Structure-Based Optimization of a Non-\u3b2-lactam Lead Results in Inhibitors That Do Not Up-Regulate \u3b2-Lactamase Expression in Cell Culture

Bacterial expression of \u3b2-lactamases is the most widespread resistance mechanism to \u3b2 -lactam antibiotics. There is a pressing need for novel, non-\u3b2-lactam inhibitors of these enzymes. Our lead, compound 1, is chemically dissimilar to \u3b2 -lactams and is a noncovalent, competitive inhibitor of the enzyme. However, at 26 \u3bcM its activity is modest (Figure 1). Using the X-ray structure of the AmpC/1 complex as a template, 14 analogues were designed and synthesized. Among these, compound 10, had a Ki of 1 \u3bcM, 26-fold better than the lead. The structures of AmpC in complex with compound 10 and an analogue, compound 11, were determined by X-ray crystallography to 1.97 and 1.96 \uc5, respectively. Compound 10 was active in cell culture, reversing resistance to the third generation cephalosporin ceftazidime in bacterial pathogens expressing AmpC. In contrast to \u3b2-lactam-based inhibitors compound 10 did not up-regulate \u3b2-lactamase expression in cell culture but simply inhibited the enzyme expressed by the resistant bacteria. Its escape from this resistance mechanism derives from its dissimilarity to \u3b2 -lactam antibiotics

Predicting and harnessing protein flexibility in the design of species-specific inhibitors of thymidylate synthase1,21Escherichia coli thymidylate synthase numbering is used unless otherwise noted.2PDB coordinates have been deposited with the RCSB with accession ID: 1JG0.

AbstractBackground: Protein plasticity in response to ligand binding abrogates the notion of a rigid receptor site. Thus, computational docking alone misses important prospective drug design leads. Bacterial-specific inhibitors of an essential enzyme, thymidylate synthase (TS), were developed using a combination of computer-based screening followed by in-parallel synthetic elaboration and enzyme assay [Tondi et al. (1999) Chem. Biol. 6, 319–331]. Specificity was achieved through protein plasticity and despite the very high sequence conservation of the enzyme between species.Results: The most potent of the inhibitors synthesized, N,O-didansyl-L-tyrosine (DDT), binds to Lactobacillus casei TS (LcTS) with 35-fold higher affinity and to Escherichia coli TS (EcTS) with 24-fold higher affinity than to human TS (hTS). To reveal the molecular basis for this specificity, we have determined the crystal structure of EcTS complexed with DDT and 2′-deoxyuridine-5′-monophosphate (dUMP). The 2.0 Å structure shows that DDT binds to EcTS in a conformation not predicted by molecular docking studies and substantially differently than other TS inhibitors. Binding of DDT is accompanied by large rearrangements of the protein both near and distal to the enzyme’s active site with movement of Cα carbons up to 6 Å relative to other ternary complexes. This protein plasticity results in novel interactions with DDT including the formation of hydrogen bonds and van der Waals interactions to residues conserved in bacterial TS but not hTS and which are hypothesized to account for DDT’s specificity. The conformation DDT adopts when bound to EcTS explains the activity of several other LcTS inhibitors synthesized in-parallel with DDT suggesting that DDT binds to the two enzymes in similar orientations.Conclusions: Dramatic protein rearrangements involving both main and side chain atoms play an important role in the recognition of DDT by EcTS and highlight the importance of incorporating protein plasticity in drug design. The crystal structure of the EcTS/dUMP/DDT complex is a model system to develop more selective TS inhibitors aimed at pathogenic bacterial species. The crystal structure also suggests a general formula for identifying regions of TS and other enzymes that may be treated as flexible to aid in computational methods of drug discovery

Structure-based discovery and in-parallel optimization of novelcompetitive inhibitors of thymidylate synthase

