scholarly journals Exploring the Landscape of Diazabicyclooctane (DBO) Inhibition: Avibactam Inactivation of PER-2 β-Lactamase

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Melina Ruggiero ◽  
Krisztina M. Papp-Wallace ◽  
Magdalena A. Taracila ◽  
Maria F. Mojica ◽  
Christopher R. Bethel ◽  
...  
Keyword(s):  
Active Site ◽  
Therapeutic Option ◽  
Structural Features ◽  
Atomic Mass ◽  
Stable Complex ◽  
Water Molecules ◽  
Gram Negative Bacteria ◽  
Biophysical Properties ◽  
Content Type ◽  
Class A

ABSTRACT PER β-lactamases are an emerging family of extended-spectrum β-lactamases (ESBL) found in Gram-negative bacteria. PER β-lactamases are unique among class A enzymes as they possess an inverted omega (Ω) loop and extended B3 β-strand. These singular structural features are hypothesized to contribute to their hydrolytic profile against oxyimino-cephalosporins (e.g., cefotaxime and ceftazidime). Here, we tested the ability of avibactam (AVI), a novel non-β-lactam β-lactamase inhibitor to inactivate PER-2. Interestingly, the PER-2 inhibition constants (i.e., k 2/K = 2 × 103 ± 0.1 × 103 M−1 s−1, where k 2 is the rate constant for acylation (carbamylation) and K is the equilibrium constant) that were obtained when AVI was tested were reminiscent of values observed testing the inhibition by AVI of class C and D β-lactamases (i.e., k 2/K range of ≈103 M−1 s−1) and not class A β-lactamases (i.e., k 2/K range, 104 to 105 M−1 s−1). Once AVI was bound, a stable complex with PER-2 was observed via mass spectrometry (e.g., 31,389 ± 3 atomic mass units [amu] → 31,604 ± 3 amu for 24 h). Molecular modeling of PER-2 with AVI showed that the carbonyl of AVI was located in the oxyanion hole of the β-lactamase and that the sulfate of AVI formed interactions with the β-lactam carboxylate binding site of the PER-2 β-lactamase (R220 and T237). However, hydrophobic patches near the PER-2 active site (by Ser70 and B3-B4 β-strands) were observed and may affect the binding of necessary catalytic water molecules, thus slowing acylation (k 2/K) of AVI onto PER-2. Similar electrostatics and hydrophobicity of the active site were also observed between OXA-48 and PER-2, while CTX-M-15 was more hydrophilic. To demonstrate the ability of AVI to overcome the enhanced cephalosporinase activity of PER-2 β-lactamase, we tested different β-lactam–AVI combinations. By lowering MICs to ≤2 mg/liter, the ceftaroline-AVI combination could represent a favorable therapeutic option against Enterobacteriaceae expressing bla PER-2. Our studies define the inactivation of the PER-2 ESBL by AVI and suggest that the biophysical properties of the active site contribute to determining the efficiency of inactivation.

scholarly journals Relebactam Is a Potent Inhibitor of the KPC-2 β-Lactamase and Restores Imipenem Susceptibility in KPC-Producing Enterobacteriaceae

2018 ◽  
Vol 62 (6) ◽  
Author(s):  
Krisztina M. Papp-Wallace ◽  
Melissa D. Barnes ◽  
Jim Alsop ◽  
Magdalena A. Taracila ◽  
Christopher R. Bethel ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Rate Constant ◽  
Active Site ◽  
Klebsiella Oxytoca ◽  
Enterobacter Aerogenes ◽  
Clinical Isolates ◽  
Piperidine Ring ◽  
Water Molecules ◽  
Content Type ◽  
Site Water

