Bicyclic Boronate β-Lactamase Inhibitors: The Present Hope against Deadly Bacterial Pathogens

PROGRESS REPORT www.advtherap.com Bicyclic Boronate 𝜷-Lactamase Inhibitors: The Present Hope against Deadly Bacterial Pathogens Emilio Lence and Concepción González-Bello* Dedicated to Prof. Javier Benavente on the occasion of his retirement. The use of 𝜷-lactamase inhibitors in combination with 𝜷-lactam antibiotics is an emerging area in drug discovery. This strategy allows the restoration of the therapeutic efficacy of these antibiotics in clinical use against multiresistant bacteria. These pathogens are drug resistant because they express 𝜷-lactamase enzymes, which prevent the antibiotic therapeutic action by catalyzing the hydrolysis of the 𝜷-lactam ring. These enzymes are quite diverse in both their structural architecture and hydrolytic capability, as well as in the mechanism of action. The ever-increasing emergence of pathogens that are capable of coproducing different types of 𝜷-lactamases has triggered the search for ultrabroad-spectrum inhibitors capable of deactivating both serineand metallo-𝜷-lactamases. A recent breakthrough in this long-pursued and unmet need is the discovery of bicyclic boronate inhibitors, specifically taniborbactam, VNRX-7145, and QPX7728, which are currently under clinical development in combination with cefepime, ceftibuten, and QPX2014, respectively. The present article highlights the therapeutic potential of these inhibitors and their spectrum of efficacy is compared with those of other 𝜷-lactam/𝜷-lactamase inhibitor combinations recently approved by the food and drug administration. The molecular basis of the ultrabroadspectrum of activity of boron-based inhibitors is also discussed, on the basis of the available crystal structures and the results of computational studies. 1. Resistance Breakers: The Right Antibiotic Partner for Reversing Antibiotic Drug Resistance The ability of antibiotics to cure bacterial infections is nowadays in serious danger due to the emergence and dissemination worldwide of multidrug-resistant bacteria.[1–3] These pathogens have evolved by developing highly sophisticated, and sometimes Dr. E. Lence, Prof. C. González-Bello Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CiQUS), Departamento de Química Orgánica Universidade de Santiago de Compostela calle Jenaro de la Fuente s/n, Santiago de Compostela 15782, Spain E-mail: [email protected] © 2021 The Authors. Advanced Therapeutics published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. DOI: 10.1002/adtp.202000246 unpredictable, mechanisms to avoid the action of antibiotics.[4] The increasing impact of these deadly pathogens in healthcare systems is worrisome, since in these cases the compromised immune system of patients facilitates the pathogenicity. Resistance to antibiotics is reaching such dangerous levels that the World Health Organization (WHO) estimates that by 2050 deaths from antibiotic resistance will exceed those caused by cancer, and around ten million people could die every year because of this problem.[5] Despite this alarming estimate, the number of new classes of antibiotics approved in the last 50 years, either by disabling unexploited bacterial targets or with a new mechanism of action, is rather low.[6] Thus, most of the antibacterial drugs in the pipeline involve chemical modifications of earlier discoveries to make them more efficient against resistant bacteria.[7] The limited profit margin of the anti-infective therapies (short-term treatments) hinders the recovery of the huge investment costs required to identify research niches and develop small molecules that target them, and this seems to be the main reason why antibiotic discovery programs have been discontinued by the big pharmaceutical companies. In fact, most of the new compounds in this area are being developed by small biotechnology companies. Another drawback is the policy of minimizing the use of the latest generation antibiotics because it goes against the basic principle of any company, i.e., the need to sell drugs to make money and be profitable. A reimbursement reform is also needed to make pharmaceutical companies more attractive businesses for investors. A review of the clinical antibacterial drug pipeline reveals that current research efforts are focused on restoring the efficacy of antibiotics in clinical use, which have proven to be safe and effective over the years. This successful and growing area of investigation involves the administration of the antibiotic in combination with a compound that either blocks a certain bacterial resistance mechanism or potentiates the action of the drug by facilitating its permeabilization into the bacterium. These compounds are known as “resistance breakers,” “antibiotic adjuvants” or “antibiotic potentiators,” and they usually lack relevant bactericidal activity in the dose that is administered.