AbstractBackground:The substrate sites of enzymes are attractive targets for structurebased inhibitor design. Two difficulties hinder efforts to discover and elaborate new (nonsubstrate-like) inhibitors for these sites. First, novel inhibitors often bind at nonsubstrate sites. Second, a novel scaffold introduces chemistry that is frequently unfamiliar, making synthetic elaboration challenging.Results:In an effort to discover and elaborate a novel scaffold for a substrate site, we combined structure-based screening with in-parallel synthetic elaboration. These techniques were used to find new inhibitors that bound to the folate site of Lactobacillus casei thymidylate synthase (LcTS), an enzyme that is a potential target for proliferative diseases, and is highly studied. The available chemicals directory was screened, using a molecular-docking computer program, for molecules that complemented the three-dimensional structure of this site. Five high-ranking compounds were selected for testing. Activity and clocking studies led to a derivative of one of these, dansyltyrosine (Ki 65 μM. Using solid-phase in-parallel techniques 33 derivatives of this lead were synthesized and tested. These analogs are dissimilar to the substrate but bind competitively with it. The most active analog had a Ki of 1.3 μM. The tighter binding inhibitors were also the most specific for LcTS versus related enzymes.Conclusions:TS can recognize inhibitors that are dissimilar to, but that bind competitively with, the folate substrate. Combining structure-based discovery with in-parallel synthetic techniques allowed the rapid elaboration of this series of compounds. More automated versions of this approach can be envisaged

Structure-based design and in-parallel synthesis of inhibitors of AmpC beta-lactamase

Background: Group I p-lactamases are a major cause of antibiotic resistance to beta -lactams such as penicillins and cephalosporins. These enzymes are only modestly affected by classic beta -lactam-based inhibitors, such as clavulanic acid. Conversely, small arylboronic acids inhibit these enzymes at sub-micromolar concentrations. Structural studies suggest these inhibitors bind to a well-defined cleft in the group I beta -lactamase AmpC; this cleft binds the ubiquitous R1 side chain of beta -lactams. Intriguingly, much of this cleft is left unoccupied by the small arylboronic acids. Results: To investigate if larger boronic acids might take advantage of this cleft, structure-guided in-parallel synthesis was used to explore new inhibitors of AmpC. Twenty-eight derivatives of the lead compound, 3-aminophenylboronic acid, led to an inhibitor with 80-fold better binding (2; K-i 83 nM). Molecular docking suggested orientations for this compound in the R1 cleft. Based on the docking results, 12 derivatives of 2 were synthesized, leading to inhibitors with iii values of 60 nM and with improved solubility. Several of these inhibitors reversed the resistance of nosocomial Gram-positive bacteria, though they showed little activity against Gram-negative bacteria. The X-ray crystal structure of compound 2 in complex with AmpC was subsequently determined to 2.1 Angstrom resolution. The placement of the proximal two-thirds of the inhibitor in the experimental structure corresponds with the docked structure, but a bond rotation leads to a distinctly different placement of the distal part of the inhibitor. In the experimental structure, the inhibitor interacts with conserved residues in the R1 cleft whose role in recognition has not been previously explored. Conclusions: Combining structure-based design with in-parallel synthesis allowed for the rapid exploration of inhibitor functionality in the R1 cleft of AmpC. The resulting inhibitors differ considerably from beta -lactams but nevertheless inhibit the enzyme well. The crystal structure of 2 (K-i 83 nM) in complex with AmpC may guide exploration of a highly conserved, largely unexplored cleft, providing a template for further design against AmpC beta -lactamase

In silico identification and experimental validation of hits active against KPC-2 \u3b2-lactamase

Bacterial resistance has become a worldwide concern, particularly after the emergence of resistant strains overproducing carbapenemases. Among these, the KPC-2 carbapenemase represents a significant clinical challenge, being characterized by a broad substrate spectrum that includes aminothiazoleoxime and cephalosporins such as cefotaxime. Moreover, strains harboring KPC-type \u3b2-lactamases are often reported as resistant to available \u3b2-lactamase inhibitors (clavulanic acid, tazobactam and sulbactam). Therefore, the identification of novel non \u3b2-lactam KPC-2 inhibitors is strongly necessary to maintain treatment options. This study explored novel, non-covalent inhibitors active against KPC-2, as putative hit candidates. We performed a structure-based in silico screening of commercially available compounds for non-\u3b2-lactam KPC-2 inhibitors. Thirty-two commercially available high-scoring, fragment-like hits were selected for in vitro validation and their activity and mechanism of action vs the target was experimentally evaluated using recombinant KPC-2. N-(3-(1H-tetrazol-5-yl)phenyl)-3-fluorobenzamide (11a), in light of its ligand efficiency (LE = 0.28 kcal/mol/non-hydrogen atom) and chemistry, was selected as hit to be directed to chemical optimization to improve potency vs the enzyme and explore structural requirement for inhibition in KPC-2 binding site. Further, the compounds were evaluated against clinical strains overexpressing KPC-2 and the most promising compound reduced the MIC of the \u3b2-lactam antibiotic meropenem by four fold

\u201cA step further in the discovery of phthalein derivatives as Thymidylate Synthase inhibitors\u201d

