1. |
- Benediktsdottir, Andrea, 1990-, et al.
(författare)
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Antibacterial sulfonimidamide-based oligopeptides as type I signal peptidase inhibitors : Synthesis and biological evaluation
- 2021
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Ingår i: European Journal of Medicinal Chemistry. - : Elsevier. - 0223-5234 .- 1768-3254. ; 224
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Tidskriftsartikel (refereegranskat)abstract
- Oligopeptide boronates with a lipophilic tail are known to inhibit the type I signal peptidase in E. coli, which is a promising drug target for developing novel antibiotics. Antibacterial activity depends on these oligopeptides having a cationic modification to increase their permeation. Unfortunately, this modification is associated with cytotoxicity, motivating the need for novel approaches. The sulfonimidamide functionality has recently gained much interest in drug design and discovery, as a means of introducing chirality and an imine-handle, thus allowing for the incorporation of additional substituents. This in turn can tune the chemical and biological properties, which are here explored. We show that introducing the sulfonimidamide between the lipophilic tail and the peptide in a series of signal peptidase inhibitors resulted in antibacterial activity, while the sulfonamide isostere and previously known non-cationic analogs were inactive. Additionally, we show that replacing the sulfonamide with a sulfonimidamide resulted in decreased cytotoxicity, and similar results were seen by adding a cationic sidechain to the sulfonimidamide motif. This is the first report of incorporation of the sulfonimidamide functional group into bioactive peptides, more specifically into antibacterial oligopeptides, and evaluation of its biological effects.
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2. |
- Benediktsdottir, Andrea, et al.
(författare)
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Design, synthesis, and in vitro biological evaluation of meta-sulfonamidobenzamide-based antibacterial LpxH inhibitors
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- New antibacterial compounds are urgently needed, especially for infections caused by the top-priority Gram-negative bacteria that are increasingly difficult to treat. Lipid A is a key component of the Gram-negative outer membrane and the LpxH enzyme plays an important role in its biosynthesis, making it an ideal antibacterial target. Inspired by previously reported ortho-N-methyl-sulfonamidobenzamide-based LpxH inhibitors, novel benzamide substitutions were explored in this work to assess their in vitro activity. Our findings reveal that maintaining wild-type antibacterial activity necessitates N-methyl removal when shifting the ortho-N-methyl-sulfonamide to the meta-position. This discovery led to the synthesis of meta-sulfonamidobenzamide analogs with potent antibacterial activity and enzyme inhibition. Moreover, we demonstrate that modifying the benzamide scaffold can alter hERG blocking. Furthermore, a LpxH-bound X-ray structure of the meta-sulfonamidobenzamide analog facilitated comparison with complexes with ortho-N-methyl-sulfonamidobenzamide analogs, and with the natural enzymatic reaction product lipid X, providing new insights into the enzyme-ligand interactions. Overall, our study has identified meta-sulfonamidobenzamide derivatives as promising LpxH-targeting hits with the potential for optimization in future antibacterial hit-to-lead programs.
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3. |
- Courtiol-Legourd, Stephanie, et al.
(författare)
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Synthesis and kinetic evaluation of phosphomimetic inhibitors targeting type B ribose-5-phosphate isomerase from Mycobacterium tuberculosis
- 2024
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Ingår i: Bioorganic & Medicinal Chemistry Letters. - : Elsevier. - 0960-894X .- 1464-3405. ; 102
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Tidskriftsartikel (refereegranskat)abstract
- Because tuberculosis is still a major health threat worldwide, identification of new drug targets is urgently needed. In this study, we considered type B ribose -5 -phosphate isomerase from Mycobacterium tuberculosis as a potential target, and addressed known problems of previous inhibitors in terms of their sensitivity to hydrolysis catalyzed by phosphatase enzymes, which impaired their potential use as drugs. To this end, we synthesized six novel phosphomimetic compounds designed to be hydrolytically stable analogs of the substrate ribose 5 -phosphate and the best known inhibitor 5-phospho-D-ribonate. The phosphate function was replaced by phosphonomethyl, sulfate, sulfonomethyl, or malonate groups. Inhibition was evaluated on type A and type B ribose -5phosphate isomerases, and stability towards hydrolysis using alkaline phosphatase and veal serum was assessed. One of the phosphomimetic analogs, 5-deoxy-5-phosphonomethyl-D-ribonate, emerged as the first strong and specific inhibitor of the M. tuberculosis enzyme that is resistant to hydrolysis.
