GPU Accelerated Quantum Virtual Screening: Application for the Natural Inhibitors of New Dehli Metalloprotein (NDM-1)

Quantum mechanical approaches for the massive computation on large biological system such as virtual screening in drug design and development have presented a challenge to computational chemists for many years. In this study, we demonstrated that by taking advantage of rapid growth of GPU-based hardware and software (i.e., teraChem), it is feasible to perform virtual screening of a refined chemical library at quantum mechanical level in order to identify the lead compounds with improved accuracy, especially for the drug targets such as metalloproteins in which significant charge transfer and polarization occur amongst the metal ions and their coordinated amino acids. Our calculations predicted four nature compounds (i.e., Curcumin, Catechin, menthol, and Ferulic acid) as the suitable inhibitors for antibiotics resistance against New Delhi Metallo-β-lactamase-1 (NDM-1). Molecular orbitals (MOs) of the QM region of metal ions and their coordinated residues indicate that the bridged hydroxide ion delocalized the electron over the Zn-OH-Zn group at HOMO, different from MOs when the OH− is not presented in NDM-1. This indicates that the bridged hydroxide ion plays an important role in the design of antibiotics and other inhibitors targeting the metalloproteins

DEVELOPMENT OF OPEN LACTAM AND CAPTOPRIL ANALOGUES FOR THE COVALENT INHIBITION OF METALLO-β-LACTAMASES

ABSTRACT DEVELOPMENT OF OPEN LACTAM AND CAPTOPRIL ANALOGUES FOR THE COVALENT INHIBITION OF METALLO-β-LACTAMASES ByMARIE-JOSIANE OHOUEU University of New Hampshire, May 2021 The synthesis of a series of compounds designed to act as inhibitors of metallo-β-lactamase enzymes (MBLs), a sub-class of β-lactamases found in several clinically difficult to treat bacteria that are responsible for the widespread β-lactam antibiotic resistance, are described. The strategy involves the introduction of a functional group, such as an epoxide or thiirane, in the designed inhibitors capable of covalently binding the MBL targets and shutting them down irreversibly. This would prevent the enzymes from hydrolyzing the antibiotic drugs which would maintain their efficacy as a form of treatment. This was first attempted through the development of a convergent synthesis which involved the formation of L- and D-vinylglycine methyl ester, serving for the incorporation of the strained member ring, in a five-step synthetic pathway. This was subsequently introduced using coupling chemistry to a dipeptide. The intermediate dipeptide precursor synthesized through amino acid coupling was phenylglycine-serine (Phe-Ser) followed by a phenylacetic acid-serine (PAA-Ser), which both mimic an open lactam structure. They were subjected to halogenation to convert the serine alcohol functional group to a bromide for the alkylation reaction with the amino group contained in the protected vinylglycine. However, the bromination of Phe-Ser proved to be difficult while the formation of the desired tripeptide with the brominated PAA-Ser was not observed. Evidence of an alkene product was observed which was attributed to the acidic proton at the α-position favoring the elimination of the bromine. Those limitations led to the modification of the serine core to aspartic acid which was thought to circumvent the elimination issue by introducing the vinylglycine by amide bond formation rather than alkylation. Investigation with the phenylacetic-acid-aspartic acid dipeptide led to a promising route in which the coupling of the vinylglycine was achieved efficiently. The subsequent last steps of epoxidation of the alkene and deprotection seemed to be successful although optimization of these is still required. Another strategy for the development of covalent inhibitors was the synthesis of compounds inspired from L-captopril, an inhibitor of angiotensin converting enzyme (ACE inhibitors) which plays a role in heart attack. Here, the strategy involves the synthesis of an alkene-containing intermediate with 2-methylprop-2-enoic acid or 2-methyl-3-butenoic acid through acylation of proline ethyl ester with the corresponding acyl chlorides. The intermediates were successfully obtained enabling the formation of the epoxide and thiirane compounds. Subsequently, the ethyl ester hydrolysis was done to get the final derivatives 1-(2-methyloxirane-2-carbonyl) pyrrolidine-2-carboxylic acid (82) and 1-(2-methylthiirane-2-carbonyl) pyrrolidine-2-carboxylic acid (83) with evidence of the formation of the desired 82 and 83. In the case of the longer chain analogues, 1-[2-(oxirane-2yl)propanoyl] pyrrolidine-2-carboylic acid (84) and 1-[2-(thiirane-2yl)propanoyl] pyrrolidine-2-carboylic acid (85), the deprotection led to the isolation of the final thiirane compound 85 in an overall 5% yield while this last deprotection step remains to be optimized to obtain 84. The synthetic pathway of the open lactam derivatives was overall successful with only the last two steps requiring further optimization which would provide a new class of β-lactamase inhibitors. The pathway for the development of the proline derivatives afforded efficiently one of the desired captopril derivatives while the purification of last step to isolate the remaining compounds needs to be improved. The strategy presented could be used in the future to provide further library compounds for MBL inhibition for further studies