Phenolphthalein (Pth) was discovered as a low micromolar inhibitor of the enzyme ThymidylateSynthase (TS), an important target for anticancer chemotherapy. In the present work, a newseries of Pth derivatives have been designed and synthesized. All the compounds have beencharacterized through NMR techniques. A set of twelve Pth derivatives has been tested againstthree TS enzymes and their bio-profiles obtained. The bio-profiling studies suggest that theinhibitory potency of the compounds has been improved of about fifty times againstLactobacillus casei TS (LcTS) and five times against humant TS (hTS) with respect to the lead.The most active compound shows an inhibition constant (Ki) of 70 nM against Escherichia coliTS (EcTS)

Protocetraric and Salazinic Acids as Potential Inhibitors of SARS-CoV-2 3CL Protease: Biochemical, Cytotoxic, and Computational Characterization of Depsidones as Slow-Binding Inactivators

The study investigated the inhibitory activity of protocetraric and salazinic acids against SARS-CoV-2 3CL(pro). The kinetic parameters were determined by microtiter plate-reading fluorimeter using a fluorogenic substrate. The cytotoxic activity was tested on murine Sertoli TM4 cells. In silico analysis was performed to ascertain the nature of the binding with the 3CL(pro). The compounds are slow-binding inactivators of 3CL(pro) with a K(i) of 3.95 μM and 3.77 μM for protocetraric and salazinic acid, respectively, and inhibitory efficiency k(inact)/K(i) at about 3 × 10(−5) s(−1)µM(−1). The mechanism of inhibition shows that both compounds act as competitive inhibitors with the formation of a stable covalent adduct. The viability assay on epithelial cells revealed that none of them shows cytotoxicity up to 80 μM, which is well below the K(i) values. By molecular modelling, we predicted that the catalytic Cys145 makes a nucleophilic attack on the carbonyl carbon of the cyclic ester common to both inhibitors, forming a stably acyl-enzyme complex. The computational and kinetic analyses confirm the formation of a stable acyl-enzyme complex with 3CL(pro). The results obtained enrich the knowledge of the already numerous biological activities exhibited by lichen secondary metabolites, paving the way for developing promising scaffolds for the design of cysteine enzyme inhibitors

4-Amino-1,2,4-triazole-3-thione as a Promising Scaffold for the Inhibition of Serine and Metallo-\u3b2-Lactamases

The emergence of bacteria that co-express serine- and metallo- carbapenemases is a threat to the efficacy of the available \u3b2-lactam antibiotic armamentarium. The 4-amino-1,2,4-triazole-3-thione scaffold has been selected as the starting chemical moiety in the design of a small library of \u3b2-Lactamase inhibitors (BLIs) with extended activity profiles. The synthesised compounds have been validated in vitro against class A serine \u3b2 12Lactamase (SBLs) KPC-2 and class B1 metallo \u3b2 12Lactamases (MBLs) VIM-1 and IMP-1. Of the synthesised derivatives, four compounds showed cross-class micromolar inhibition potency and therefore underwent in silico analyses to elucidate their binding mode within the catalytic pockets of serine- and metallo-BLs. Moreover, several members of the synthesised library have been evaluated, in combination with meropenem (MEM), against clinical strains that overexpress BLs for their ability to synergise carbapenems

Structure-Based Optimization of 1,2,4-Triazole-3-Thione Derivatives: Improving Inhibition of NDM-/VIM-Type Metallo-β-Lactamases and Synergistic Activity on Resistant Bacteria

The worldwide emergence and dissemination of Gram-negative bacteria expressing metallo-beta-lactamases (MBLs) menace the efficacy of all beta-lactam antibiotics, including carbapenems, a last-line treatment usually restricted to severe pneumonia and urinary tract infections. Nonetheless, no MBL inhibitor is yet available in therapy. We previously identified a series of 1,2,4-triazole-3-thione derivatives acting as micromolar inhibitors of MBLs in vitro, but devoid of synergistic activity in microbiological assays. Here, via a multidisciplinary approach, including molecular modelling, synthesis, enzymology, microbiology, and X-ray crystallography, we optimized this series of compounds and identified low micromolar inhibitors active against clinically relevant MBLs (NDM-1- and VIM-type). The best inhibitors increased, to a certain extent, the susceptibility of NDM-1- and VIM-4-producing clinical isolates to meropenem. X-ray structures of three selected inhibitors in complex with NDM-1 elucidated molecular recognition at the base of potency improvement, confirmed in silico predicted orientation, and will guide further development steps