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4. |
- De Rosa, Maria, et al.
(författare)
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Design, synthesis and in vitro biological evaluation of oligopeptides targeting E. coli type I signal peptidase (LepB)
- 2017
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Ingår i: Bioorganic & Medicinal Chemistry. - : Elsevier BV. - 0968-0896 .- 1464-3391. ; 25:3, s. 897-911
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Tidskriftsartikel (refereegranskat)abstract
- Type I signal peptidases are potential targets for the development of new antibacterial agents. Here we report finding potent inhibitors of E. coli type I signal peptidase (LepB), by optimizing a previously reported hit compound, decanoyl-PTANA-CHO, through modifications at the N- and C-termini. Good improvements of inhibitory potency were obtained, with IC50s in the low nanomolar range. The best inhibitors also showed good antimicrobial activity, with MICs in the low μg/mL range for several bacterial species. The selection of resistant mutants provided strong support for LepB as the target of these compounds. The cytotoxicity and hemolytic profiles of these compounds are not optimal but the finding that minor structural changes cause the large effects on these properties suggests that there is potential for optimization in future studies.
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5. |
- Lu, Lu, 1984-, et al.
(författare)
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MEPicides : α,β-Unsaturated Fosmidomycin N-acyl Analogsas inhibitors that selectively target DXR from Plasmodium falciparum, the deadliest causative parasite of human Malaria
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Fosmidomycin and FR-9000098 have been confirmed to show parasiticidal activity against Plasmodium falciparum, targeting DXR involved in the MEP pathway. We designed a construct of PfDXR that has successfully been overexpressed in E. coli BL21(DE3) C43, and purified by IMAC and SEC, with the final yield of 1.2 mg/ 8 L culture. PfDXR was concentrated to 20 mg/ml, and co-crystallized with previously tested inhibitors in the FR-9000098 scaffold in the presence of Mn2+. Three FR-9000098 analogues with double-bonded Ca-Cband/or a phenyl ring with various lengths to N1, showed inhibitory activities with IC50s roughly 50 nM. Three crystals were in triclinic P1space group, with similar dimensions in the unit cell (51Å, 56Å, 86Å, 103°, 103°, 101°). All four complex structures have been crystallographically determined at resolutions in the range 1.86 Å, 2.45 Å, 2.13Å, 2.05 Å. Given the high similarity in structures, the initial phases were determined by rigid body refinement with search model PfDXR-FN3 complex, followed by restrained refinement in refmac5. Subsequently, the ligands and surrounding amino acid residues were manually rebuilt with theqdstools in O. the Ca-Cbbonds of the three ligands were altered from a single to double bond based on the structure of FR9000098. In addition, two ligands were extended at the Cdwith a phenyl group, and with the benzyl group connected by two carbons. N-terminal NADPH binding domains from four complexes undergo minor rigid body movement, and more details of conformational changes in the flap region are discussed.