Virtual screening identifies broad-spectrum \u3b2-lactamase inhibitors with activity on clinically relevant serine- and metallo-carbapenemases

Bacteria are known to evade \u3b2-lactam antibiotic action by producing \u3b2-lactamases (BLs), including carbapenemases, which are able to hydrolyze nearly all available \u3b2-lactams. The production of BLs represents one of the best known and most targeted mechanisms of resistance in bacteria. We have performed the parallel screening of commercially available compounds against a panel of clinically relevant BLs: class A CTX-M-15 and KPC-2, subclass B1 NDM-1 and VIM-2 MBLs, and the class C P. aeruginosa AmpC. The results show that all BLs prefer scaffolds having electron pair donors: KPC-2 is preferentially inhibited by sulfonamide and tetrazole-based derivatives, NDM-1 by compounds bearing a thiol, a thiosemicarbazide or thiosemicarbazone moiety, while VIM-2 by triazole-containing molecules. Few broad-spectrum BLs inhibitors were identified; among these, compound 40 potentiates imipenem activity against an NDM-1-producing E. coli clinical strain. The binary complexes of the two most promising compounds binding NDM-1 and VIM-2 were obtained at high resolution, providing strong insights to improve molecular docking simulations, especially regarding the interaction of MBLs with inhibitors

Biochemical and Microbiological Investigations of Inhibitors for β-Lactamases from Human Pathogenic Bacteria

The majority of antibiotics prescribed for treatment of bacterial infections are β-lactam antibiotics. Resistance to these has evolved in a few different ways, notably by regulating permeability and by the expression of β-lactamases which hydrolyze the antibiotic before it reaches its target. Three of the classes of β-lactamases (classes A, C and D) are serine-β-lactamases (SBLs) and the fourth class (Class B) consists of metallo-β-lactamases (MBLs) that rely on one or two zinc ions for their catalytic activity. Bacteria producing β-lactamases that are capable of hydrolyzing the β-lactam bond in all of the classes of β-lactam antibiotics including penicillins, monobactams, cephalosporins and carbapenems are of great clinical concern. As a consequence of the increasing prevalence of resistance, there is much interest in the discovery of inhibitors for such clinically important β-lactamases as well as in the discovery of β-lactam antibiotics that are less susceptible to inactivation by β-lactamases. Described in this thesis are the kinetic properties of new chromogenic cephalosporin-type substrates that are susceptible to hydrolysis by clinically important SBLs and MBLs but that exhibit a much more pronounced colour change upon hydrolysis than does the commercially available and widely used chromogenic cephalosporin called nitrocefin. Some of these substrates also offer more favourable kinetic properties for assaying MBLs in vivo. Also in this thesis, biochemical as well as microbiological investigations of several classes of SBL and MBL inhibitors are describes as well as one class of cephalosporins that exhibit inhibition of MBLs and surprising antibacterial potency against certain clinically significant MBL-producing Gram negative bacteria. More specifically, 6-phosphonomethylpyridine-2-carboxylates (PMPCs) and a number of derivatives thereof, synthesized previously in this research group have been shown in this thesis research to be potent inhibitors (low to submicromolar Ki) of the major Class B1 MBLs, IMP-1, VIM-2, NDM-1 and SPM-1 as well as the Class B3 MBL L1, all of which are dizinc enzymes, and somewhat less potent inhibitors of the monozinc Class B2 MBL, SFH-1, which is of lesser clinical significance. These compounds that are expected to exhibit metal-binding characteristics were found to exhibit a time-dependent inhibition mechanism which fits a kinetic mechanism that is consistent with slow binding to the active site and even slower release of the inhibitor without expulsion of the metal ions form the MBL active site. Microbiological investigations were also carried out involving combinations of PMPCs with the carbapenem antibiotic meropenem and demonstrated an ability of the PMPCs to lower the MICs of meropenem against of MBL-producing clinical strains of Eschericia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii and Stenotrophomonas maltophilia thus encouraging further research on even more potent PMPCs for potential clinical use in combination with carbapenems. Another class of MBL inhibitors that was studied consist of cephalosporin derivatives that incorporate an aromatic thioester linked to C3ʹ of the cephalosporin core. It was found that inhibition of the Class B3 MBL L1 was likely the consequence largely of the binding of an arylthioacid conjugate base to the active site zinc ions after its expulsion from the hydrolysis product of the cephalosporin. This led to a study of the MBL-inhibitory properties of a series of synthetic arylthioacids, a class of compounds that have not been well studied as metallo enzyme inhibitors. These compounds were found to be poor inhibitors of Class B1 MBLs (IMP-1, VIM-2, NDM-2 and SPM-1) but good inhibitors of the B3 MBL L1. These observations are consistent with inhibition of Class B1 MBLs arising not from the arylthicarboxylate released but from binding the cephalosporin-derived metal-binding species formed upon expulsion of the arylthiocarboylate from the active site. A further enzyme kinetic study revealed that synthetic samples of pyridine-2,6-bis(carbothioic) acid and 6-thiocarboxy-picolinic acid, previously known as Fe3+-binding siderophores produced by some species of Pseudomonas, were good inhibitors of Class B1 and B3 MBLs and also exhibited an ability to lower the MIC of meropenem against MBL-producing clinically important Gram negative bacteria. One cephalosporin called UW-123 with at C3ʹ-arylacylthio group and a siderophore mimic attached to the amide group at C7 was studied in some detail microbiologically and found to exhibit good standalone antibiotic activity against certain MBL-producing Gram-negative bacteria especially those producing the widespread MBL VIM-2. UW-123 was also shown to be bacteriostatic and to bind preferentially to induce filamentation of E. coli cells. It was found to bind most strongly to PBP 3 and 1a but also significantly to PBP1b and 4 in P. aeruginosa. In E. coli, UW-123 bound most tightly to PBP3 but also significantly to PBP1a/b and PBP2. Finally, the ability of a cyclobutanone mimic of penem antibiotics JJ05-1058, previously prepared in this laboratory, to inhibit both SBLs and MBLs was demonstrated and the ability of this compound to bind to the low molecular weight penicillin binding proteins was observed suggesting that such compound may have some promise as broad spectrum MBL/SBL inhibitors and possible also as antibiotics that inhibit penicillin binding proteins