ABSTRACT The imipenem-relebactam combination is in development as a potential treatment regimen for infections caused by Enterobacteriaceae possessing complex β-lactamase backgrounds. Relebactam is a β-lactamase inhibitor that possesses the diazabicyclooctane core, as in avibactam; however, the R1 side chain of relebactam also includes a piperidine ring, whereas that of avibactam is a carboxyamide. Here, we investigated the inactivation of the Klebsiella pneumoniae carbapenemase KPC-2, the most widespread class A carbapenemase, by relebactam and performed susceptibility testing with imipenem-relebactam using KPC-producing clinical isolates of Enterobacteriaceae . MIC measurements using agar dilution methods revealed that all 101 clinical isolates of KPC-producing Enterobacteriaceae ( K. pneumoniae , Klebsiella oxytoca , Enterobacter cloacae , Enterobacter aerogenes , Citrobacter freundii , Citrobacter koseri , and Escherichia coli ) were highly susceptible to imipenem-relebactam (MICs ≤ 2 mg/liter). Relebactam inhibited KPC-2 with a second-order onset of acylation rate constant ( k 2 / K ) value of 24,750 M −1 s −1 and demonstrated a slow off-rate constant ( k off ) of 0.0002 s −1 . Biochemical analysis using time-based mass spectrometry to map intermediates revealed that the KPC-2–relebactam acyl-enzyme complex was stable for up to 24 h. Importantly, desulfation of relebactam was not observed using mass spectrometry. Desulfation and subsequent deacylation have been observed during the reaction of KPC-2 with avibactam. Upon molecular dynamics simulations of relebactam in the KPC-2 active site, we found that the positioning of active-site water molecules is less favorable for desulfation in the KPC-2 active site than it is in the KPC-2–avibactam complex. In the acyl complexes, the water molecules are within 2.5 to 3 Å of the avibactam sulfate; however, they are more than 5 to 6 Å from the relebactam sulfate. As a result, we propose that the KPC-2–relebactam acyl complex is more stable than the KPC-2–avibactam complex. The clinical implications of this difference are not currently known.

scholarly journals Membrane Protein Complex ExbB4-ExbD1-TonB1from Escherichia coli Demonstrates Conformational Plasticity

2015 ◽  
Vol 197 (11) ◽  
pp. 1873-1885 ◽  
Author(s):  
Aleksandr Sverzhinsky ◽  
Jacqueline W. Chung ◽  
Justin C. Deme ◽  
Lucien Fabre ◽  
Kristian T. Levey ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Iron Acquisition ◽  
Proton Translocation ◽  
Three Dimensional ◽  
Structural Data ◽  
Structural Features ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type

ABSTRACTIron acquisition at the outer membrane (OM) of Gram-negative bacteria is powered by the proton motive force (PMF) of the cytoplasmic membrane (CM), harnessed by the CM-embedded complex of ExbB, ExbD, and TonB. Its stoichiometry, ensemble structural features, and mechanism of action are unknown. By panning combinatorial phage libraries, periplasmic regions of dimerization between ExbD and TonB were predicted. Using overexpression of full-length His6-taggedexbB-exbDand S-taggedtonB, we purified detergent-solubilized complexes of ExbB-ExbD-TonB fromEscherichia coli. Protein-detergent complexes of ∼230 kDa with a hydrodynamic radius of ∼6.0 nm were similar to previously purified ExbB4-ExbD2complexes. Significantly, they differed in electronegativity by native agarose gel electrophoresis. The stoichiometry was determined to be ExbB4-ExbD1-TonB1. Single-particle electron microscopy agrees with this stoichiometry. Two-dimensional averaging supported the phage display predictions, showing two forms of ExbD-TonB periplasmic heterodimerization: extensive and distal. Three-dimensional (3D) particle classification showed three representative conformations of ExbB4-ExbD1-TonB1. Based on our structural data, we propose a model in which ExbD shuttles a proton across the CM via an ExbB interprotein rearrangement. Proton translocation would be coupled to ExbD-mediated collapse of extended TonB in complex with ligand-loaded receptors in the OM, followed by repositioning of TonB through extensive dimerization with ExbD. Here we present the first report for purification of the ExbB-ExbD-TonB complex, molar ratios within the complex (4:1:1), and structural biology that provides insights into 3D organization.IMPORTANCEReceptors in the OM of Gram-negative bacteria allow entry of iron-bound siderophores that are necessary for pathogenicity. Numerous iron-acquisition strategies rely upon a ubiquitous and unique protein for energization: TonB. Complexed with ExbB and ExbD, the Ton system links the PMF to OM transport. Blocking iron uptake by targeting a vital nanomachine holds promise in therapeutics. Despite much research, the stoichiometry, structural arrangement, and molecular mechanism of the CM-embedded ExbB-ExbD-TonB complex remain unreported. Here we demonstratein vitroevidence of ExbB4-ExbD1-TonB1complexes. Using 3D EM, we reconstructed the complex in three conformational states that show variable ExbD-TonB heterodimerization. Our structural observations form the basis of a model for TonB-mediated iron acquisition.

