screening of 57 natural products for in vitro inhibition of Leishmania major DHODH (LmDHODH) was carried out, including cross validation against the human DHODH (HsDHODH). A subset of natural products consisting of 21 sesquiterpene lactones (STLs) was submitted to QSAR studies. Additionally, thermostability studies by differential scanning fluorimetry (DSF) were performed to determine whether the STLs are effectively or not binding to the enzyme. The IC50 values against LmDHODH varied from 27 to 1200 µM; only irrelevant inhibition was obtained on HsDHODH. DSF assays confirmed binding of STLs to LmDHODH; moreover, it is suggested that such inhibitors might act in a different site other than the active site. A reliable QSAR model based on molecular descriptors was obtained (R2: 0.83; Q2CV: 0.69 and Q2EXT/F2: 0.66) indicating that stronger inhibition requires a balanced distribution of the hydrophobic regions across the molecular surface, as well as higher width and lower hydrophobicity of the molecules. A pharmacophore-based 3D-QSAR approach also afforded a useful model (R2: 0.72; Q2CV: 0.50 and Q2EXT/F2: 0.62), which confirmed the importance of proper orientation of the ligands, molecular surface features and shape for stronger inhibition, reflecting properties of a putative common binding site. These data indicated for the first time that natural products can actually inhibit LmDHODH and highlighted some metabolites as potentially interesting starting points for the discovery of more potent LmDHODH inhibitors, ultimately aiming at new effective therapeutic alternatives for leishmaniasis and, possibly, other NDs caused by trypanosomatids.

Plasmodium falciparum dihydroorotate dehydrogenase: a drug target against malaria.

Malaria remains one of the most lethal infectious diseases worldwide, and the most severe form is caused by Plasmodium falciparum. In recent decades, the major challenge to treatment of this disease has been the ability of the protozoan parasite to develop resistance to the drugs that are currently in use. Among P. falciparum enzymes, P. falciparum dihydroorotate dehydrogenase has been identified as an important target in drug discovery. Interference with the activity of this enzyme inhibits de novo pyrimidine biosynthesis and consequently prevents malarial infection. Organic synthesis, x-ray crystallography, high-throughput screening and molecular modeling methods such as molecular docking, quantitative structure-activity relationships, structure-based pharmacophore mapping and molecular dynamics simulations have been applied to the discovery of new inhibitors of P. falciparum dihydroorotate dehydrogenase.

Discovery of Antimalarial Azetidine-2-carbonitriles That Inhibit P. falciparum Dihydroorotate Dehydrogenase.

Dihydroorotate dehydrogenase (DHODH) is an enzyme necessary for pyrimidine biosynthesis in protozoan parasites of the genus Plasmodium, the causative agents of malaria. We recently reported the identification of novel compounds derived from diversity-oriented synthesis with activity in multiple stages of the malaria parasite life cycle. Here, we report the optimization of a potent series of antimalarial inhibitors consisting of azetidine-2-carbonitriles, which we had previously shown to target P. falciparum DHODH in a biochemical assay. Optimized compound BRD9185 (27) has in vitro activity against multidrug-resistant blood-stage parasites (EC50 = 0.016 µM) and is curative after just three doses in a P. berghei mouse model. BRD9185 has a long half-life (15 h) and low clearance in mice and represents a new structural class of DHODH inhibitors with potential as antimalarial drugs.