Structural Dynamics of L1 and L2 β-lactamase

Stenotrophomonas maltophilia is a Gram-negative bacterium, found in several different environments, such as soil, water and hospital. It can cause multiple infections but also has strong resistance to many antibiotics such as cephalosporins, carbapenems, and aminoglycosides. S. maltophilia confers antibiotic resistance through expression of two different β-lactamases: L1-metallo-β-lactamase (L1 MBL) and L2 β-lactamase. L1 MBL is a class B3 β-lactamase and is the only known tetrameric β-lactamase known to humans. L2 is a class A β-lactamase which has been recently identified. In L1 MBL, there are, two loops (α3-β7 and β12-α5) known as the gating loops, that enclose the active site. The “open” and “close” conformations of these two loops were observed in the molecular dynamic simulation. These two conformations allow the gate loops have the ability of controlling the volume of the zinc binding pocket. The pocket size affects the substrate binding and further influence the catalytic activity of the whole protein. Therefore, gating loops are thought to have an important role in substrate binding and catalysis. In this thesis, the dynamics of the gating loops is explored through Markov state models. The “open” and “closed” confirmations are defined and three key interactions (salt bridge between R236 and D150c, the π–π stacking between H151 and Y227 and the orientation of P225) were identified that play an important role in controlling the conformation of the gating loops. Furthermore, as a tetramer, the correlation between the four subunits was also explored through CVAE-based deep learning and network analysis. The results revealed a ‘dimer of dimer’ dynamics in L1 MBL. The second part of the thesis focuses on exploring the dynamics of L2 β-lactamase family consisting of L2a, L2b, L2c and L2d enzymes. Homology modelling, MDLovofit, Markov state models, dynamic cross-correlation analysis and CVAE-based deep learning were employed for identifying potential key interactions and dynamic correlations between each subtype. Two dynamic combinations regions were revealed (α1 helix/N-terminal, β9-α15 loop, β7-β8 loop, hinge region, and C terminal, β1-β2 loop, β8-β9 loop) which exist in all four L2 β-lactamases. Stabilising these two combinations could possibly help inhibit the function of L2 β-lactamases. Besides, several potential key residues which result in high dynamic regions were also identified. Since very few research targeted on L2 β-lactamases, this work could be a starting point for the following-up work. The improved understanding of the dynamics of L1 and L2 β-lactamases will help in the design of their inhibitors and discovery of novel resistance breakers