[8–20] Among them, 𝛽lactamase inhibitors are the most prominent as they have proven to be the most successful compounds in restoring the efficacy of Adv. Therap. 2021,4, 2000246 2000246 (1 of 21) © 2021 The Authors. Advanced Therapeutics published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com Figure 1. A) Enzymatic inactivation of carbapenems. B) Bacterial pathogens of critical priority according to the WHO. C) Most relevant bicyclic boronate inhibitors under clinical development. D) Chemical structure of vaborbactam that is the first boron-based 𝛽-lactamase inhibitor approved by the food and drug administration (FDA) and general structure of 1,6-diazabicyclo[3.2.1]octane (DBO) inhibitors. The combination therapy and the development state are indicated in brackets. 𝛽-lactam antibiotics (penicillins, cephalosporins, monobactams, and carbapenems), which compromise 70% of all antibacterial drugs in clinical use.[21–23] These life-saving drugs are safe, very effective, and well tolerated by most patients, with only few cases of allergic reactions.[24] The mechanism of action involves inhibition of the growth of the bacterial wall, specifically the biosynthesis of the peptidoglycan catalyzed by PBPs (penicillinbinding proteins), which imparts rigidity to this essential structure for bacterial survival.[25] The alteration of the natural balance between the synthesis of peptidoglycan and its hydrolysis, which is catalyzed by murein hydrolases, in the regeneration of the bacterial wall is responsible for the bactericidal effect. The utility of 𝛽-lactam antibiotics is being threatened by the ever-increasing production and dissemination worldwide of 𝛽-lactamases.[26,27] These enzymes confer resistance to 𝛽-lactam antibiotics through hydrolysis of the 𝛽-lactam ring to afford inactive products, thus preventing the inhibition of their therapeutic target, i.e., PBPs (Figure 1A). This inactivation process is one of the most relevant resistance mechanisms in Gram-negative bacteria, including the multidrug-resistant pathogens highlighted by the WHO, namely, Acinetobacter baumannii,Pseudomonas aeruginosa,andEntereobacteriaceae (Figure 1B).[28] Among the different types of 𝛽-lactamase inhibitors, significant effort is currently being devoted to the development of compounds with an ultrabroad-spectrum activity, i.e., activity against the four types of 𝛽-lactamase enzymes, which are characterized as having quite distinct hydrolytic capabilities. This goal has proven to be challenging given the variety of structural topologies of these enzymes and the markedly different mechanisms of action of the serine-based enzymes when compared with the zinc-dependent hydrolases. In particular, the search for efficient inhibitors against the metallo-dependent enzymes, for which effective therapies in clinical practice are currently not available, is one of the field’s biggest unmet needs. The search for ultrabroad-spectrum inhibitors that are able to inhibit serineand metallo-𝛽-lactamase enzymes is an emerging area in antibacterial drug discovery. The present article is focused on the therapeutic potential of recently developed combination therapies in which the latest bicyclic boronates (monoester form) are used, specifically taniborbactam (formerly VNRX-5133, 1), the orally bioavailable inhibitor VNRX-7145 (2) and QPX7728 (3), which are in advanced clinical development (Figure 1C). These resistance breakers are the present hope against 𝛽-lactamase-producing carbapenemresistant superbugs that produce either serineor/and metallo𝛽-lactamase enzymes. In order to gain an insight into the scope of the bicyclic boronates currently under clinical study, this article also provides a brief overview of the susceptibility spectrum of the most recent combination therapies approved by the FDA involving other types of 𝛽-lactamase inhibitors, such as vaborbactam and 1,6-diazabicyclo[3.2.1]octanes (DBOs) (Figure 1D). Finally, the molecular basis of the ultrabroad-spectrum efficacy of taniborbactam and QPX7728 is also discussed based on the available crystal structures of the enzyme complexes and the Molecular Dynamics (MD) simulation studies discussed here. 2. 