scholarly journals Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate

2016 ◽  
Vol 61 (1) ◽  
Author(s):  
Venu Gopal Vandavasi ◽  
Patricia S. Langan ◽  
Kevin L. Weiss ◽  
Jerry M. Parks ◽  
Jonathan B. Cooper ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Active Site Residues ◽  
Hydrogen Atoms ◽  
Content Type ◽  
X Ray Crystallography ◽  
General Base ◽  
Class A ◽  
Extended Spectrum ◽  
Enzyme Intermediate

ABSTRACT The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. The protonation states of active-site residues that are responsible for hydrolysis have been determined previously for the apo form of a CTX-M β-lactamase but not for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a class A β-lactamase in an acyl-enzyme complex with aztreonam, we directly observed most of the hydrogen atoms (as deuterium) within the active site. Although Lys 234 is fully protonated in the acyl intermediate, we found that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, as previously proposed.

scholarly journals Outer Membrane Targeting of Pseudomonas aeruginosa Proteins Shows Variable Dependence on the Components of Bam and Lol Machineries

mBio ◽  
2011 ◽  
Vol 2 (6) ◽  
Author(s):  
Hanh H. Hoang ◽  
Nicholas N. Nickerson ◽  
Vincent T. Lee ◽  
Anastasia Kazimirova ◽  
Mohamed Chami ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Protein Translocation ◽  
Lipid Vesicles ◽  
Structural Features ◽  
Gram Negative Bacteria ◽  
Variable Effect ◽  
Gram Negative ◽  
Content Type

ABSTRACT In Gram-negative bacteria, the Lol and Bam machineries direct the targeting of lipidated and nonlipidated proteins, respectively, to the outer membrane (OM). Using Pseudomonas aeruginosa strains with depleted levels of specific Bam and Lol proteins, we demonstrated a variable dependence of different OM proteins on these targeting pathways. Reduction in the level of BamA significantly affected the ability of the β-barrel membrane protein OprF to localize to the OM, while the targeting of three secretins that are functionally related OM proteins was less affected (PilQ and PscC) or not at all affected (XcpQ). Depletion of LolB affected all lipoproteins examined and had a variable effect on the nonlipidated proteins. While the levels of OprF, PilQ, and PscC were significantly reduced by LolB depletion, XcpQ was unaffected and was correctly localized to the OM. These results suggest that certain β-barrel proteins such as OprF primarily utilize the complete Bam machinery. The Lol machinery participates in the OM targeting of secretins to variable degrees, likely through its involvement in the assembly of lipidated Bam components. XcpQ, but not PilQ or PscC, was shown to assemble spontaneously into liposomes as multimers. This work raises the possibility that there is a gradient of utilization of Bam and Lol insertion and targeting machineries. Structural features of individual proteins, including their β-barrel content, may determine the propensity of these proteins for folding (or misfolding) during periplasmic transit and OM insertion, thereby influencing the extent of utilization of the Bam targeting machinery, respectively. IMPORTANCE Targeting of lipidated and nonlipidated proteins to the outer membrane (OM) compartment in Gram-negative bacteria involves the transfer across the periplasm utilizing the Lol and Bam machineries, respectively. We show that depletion of Bam and Lol components in Pseudomonas aeruginosa does not lead to a general OM protein translocation defect, but the severity (and therefore, Lol and Bam dependence), varies with individual proteins. XcpQ, the secretin component of the type II secretion apparatus, is translocated into the OM without the assistance of Bam or Lol machineries. The hypothesis that XcpQ, after secretion across the cytoplasmic membrane, does not utilize the OM targeting machineries was supported by demonstrating that in vitro-synthesized XcpQ (but not the other P. aeruginosa secretins) can spontaneously incorporate into lipid vesicles. Therefore, the requirement for ancillary factors appears to be, in certain instances, dictated by the intrinsic properties of individual OM proteins, conceivably reflecting their propensities to misfold during periplasmic transit.