Paramagnetism & structural biology : biochemical & biophysical analysis of imp-1 metallo-beta-lactamase

Resistance to beta-lactam antibiotics by pathogenic bacteria is a global concern. Typically arising between 2 and 3 years after the introduction of the antibiotic into clinical use, it is usually due to beta-lactamase activity. The gene for IMP-1, a metallo-beta-lactamase with two catalytic metal ions, is located on an extremely mobile integron element, enabling the rapid horizontal transfer of beta-lactam resistance between bacterial species, including genera of pathogenically relevant bacteria. Along with the absence of any viable metallo-beta-lactamase inhibitors, this gene mobility makes IMP-1 particularly problematic in the clinical environment. Chapter two of this thesis reports nuclear magnetic resonance (NMR) analysis of IMP-1. 90% of the backbone amide resonances of IMP-1 were assigned using conventional 3D NMR experiments along with selective isotope labelling using cell-free methods. IMP-1 was found to have a high affinity for iron through the course of this study. The iron form was structurally and biochemically characterised using several techniques. A 1.8 Angstrom crystal structure of the iron variant was solved, showing a tertiary structure almost identical to the previously solved X-ray structures of the di-zinc form. NMR analysis suggested subtle differences in structure and/or mobility between the two species. Observed paramagnetic NMR effects induced by the iron centre included paramagnetic relaxation enhancement (PRE), which located the metal centre in the active site. Pseudocontact shifts (PCS) were also evident and a magnetic susceptibility anisotropy tensor was calculated. Paramagnetic NMR methods concurred with anomalous X-ray scattering experiments, locating the metal-binding site of the iron ion. A protocol for the use of paramagnetic effects from the iron centre in a high-throughput drug screening is proposed. A novel method of generating perdeuterated proteins using cell-free protein synthesis with isotope-labelled amino acids is described in chapter three. Performing the cell-free reaction in H2O-based buffers avoids the need for back-exchange of protons onto the backbone amides, which would be required following expressions in D2O. This is particularly useful for cases where protein refolding is impossible, such as the IMP-1 metallo-beta-lactamase. For proteins that can be expressed in good yields, the cost difference compared to conventional isotope-labelling methods is minimal. This chapter includes a reproduction of the application note produced for Cambridge Isotope Laboratories. The use of hyperpolarising agents such as para-hydrogen has recently engendered much interest in the NMR community. This methodology promises up to 350-fold signal-to-noise improvements over conventional experiments that start with a Boltzmann thermal population. Chapter four of this thesis briefly documents the establishment of an in-house built para-hydrogen producing rig in preparation for future studies into hyperpolarisation techniques. A standard operating procedure for obtaining optimal yields is also included. Structure determination of chemical products isolated from natural sources is an essential part of the natural drug discovery process. Chapter five briefly documents how NMR was used in this step for a natural product with pro-angiogenic biological properties extracted from soybean extracellular fluids that proved difficult to identify via other methods

Application of molecular mechanics polarization to fragment based drug design.

Polarization is a term that is often excluded from almost all virtual screening. Polarizability helps explain interactions between nonpolar atoms and electrically charged species. When studying fragments in FBDD these minor interactions could have large effect in changing how well a ligand will bind to its target. After including the polarizability terms in docking a validation set of ligands (Favia et al., 2011) with GLIDE, it improved the results the amount good docked poses (< 2 Å RMSD) by up to 12%. However some ligands were bound in incorrect poses. Further investigation was carried out with MD to observe if given enough time ligands bound in an incorrect pose would return to the binding site. In the first stages of investigating MD we ascertained if we could use GPUs to simulate larger systems and faster. After some performing some MD simulations in GROMACs we found that GPUs were an improved option and thus continued the simulation work with ACEMD which allowed multiple GPUs in tandem. After running the MD simulations for 200ns with atomic charges generated from the polarization the results we found were quite interesting. Some ligands would be trapped in their binding site but would fluctuate quite readily such as 2GVV. Some ligands showed that despite low RMSD they would be ejected from the binding site. In some cases the ligands would then attempt to return to their binding site. Ligands such as in 2CIX would show binding based on the breathing movement of the protein. Some ligands such as 1F5F or 1F8E bound tightly to their binding site during the MD, these ligands also enjoyed improved docking polarization with 0.1 – 1.0 Å improvement. These could be carried forward to become good candidates for experimental testing. Polarization is shown to have an overall positive effect improving binding data and if implemented with simple methods would have little opportunity cost to be added to modern FBDD methods

A Computational perspective on the concerted cleavage mechanism of the natural targets of HIV-1 protease.