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6. |
- Lu, Lu, 1984-
(författare)
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Structural and Biochemical Characterizations of Three Potential Drug Targets from Pathogens
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- As antibiotic resistance of various pathogens emerged globally, the need for new effective drugs with novel modes of action became urgent. In this thesis, we focus on infectious diseases, e.g. tuberculosis, malaria, and nosocomial infections, and the corresponding causative pathogens, Mycobacterium tuberculosis, Plasmodium falciparum, and the Gram-negative ESKAPE pathogens that underlie so many healthcare-acquired diseases. Following the same-target-other-pathogen (STOP) strategy, we attempted to comprehensively explore the properties of three promising drug targets.Signal peptidase I (SPase I), existing both in Gram-negative and Gram-positive bacteria, as well as in parasites, is vital for cell viability, due to its critical role in signal peptide cleavage, thus, protein maturation, and secreted protein transport. Three factors, comprising essentiality, a unique mode of action, and easy accessibility, make it an attractive drug target. We have established a platform, investigating the protein purification, enzymatic kinetics, and inhibition. A full-length SPase I from E. coli, including two transmembrane segments, was produced and purified in the presence of 0.5 % Triton X-100. In the in vitro biochemical assay, it exhibits proteolytic activity on antigen 85A from M. tuberculosis, with a Km of 20 µM and a kcat of 135 s-1. A series of macrocyclic oligopeptides that have been proven inhibitory to E. coli SPase I also showed potency against a panel of Gram-negative bacteria. 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is responsible for the production of methylerythritol phosphate (MEP) in the non-mevalonate pathway of isoprenoid biosynthesis, and is thus essential for cell growth. DXRs from M. tuberculosis and P. falciparum have been under investigation in our lab for years. I addressed structural and biochemical characterizations of PfDXR with analogs of 3-(N-formyl-N-hydroxyamino)propyl- phosphonate (fosmidomycin) and 3-(N-acetyl-N-hydroxyamino)propyl- phosphonate (FR-9000098), two natural products showing potency against P. falciparum. Chemical modifications, methylation at Cg, and double bond formation between Ca and Cb, were investigated to increase the pathogenicidal activity. Crystallographic complex structures of PfDXR and four novel compounds inhibitory to PfDXR in a dose-dependent manner were solved, and ligand binding will be discussed in detail.Type II NADH dehydrogenase (NDH-2) is an essential component in the respiratory chain, playing an important role in electron transfer. Biomembrane-bound NDH-2 from M. tuberculosis was over-expressed in E. coli, as well as the homolog from M. smegmatis. The purified NDH-2s were kinetically characterized, and showed a similar affinity to previously reported NDH-2s expressed M. smegmatis. A collection of novel inhibitors in the scaffold of quinolinyl pyrimidines were synthesized and tested for inhibition in a biochemical assay.
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7. |
- Lu, Lu, 1984-, et al.
(författare)
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Synthesis and in vitro biological evaluation of quinolinyl pyrimidines targeting type II NADH-dehydrogenase (NDH-2)
- 2022
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Ingår i: ACS - Infectious Diseases. - : American Chemical Society (ACS). - 2373-8227. ; 8:3, s. 482-498
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Tidskriftsartikel (refereegranskat)abstract
- Type II NADH dehydrogenase (NDH-2) is an essential component of electron transfer in many microbial pathogens but has remained largely unexplored as a potential drug target. Previously, quinolinyl pyrimidines were shown to inhibit Mycobacterium tuberculosis NDH-2, as well as the growth of the bacteria [Shirude, P. S.; ACS Med. Chem. Lett. 2012, 3, 736−740]. Here, we synthesized a number of novel quinolinyl pyrimidines and investigated their properties. In terms of inhibition of the NDH-2 enzymes from M. tuberculosis and Mycobacterium smegmatis, the best compounds were of similar potency to previously reported inhibitors of the same class (half-maximal inhibitory concentration (IC50) values in the low-μM range). However, a number of the compounds had much better activity against Gram-negative pathogens, with minimum inhibitory concentrations (MICs) as low as 2 μg/mL. Multivariate analyses (partial least-squares (PLS) and principle component analysis (PCA)) showed that overall ligand charge was one of the most important factors in determining antibacterial activity, with patterns that varied depending on the particular bacterial species. In some cases (e.g., mycobacteria), there was a clear correlation between the IC50 values and the observed MICs, while in other instances, no such correlation was evident. When tested against a panel of protozoan parasites, the compounds failed to show activity that was not linked to cytotoxicity. Further, a strong correlation between hydrophobicity (estimated as clog P) and cytotoxicity was revealed; more hydrophobic analogues were more cytotoxic. By contrast, antibacterial MIC values and cytotoxicity were not well correlated, suggesting that the quinolinyl pyrimidines can be optimized further as antimicrobial agents.
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8. |
- Singh, Vinayak, et al.
(författare)
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Identification of aminopyrimidine-sulfonamides as potent modulators of Wag31-mediated cell elongation in mycobacteria.