scholarly journals Insights Into the Inhibition of MOX-1 β-Lactamase by S02030, a Boronic Acid Transition State Inhibitor

2021 ◽  
Vol 12 ◽  
Author(s):  
Tatsuya Ishikawa ◽  
Nayuta Furukawa ◽  
Emilia Caselli ◽  
Fabio Prati ◽  
Magdalena A. Taracila ◽  
...  
Keyword(s):  
Transition State ◽  
Active Site ◽  
Boronic Acid ◽  
Laboratory Strain ◽  
Multidrug Resistant ◽  
Mechanisms Of Action ◽  
Gram Negative Bacteria ◽  
Class A ◽  
Fixed Concentration ◽  
Active Site Cavity

The rise of multidrug resistant (MDR) Gram-negative bacteria has accelerated the development of novel inhibitors of class A and C β-lactamases. Presently, the search for novel compounds with new mechanisms of action is a clinical and scientific priority. To this end, we determined the 2.13-Å resolution crystal structure of S02030, a boronic acid transition state inhibitor (BATSI), bound to MOX-1 β-lactamase, a plasmid-borne, expanded-spectrum AmpC β-lactamase (ESAC) and compared this to the previously reported aztreonam (ATM)-bound MOX-1 structure. Superposition of these two complexes shows that S02030 binds in the active-site cavity more deeply than ATM. In contrast, the SO3 interactions and the positional change of the β-strand amino acids from Lys315 to Asn320 were more prominent in the ATM-bound structure. MICs were performed using a fixed concentration of S02030 (4 μg/ml) as a proof of principle. Microbiological evaluation against a laboratory strain of Escherichia coli expressing MOX-1 revealed that MICs against ceftazidime are reduced from 2.0 to 0.12 μg/ml when S02030 is added at a concentration of 4 μg/ml. The IC50 and Ki of S02030 vs. MOX-1 were 1.25 ± 0.34 and 0.56 ± 0.03 μM, respectively. Monobactams such as ATM can serve as informative templates for design of mechanism-based inhibitors such as S02030 against ESAC β-lactamases.

scholarly journals Molecular Basis of Class A β-Lactamase Inhibition by Relebactam

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Catherine L. Tooke ◽  
Philip Hinchliffe ◽  
Pauline A. Lang ◽  
Adrian J. Mulholland ◽  
Jürgen Brem ◽  
...  
Keyword(s):  
Resistance Mechanism ◽  
Multidrug Resistant ◽  
Piperidine Ring ◽  
Gram Negative Bacteria ◽  
Important Class ◽  
Phase 3 ◽  
Content Type ◽  
X Ray ◽  
Class A ◽  
Comprehensive Comparison

ABSTRACT β-Lactamase production is the major β-lactam resistance mechanism in Gram-negative bacteria. β-Lactamase inhibitors (BLIs) efficacious against serine β-lactamase (SBL) producers, especially strains carrying the widely disseminated class A enzymes, are required. Relebactam, a diazabicyclooctane (DBO) BLI, is in phase 3 clinical trials in combination with imipenem for the treatment of infections by multidrug-resistant Enterobacteriaceae. We show that relebactam inhibits five clinically important class A SBLs (despite their differing spectra of activity), representing both chromosomal and plasmid-borne enzymes, i.e., the extended-spectrum β-lactamases L2 (inhibition constant 3 μM) and CTX-M-15 (21 μM) and the carbapenemases KPC-2, -3, and -4 (1 to 5 μM). Against purified class A SBLs, relebactam is an inferior inhibitor compared with the clinically approved DBO avibactam (9- to 120-fold differences in half maximal inhibitory concentration [IC50]). MIC assays indicate relebactam potentiates β-lactam (imipenem) activity against KPC-producing Klebsiella pneumoniae, with similar potency to avibactam (with ceftazidime). Relebactam is less effective than avibactam in combination with aztreonam against Stenotrophomonas maltophilia K279a. X-ray crystal structures of relebactam bound to CTX-M-15, L2, KPC-2, KPC-3, and KPC-4 reveal its C2-linked piperidine ring can sterically clash with Asn104 (CTX-M-15) or His/Trp105 (L2 and KPCs), rationalizing its poorer inhibition activity than that of avibactam, which has a smaller C2 carboxyamide group. Mass spectrometry and crystallographic data show slow, pH-dependent relebactam desulfation by KPC-2, -3, and -4. This comprehensive comparison of relebactam binding across five clinically important class A SBLs will inform the design of future DBOs, with the aim of improving clinical efficacy of BLI–β-lactam combinations.