Doctoral Degree. University of KwaZulu-Natal, Durban.One infectious disease that has had both a profound health and cultural impact on the human race in recent decades is the Acquired Immune Deficiency Syndrome (AIDS) caused by the Human Immunodeficiency Virus (HIV). A major breakthrough in the treatment of HIV-1 was the use of drugs inhibiting specific enzymes necessary for the replication of the virus. Among these enzymes is HIV-1 protease (PR), which is an important degrading enzyme necessary for the proteolytic cleavage of the Gag and Gag-Pol polyproteins, required for the development of mature virion proteins. The mechanism of action of the HIV-1 PR on the proteolysis of these polyproteins has been a subject of research over the past three decades. Most investigations on this subject have been dedicated to exploring the reaction mechanism of HIV-1 PR on its targets as a stepwise general acid-base process with little attention on a concerted model. One of the shortcomings of the stepwise reaction pathway is the existence of more than two TS moieties, which have led to varying opinions on the exact rate-determining step of the reaction and the protonation pattern of the catalytic aspartate group at the HIV-1 PR active site. Also, there is no consensus on the actual recognition mechanism of the natural substrates by the HIV-1 PR. By means of concerted transition state (TS) structural models, the recognition mode and the reaction mechanism of HIV-1 PR with its natural targets were investigated in this present study. The investigation was designed to elucidate the cleavage of natural substrates by HIV-1 PR using the concerted TS model through the application of computational methods to unravel the recognition and reaction process, compute activation parameters and elucidate quantum chemical properties of the system. Quantum mechanics (QM) methods including the density functional theory (DFT) models and Hartree-Fock (HF), molecular mechanics (MM) and hybrid QM/MM were employed to provide better insight in this topic. Based on experience with concerted TS modelling, the six-membered ring TS structure was proposed. Using a small model system and QM methods (DFT and HF), the enzymatic mechanism of HIV-1 PR was studied as a general acid-base model having both catalytic aspartate group participating and water molecule attacking the natural substrate synchronously. The natural substrate scissile bond strength was also investigated via changes of electronic effects. The proposed concerted six-membered ring TS mechanism of the natural substrate within the entire enzyme was studied using hybrid QM/MM; “Our own N-layered Integrated molecular Orbital and molecular Mechanics” (ONIOM) method. This investigation led us to a new perspective in which an acyclic concerted pathway provided a better approach to the subject than the proposed six-membered model. The natural substrate recognition pattern was therefore investigated using the concerted acyclic TS modelling to examine if HIV-1 (South Africa subtype C, C-SA and subtype B) PRs recognize their substrates in the same manner using ONIOM approach. A major outcome in the present investigation is the computational modelling of a new, potentially active, substrate-based inhibitor through the six-membered concerted cyclic TS modelling and a small system. By modelling the entire enzyme—substrate system using a hybrid QM/MM (ONIOM) method, three different pathways were obtained. (1) A concerted acyclic TS structure, (2) a concerted six-membered cyclic TS model and (3) another sixmembered ring TS model involving two water molecules. The activation free energies obtained for the first and the last pathways were in agreement with in vitro HIV-1 PR hydrolysis data. The mechanism that provides marginally the lowest activation barrier involves an acyclic TS model with one water molecule at the HIV-1 PR active site. The outcome of the study provides a plausible theoretical benchmark for the concerted enzymatic mechanism of HIV-1 PRs which could be applied to related homodimeric protease and perhaps other enzymatic processes. Applying the one-step concerted acyclic catalytic mechanism for two HIV-1 PR subtypes, the recognition phenomena of both enzyme and substrate were studied. It was observed that the studied HIV-1 PR subtypes (B and C-SA) recognize and cleave at both scissile and non-scissile regions of the natural substrate sequences and maintaining preferential specificity for the scissile bonds with characteristic lower activation free energies. Future studies on the reaction mechanism of HIV-1 PR and natural substrates should involve the application of advanced computational techniques to provide plausible answers to some unresolved perspectives. Theoretical investigations on the enzymatic mechanism of HIV-1 PR— natural substrate in years to come, would likely involve the application of sophisticated computational techniques aimed at exploring more than the energetics of the system. The possibility of integrated computational algorithms which do not involve partitioning/restraining/constraining/cropped model systems of the enzyme—substrate mechanism would likely surface in future to accurately elucidate the HIV-1 PR catalytic process on natural substrates/ligands