- 2017
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 103:1, s. 13-25
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Tidskriftsartikel (refereegranskat)abstract
- There is an urgent need to discover new anti-tubercular agents with novel mechanisms of action in order to tackle the scourge of drug-resistant tuberculosis. Here, we report the identification of such a molecule - an AminoPYrimidine-Sulfonamide (APYS1) that has potent, bactericidal activity against M. tuberculosis. Mutations in APYS1-resistant M. tuberculosis mapped exclusively to wag31, a gene that encodes a scaffolding protein thought to orchestrate cell elongation. Recombineering confirmed that a Gln201Arg mutation in Wag31 was sufficient to cause resistance to APYS1, however, neither overexpression nor conditional depletion of wag31 impacted M. tuberculosis susceptibility to this compound. In contrast, expression of the wildtype allele of wag31 in APYS1-resistant M. tuberculosis was dominant and restored susceptibility to APYS1 to wildtype levels. Time-lapse imaging and scanning electron microscopy revealed that APYS1 caused gross malformation of the old pole of M. tuberculosis, with eventual lysis. These effects resembled the morphological changes observed following transcriptional silencing of wag31 in M. tuberculosis. These data show that Wag31 is likely not the direct target of APYS1, but the striking phenotypic similarity between APYS1 exposure and genetic depletion of Wag31 in M. tuberculosis suggests that APYS1 might indirectly affect Wag31 through an as yet unknown mechanism.
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9. |
- Sooriyaarachchi, Sanjeewani, et al.
(författare)
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Conformational Changes and Ligand Recognition of Escherichia coli d-Xylose Binding Protein Revealed
- 2010
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 402:4, s. 657-668
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Tidskriftsartikel (refereegranskat)abstract
- ABC transport systems account for most import of necessary nutrients in bacteria. The periplasmic binding component (or an equivalent membrane-anchored protein) is critical to recognizing the cognate ligand and directing it to the appropriate membrane permease. Here we report X-ray structures of D-xylose-binding protein from Escherichia coli in ligand-free open, ligand-bound open and ligand-bound closed forms, at 2.15, 2.2, and 2.2-Å resolution, respectively. The ligand-bound open form is the first such structure to be reported at high resolution; the combination of the three different forms from the same protein furthermore gives unprecedented detail concerning the conformational changes involved in binding protein function. As is typical for the structural family, the protein has two similar globular domains, which are connected by a three-stranded hinge region. The open liganded structure shows that xylose binds first to the C-terminal domain, with only very small conformational changes resulting. After a 34° closing motion, additional interactions are formed with the N-terminal domain; changes in this domain are larger, and serve to make the structure more ordered near the ligand. An analysis of the interactions suggests why xylose is the preferred ligand. Further, a comparison with the most closely related proteins in the structural family shows that the conformational changes are distinct in each type of binding protein, which may have implications for how the individual proteins act in concert with their respective membrane permeases.
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10. |
- Sooriyaarachchi, Sanjeewani, et al.
(författare)
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Targeting an Aromatic Hotspot in Plasmodium falciparum 1-Deoxy-d-xylulose-5-phosphate Reductoisomerase with -Arylpropyl Analogues of Fosmidomycin
- 2016
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Ingår i: ChemMedChem. - : Wiley. - 1860-7179 .- 1860-7187. ; 11:18, s. 2024-2036
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Tidskriftsartikel (refereegranskat)abstract
- Blocking the 2-C-methyl-d-erythrithol-4-phosphate pathway for isoprenoid biosynthesis offers new ways to inhibit the growth of Plasmodium spp. Fosmidomycin [(3-(N-hydroxyformamido)propyl)phosphonic acid, 1] and its acetyl homologue FR-900098 [(3-(N-hydroxyacetamido)propyl)phosphonic acid, 2] potently inhibit 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in this biosynthetic pathway. Arylpropyl substituents were introduced at the -position of the hydroxamate analogue of 2 to study changes in lipophilicity, as well as electronic and steric properties. The potency of several new compounds on the P.falciparum enzyme approaches that of 1 and 2. Activities against the enzyme and parasite correlate well, supporting the mode of action. Seven X-ray structures show that all of the new arylpropyl substituents displace a key tryptophan residue of the active-site flap, which had made favorable interactions with 1 and 2. Plasticity of the flap allows substituents to be accommodated in many ways; in most cases, the flap is largely disordered. Compounds can be separated into two classes based on whether the substituent on the aromatic ring is at the meta or para position. Generally, meta-substituted compounds are better inhibitors, and in both classes, smaller size is linked to better potency.
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