scholarly journals Ligand-Dependent Disorder of the Ω Loop Observed in Extended-Spectrum SHV-Type β-Lactamase

2011 ◽  
Vol 55 (5) ◽  
pp. 2303-2309 ◽  
Author(s):  
Jared M. Sampson ◽  
Wei Ke ◽  
Christopher R. Bethel ◽  
S. R. R. Pagadala ◽  
Michael D. Nottingham ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Gram Negative Bacteria ◽  
Structural Alterations ◽  
Critical Amino Acid ◽  
Class A ◽  
Extended Spectrum ◽  
Loop Flexibility ◽  
Bacteria Resistance ◽  
Lactamase Inhibitor

ABSTRACTAmong Gram-negative bacteria, resistance to β-lactams is mediated primarily by β-lactamases (EC 3.2.6.5), periplasmic enzymes that inactivate β-lactam antibiotics. Substitutions at critical amino acid positions in the class A β-lactamase families result in enzymes that can hydrolyze extended-spectrum cephalosporins, thus demonstrating an “extended-spectrum” β-lactamase (ESBL) phenotype. Using SHV ESBLs with substitutions in the Ω loop (R164H and R164S) as target enzymes to understand this enhanced biochemical capability and to serve as a basis for novel β-lactamase inhibitor development, we determined the spectra of activity and crystal structures of these variants. We also studied the inactivation of the R164H and R164S mutants with tazobactam and SA2-13, a unique β-lactamase inhibitor that undergoes a distinctive reaction chemistry in the active site. We noted that the reducedKivalues for the R164H and R164S mutants with SA2-13 are comparable to those with tazobactam (submicromolar). The apo enzyme crystal structures of the R164H and R164S SHV variants revealed an ordered Ω loop architecture that became disordered when SA2-13 was bound. Important structural alterations that result from the binding of SA2-13 explain the enhanced susceptibility of these ESBL enzymes to this inhibitor and highlight ligand-dependent Ω loop flexibility as a mechanism for accommodating and hydrolyzing β-lactam substrates.

scholarly journals Structural Basis of Functional Diversification of the HD-GYP Domain Revealed by the Pseudomonas aeruginosa PA4781 Protein, Which Displays an Unselective Bimetallic Binding Site

2015 ◽  
Vol 197 (8) ◽  
pp. 1525-1535 ◽  
Author(s):  
Serena Rinaldo ◽  
Alessandro Paiardini ◽  
Valentina Stelitano ◽  
Paolo Brunotti ◽  
Laura Cervoni ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Active Site ◽  
Metal Binding ◽  
Binding Mode ◽  
Structural Features ◽  
Structural Basis ◽  
Content Type ◽  
Secondary Messenger ◽  
Wide Range

ABSTRACTThe intracellular level of the bacterial secondary messenger cyclic di-3′,5′-GMP (c-di-GMP) is determined by a balance between its biosynthesis and degradation, the latter achieved via dedicated phosphodiesterases (PDEs) bearing a characteristic EAL or HD-GYP domain. We here report the crystal structure of PA4781, one of the threePseudomonas aeruginosaHD-GYP proteins, which we have previously characterizedin vitro. The structure shows a bimetallic active site whose metal binding mode is different from those of both HD-GYP PDEs characterized so far. Purified PA4781 does not contain iron in the active site as for other HD-GYPs, and we show that it binds to a wide range of transition metals with similar affinities. Moreover, the structural features of PA4781 indicate that this is preferentially a pGpG binding protein, as we previously suggested. Our results point out that the structural features of HD-GYPs are more complex than predicted so far and identify the HD-GYP domain as a conserved scaffold which has evolved to preferentially interact with a partner GGDEF but which harbors different functions obtained through diversification of the active site.IMPORTANCEIn bacteria, the capability to form biofilms, responsible for increased pathogenicity and antibiotic resistance, is almost universally stimulated by the second messenger cyclic di-GMP (c-di-GMP). To design successful strategies for targeting biofilm formation, a detailed characterization of the enzymes involved in c-di-GMP metabolism is crucial. We solved the structure of the HD-GYP domain of PA4781 fromPseudomonas aeruginosa, involved in c-di-GMP degradation. This is the third structure of this class of phosphodiesterases to be solved, and with respect to its homologues, it shows significant differences both in the nature and in the binding mode of the coordinated metals, indicating that HD-GYP proteins are able to fine-tune their function, thereby increasing the chances of the microorganism to adapt to different environmental needs.