𝜷-Lactamases: Classes and Mechanisms The overuse and misuse of 𝛽-lactam antibiotics over the last 80 years, together with the intrinsic evolutionary character of bacteria as a response to exposure to hostile environments, has led to the development of a huge number of 𝛽-lactamase enzymes. According to the Beta-lactamase database, ≈5000 𝛽-lactamases have been identified to date and these are classified into four groups (A–D) based on their sequence identities.[29–32] These enzymes have quite distinct hydrolytic capabilities and are structurally diverse, with the most frequent classes being A (≈32%) and C (≈30%), followed by D (≈20%), and B (≈14%).[29] Adv. Therap. 2021,4, 2000246 2000246 (2 of 21) © 2021 The Authors. Advanced Therapeutics published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com Figure 2. Enzymatic hydrolysis of carbapenems catalyzed by A) serine-𝛽-lactamases and B) metallo-𝛽-lactamases. R =carbapenem side chain. Classes A, C, and D are serine-𝛽-lactamase enzymes that hydrolyze the 𝛽-lactam antibiotic in a covalent-catalyzed process that involves the formation of an acyl-enzyme adduct using a catalytic serine residue, which behaves as the reactive nucleophile, and an active site residue that acts as a general base (lysine, carboxylated lysine or glutamate) (Figure 2A).[33,34] The mechanism involves two steps. i) The formation of a tetrahedral transition state 1 TTS1 that will be stabilized by strong hydrogen bonding interactions with the bottom part of the active site (the oxyanion hole). ii) The deacylation process by hydrolysis of the acyl-enzyme adduct through the formation of the tetrahedral transition state 2TTS2. Although the mechanism of action of the three types of serine-𝛽-lactamases is globally similar, the structural differences between these species are very relevant and explain their quite distinct hydrolytic capabilities and substrate preferences. Thus, class A enzymes show quite diverse activity since, even though they typically hydrolyze penicillins (in fact they are historically known as penicillinases), some of their variant enzymes have evolved to hydrolyze narrowand expanded-spectrum cephalosporins (SHV-2, TEM-10, CTX-M, GES-1) and others even hydrolyze carbapenems (KPC, SME, IMI, NMC-A, GES-2).[35,36] Class C enzymes mainly hydrolyze cephalosporins (AmpC, CMY, ACT-1, DHA, FOX). The most relevant examples of this group are the chromosomally encoded AmpC enzymes that inactivate most cephalosporins, including expanded spectrum cephalosporins (ceftazidime, cefotaxime, ceftriazone) and cephamycins (cefoxitin). Although the basal expression of AmpC-type enzymes is usually low, in most clinical isolates high levels can be induced by exposure to 𝛽-lactam antibiotics and/or by constitutive expression.[37] The AmpC-type enzymes make the bacterium intrinsically resistant to most cephalosporins. For P. aeruginosa, AmpC enzymes are known as PDC (Pseudomonas-derived cephalosporinase) and these can be induced in most clinical isolates by 𝛽-lactam antibiotics.[38] Class D enzymes (oxacillinases, OXA) hydrolyze penicillins and cloxacillin. However, OXA-type enzymes have also evolved to inactivate narrow-spectrum cephalosporins (OXA-1, OXA-10), expanded-spectrum cephalosporins (OXA-13, OXA-17) or even carbapenems (OXA-23, OXA-24/40, OXA-48), which are known as carbapenem-hydrolyzing class D 𝛽-lactamases.[31,39–47] Unlike serine-𝛽-lactamases (A, C, and D), class B enzymes inactivate 𝛽-lactam antibiotics through a completely distinct mechanism that does not involve the formation of ligandenzyme adducts (Figure 2B). Instead, these enzymes catalyze the hydrolysis of the 𝛽-lactam bond using a hydroxide anion as Adv. Therap. 2021,4, 2000246 2000246 (3 of 21) © 2021 The Authors. Advanced Therapeutics published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com Figure 3. Close view of the active site observed in X-ray crystal structures of 𝛽-lactamases of subclasses B1, B2, and B3. Structures of VIM-1 from P. aeruginosa (PDB 5N5G), CphA from Aeromonas hydrophyla (PDB 1×8G) and L1 Stenotrophomonas maltophilia (PDB 1SML) in the wild-type forms are shown. Note how B1 and B3 subclasses are di-zinc-dependent enzymes that have a hydroxide anion bridging the two cationic centers and a common Zn1 site arrangement (His-His-His triad). By contrast, only one catalytic Zn2+ion is involved in the B2 subclass. A carbonate ion coordinated to the metalisobservedinPDB1×8G. a nucleophile (noncovalent catalysis).