scholarly journals Substrate Spectrum Extension of PenA in Burkholderia thailandensis with a Single Amino Acid Deletion, Glu168del

2012 ◽  
Vol 56 (7) ◽  
pp. 4005-4008 ◽  
Author(s):  
Hyojeong Yi ◽  
Karan Kim ◽  
Kwang-Hwi Cho ◽  
Oksung Jung ◽  
Heenam Stanley Kim
Keyword(s):  
Deletion Mutation ◽  
Single Amino Acid ◽  
Functional Domain ◽  
Amino Acid Deletion ◽  
Burkholderia Thailandensis ◽  
Content Type ◽  
Class A ◽  
Omega Loop ◽  
Substitution Mutations ◽  
Substrate Spectrum

ABSTRACTWe describe a deletion mutation in a class A β-lactamase, PenA, ofBurkholderia thailandensisthat extended the substrate spectrum of the enzyme to include ceftazidime. Glu168del was located in a functional domain called the omega loop causing expansion of the space in the loop, which in turn increased flexibility at the active site. This deletion mutation represents a rare but significant alternative mechanical path to substrate spectrum extension in PenA besides more common substitution mutations.

scholarly journals Toward the Identification of Potential α-Ketoamide Covalent Inhibitors for SARS-CoV-2 Main Protease: Fragment-Based Drug Design and MM-PBSA Calculations

Processes ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 1004
Author(s):  
Mahmoud A. El Hassab ◽  
Mohamed Fares ◽  
Mohammed K. Abdel-Hamid Amin ◽  
Sara T. Al-Rashood ◽  
Amal Alharbi ◽  
...  
Keyword(s):  
Drug Design ◽  
Active Site ◽  
Binding Free Energy ◽  
Stable Complex ◽  
Active Site Residues ◽  
Structure Based Drug Design ◽  
Enzyme Active Site ◽  
Potential Inhibitor ◽  
Main Protease ◽  
Covalent Docking

Since December 2019, the world has been facing the outbreak of the SARS-CoV-2 pandemic that has infected more than 149 million and killed 3.1 million people by 27 April 2021, according to WHO statistics. Safety measures and precautions taken by many countries seem insufficient, especially with no specific approved drugs against the virus. This has created an urgent need to fast track the development of new medication against the virus in order to alleviate the problem and meet public expectations. The SARS-CoV-2 3CL main protease (Mpro) is one of the most attractive targets in the virus life cycle, which is responsible for the processing of the viral polyprotein and is a key for the ribosomal translation of the SARS-CoV-2 genome. In this work, we targeted this enzyme through a structure-based drug design (SBDD) protocol, which aimed at the design of a new potential inhibitor for Mpro. The protocol involves three major steps: fragment-based drug design (FBDD), covalent docking and molecular dynamics (MD) simulation with the calculation of the designed molecule binding free energy at a high level of theory. The FBDD step identified five molecular fragments, which were linked via a suitable carbon linker, to construct our designed compound RMH148. The mode of binding and initial interactions between RMH148 and the enzyme active site was established in the second step of our protocol via covalent docking. The final step involved the use of MD simulations to test for the stability of the docked RMH148 into the Mpro active site and included precise calculations for potential interactions with active site residues and binding free energies. The results introduced RMH148 as a potential inhibitor for the SARS-CoV-2 Mpro enzyme, which was able to achieve various interactions with the enzyme and forms a highly stable complex at the active site even better than the co-crystalized reference.

Sign in / Sign up

Export Citation Format

Share Document