[48–56] Class B enzymes are divided into three subclasses (B1, B2, and B3) based on primary amino acid sequence homology, which is relatively low between subclasses (<20%) but significantly higher within a subclass. The B1 and B3 subclasses, which are the most abundant of the three, exhibit a broad-spectrum activity as they can hydrolyze penicillins, cephalosporins, and carbapenems. All B1 and most of B3 enzymes are di-zinc-dependent hydrolases. By contrast, the B2 enzymes are mono-zinc-dependent hydrolases that specifically hydrolyze carbapenems and display poor hydrolytic capacity against penicillins and cephalosporins. For the dinuclear enzymes (B1 and B3 subclasses), the hydroxide anion is bridged by the two Zn2+ions, which are in close proximity to one another (Figure 3). One Zn2+ion is coordinated to three histidine residues (Zn1 site, tetrahedral) and the other ion is bound to a water molecule and three residues (for B1 subclass, Asp-Cys-His; and for B3 subclass, Asp-His-His) (Zn2 site, bipyrimidal). It has been proposed that the 𝛽-lactam antibiotic hydrolysis process is initiated by coordination of its carboxylate group to the Zn2 site and of its carbonyl group to the Zn1 site. This coordination triggers the nucleophilic attack of the hydroxyl group located between the two ions, thus leading to the tetrahedral transition state 3 TTS3 and the cleavage of the C─N bond (Figure 2B). An anionic intermediate is then generated and subsequently protonated to afford an enzyme/product complex before product release.[57] Based on X-ray crystal structures of NDM-1 in complex with hydrolyzed imipenem and meropenem, as well as NMR monitoring of the reaction process, significant differences in the hydrolysis of carbapenems catalyzed by metallo-𝛽-lactamases were identified compared with penicillins and cephalosporins.[58] The structures of two NDM-1/intermediate complexes (PDBs 5YPK and 5YPI) and the NDM-1/product complex (PDB 5YPL) captured revealed that, in contrast to penicillin and cephalosphorin hydrolysis (PDBs 4EYB and 4EYF),[55] the carbapenem hydrolysis mechanism would involve intermediate complex lacking of a bridging water molecule between the two Zn(II) centers. The final protonation of the hydrolyzed carbapenem intermediate would take place by reaction with a bulky water molecule located in the 𝛽-face. Among the three subclasses, the B1 enzymes are the most abundant and these include the clinically relevant and transferable IMP-, VIM-, and NDM-type enzymes. These hydrolases are the most directly related to 𝛽-lactam antibiotic resistance because they are widely found in the deadly pathogens A. baumannii,P. aeruginosa,andEnterobacteriaceae.[59,60] The B2 enzymes are chromosomally encoded and characterized by a bipyramidal coordination of the catalytic Zn2+ion as the Zn2 site arrangement.[48,61] In this case, the nucleophilic hydroxide anion would be generated by deprotonation of the bridging water molecule between the aspartate residue of the zinc coordination sphere and a histidine residue in the vicinity (Asp120 and His118, respectively, in PDB 1×8G, Figure 3).[62] It has been suggested that one of the latter two residues might act as a general base. The B2 subclass enzymes are inhibited when a second Zn2+ion binds to the Zn1 site.[63] CphA and Sfh-I are the most relevant examples of this enzyme subclass. 3. Carbapenemases: The Nightmare of Anti-Infective Therapies Based on 𝜷-Lactam Antibiotics Despite being the least common 𝛽-lactamases in deadly pathogens, class B enzymes represent a huge risk to hospitalized patients because i) they can potentially confer extremely broad-spectrum resistance to antibiotics as—with the exception of monobactams—they can hydrolyze virtually all 𝛽-lactam antibiotics, and ii) clinically approved inhibitors are not currently available. In addition, the extensive knowledge achieved over the years on the inhibition of serine-𝛽-lactamases, which will be briefly summarized in Section 4, is of little relevance in the search for new inhibitors against these zinc-dependent hydrolases. Thus, there are marked differences between these two types of enzymes, both in the mechanism of action and the structural topology, which hinders the development of effective inhibitors based on previous scaffolds.[60,64–66] Nowadays, there is a great deal of concern about the impact of the infections caused by carbapenem-resistant A. baumannii, P. aeruginosa,andEnterobacteriaceae that are frequently found in hospitalized patients fitted with invasive devices or exposed to extended antibiotic regimens.[67–70] Of particular concern is the global incidence of class A carbapenemases, such as KPC, SME, IMI, NMC-A, and GES-2, carbapenem-hydrolyzing class D𝛽-lactamases, such as OXA-23, OXA-24/40, and OXA-48, Adv. Therap. 2021,4, 2000246 2000246 (4 of 21) © 2021 The Authors. Advanced Therapeutics published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com Figure 4. Most relevant carbapenemases widely found in multidrug-resistant pathogens A. baumannii,P. aeruginosa,andEnterobacteriaceae. as well as metallo-𝛽-lactamases such as IMP, VIM, and NDM (Figure 4). The ever-increasing appearance and dissemination in clinical settings of these types of strains, which are mostly encoded by plasmids, is worrisome since it significantly narrows the therapeutic options and, in some cases, solutions are not available. This issue is further exacerbated by the existence and spread of bacterial strains that coproduce serineand metallocarbapenemases. Thus, i) class B enzymes hydrolyze approved serine-𝛽-lactamase inhibitors such as avibactam, and efficient inhibitors against them are not clinically available,[71] and ii) numerous examples of clinical isolates of Klebsiella pneumoniae strains that coexpress NDM-1 and OXA-type enzymes have already been found worldwide (India, Nepal, Italy, Korea, United Arab Emirates, Switzerland).[72–79] It is therefore not surprising that the development of carbapenemase inhibitors with an ultrabroad-spectrum activity, i.e., effective against both metalloand serine-𝛽-lactamases, has become a flourishing research area in drug discovery in recent years. 4. Overview and Spectrum Susceptibility of Non-Boron-Based 𝜷-Lactamase Inhibitors The most relevant 𝛽-lactamase inhibitors, either in clinical use or at different stages of development, are summarized in Figure 5. Clavulanic acid (4) and the penicillin-based sulfones sulbactam (5) and tazobactam (6), which were the first to be introduced in clinic in combination with various penicillins (amoxicillin, ampicillin, piperacillin, ticarcillin) as well as more recently with fourth and fifth generation cephalosporins (cefepime, ceftolozane) are only effective against bacterial strains that harbor extendedspectrum-𝛽-lactamases (ESBLs, class A).[80,81] None of these combinations, including the recently developed enmetazobactam (7, formerly AAI101)/cefepime,[82] are useful against strains that produce the most relevant serineand metallo-carbapenemases or against class C cephalosporinases (Figure 4). A huge breakthrough in 𝛽-lactamase inhibition was the development of 1,6-diazabicyclo[3.2.1]octanes (DBOs) that was triggered by the identification of its parent inhibitor, avibactam (8), which was approved in 2014 by the FDA in combination with ceftazidime.[83–85] Avibactam efficiently restores the bactericidal activity of ceftazidime against infections caused by bacterial strains producing class A carbapenemases (KPC-type), class C cephalosporinases (AmpC-type), as well as some class D enzymes (OXA-48) (Table 1). Unfortunately, the combination avibactam/ceftazidime is ineffective against relevant strains harboring carbapenem-hydrolyzing class D 𝛽-lactamases, such as OXA-23 and OXA-24/40, which are widely found in P. aeruginosa and A. baumannii, as well as Enterobacteriaceae-producing metallo-carbapenemases, such as NDM-1, IMP-1 and VIM1/VIM-2. Recently, avibactam in combination with aztreonam and metronidazol has opened new therapeutic opportunities for the treatment of infections caused by Gram-negative bacteria producing metallo-𝛽-lactamases, in particular, intra-abdominal infections and nosocomial pneumonia.[86–87] This therapy, which exploits the stability of aztreonam against class B enzymes and is currently under phase III of clinical development, is efficient against the vast majority of Enterobacteriaceae and particularly attractive for patients with a history of allergy to other 𝛽-lactam antibiotics. However, this treatment is not suitable for infections caused by Gram-positive or anaerobic bacteria, since aztreonam is inefficient against them.[88] The discovery of avibactam (9) triggered a frantic race to expand its spectrum of activity by modification of its DBO scaffold. In particular, significant attention has been devoted to expand avibactam’s activity against bacterial strains that express class D carbapenemase OXA-23 and OXA-24/40 enzymes. Efforts were initially focused on modifications of the side chain (primary amide group), either directed to introduce into the scaffold extra binding interactions with the active site and/or to improve their pharmacological properties. This research led to the discovery of relebactam (10), which was approved in 2019 by the FDA in combination with imipenem and cilastatin,[89,90] followed by zidebactam (11, formerly WCK 5222)[91–93] and nacubactam (12, formerly RG6080, OP0595),[94–96] which are both under phase I clinical studies in combination with cefepime and meropenem, respectively. Unlike the other DBO inhibitors, nacubactam has a dual mode of action because, in addition to being a serine-𝛽lactamase inhibitor, it also has bactericidal activity by targeting PBP2 in Enterobacteriaceae.[96–98] The most meaningful progress in the development of DBO inhibitors occurred with the rigidification and functionalization of the six-membered ring. This type of modification provided a solution to one of the field’s long-pursued unmet needs, namely to expand the inhibitory range of these compounds to include class D 𝛽-lactamases, such as OXA-23 and OXA-24/40, in the list of serine 𝛽-lactamases that can be blocked (Table 1). Unlike Adv. Therap. 2021,4, 2000246 2000246 (5 of 21) © 2021 The Authors. Advanced Therapeutics published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com Figure 5. Clavulanic acid (5), most relevant examples of penicillin-based sulfones and 1,6-diazabicyclo[3.2.1]octanes. The combination therapy and the development state are indicated in brackets. The chemical modifications in the parent inhibitors, sulbactam and avibactam, that have resulted in subsequent analogs are highlighted in blue. other 𝛽-lactamases, the latter class D carbapenemases have a rigid and apolar tunnel-like entrance, which is composed of a methionine and a tyrosine or phenylalanine residue, that facilitates exquisite control of the substrate conformation for the serine-catalyzed hydrolysis, thus enhancing the carbapenemase activity.[99] The most prominent inhibitor, which was developed by Entasis Therapeutics, is durlobactam (13, formerly ETX2514) and together with sulbactam (5) this proved to be an excellent therapeutic candidate for the treatment of infections caused by multidrug-resistant Acinetobacter spp.[100–102] The combination durlobactam/sulbactam is currently under phase III clinical studies. The same company also developed ETX0282 (14), an oral prodrug of another relevant DBO analog ETX1317 (15), which is a regioisomer of durlobactam (13) in which the sulfate moiety has been replaced by an (R)-2-fluoroacetate group. ETX0282 (14) in combination with cefpodoxime proxetil, which is a prodrug of cefpodoxime approved for the treatment of antibiotic-resistant Enterobacteriaceae urinary tract infection, is a promising oral therapy for infections caused by ESBLproducing and carbapenem-resistant Enterobacteriaceae.[103–106] Undoubtedly, the broad-spectrum of activity of durlobactam (13) and ETX0282 (14)—or its active form ETX1317 (15)—represents a huge advance in restoring the efficacy of carbapenems in infections caused by the WHO priority pathogens. However, like avibactam these compounds are also inefficient against metallo-𝛽-lactamases (Table 1). 5. Bicyclic Boronates: The Present Hope Toward “Pan-𝜷-Lactamase Inhibitors” In general, the ability of boronic acids to change their hybridization state between sp2 (trigonal) and sp3 (tetrahedral) forms Adv. Therap. 2021,4, 2000246 2000246 (6 of 21) © 2021 The Authors. Advanced Therapeutics published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com Table 1. Spectrum of activity of DBO inhibitors among the fourth classes of 𝛽-lactamase enzymes. Inhibitor Class A Class C Class B Class D Avibactam (9) Yes Yes No Some ESBL, KPC AmpC-type – OXA-10, OXA-48 Relebactam (10)Yes Yes No No ESBL, KPC AmpC-type – – Zidebactam (11)Yes Yes No No ESBL, KPC AmpC-type – – Nacubactam (12)Yes Yes No No ESBL, KPC AmpC-type – – Durlobactam (13) Yes Yes No Yes ESBL, KPC AmpC-type – OXA-1, OXA-10, OXA-23, OXA-24/40, OXA-48 ETX0282 (14) Yes Yes No Yes ESBL, KPC AmpC-type – OXA-1, OXA-10, OXA-23, OXA-24/40, OXA-48 in aqueous environments makes them good mimetics of the transition state for enzymes using amides, esters or lactams as substrates.[107] This feature and the improved pharmacokinetics profile of the corresponding derivatives are attractive properties that have been exploited in drug development and resulted in several approved drugs. Good examples are bortezomid (Velcade, FDA approved in 2005)[108] and ixazomid (Ninlaro, FDA approved in 2015),[109] which are proteasome inhibitors used for the treatment of myeloma, and tavaborole (Kerydin, FDA approved in 2014),[110] which is a Leucyl-tRNA synthetase inhibitor employed for the treatment of onychomycosis (a fungal infection). The investigations into the potential of boronic acids in the 𝛽-lactamase inhibition field emerged after it was demonstrated that boric acid and subsequent diverse phenylboronic acids inhibit serine-𝛽-lactamase enzymes.[33,111–113] These compounds are considered good transition state analogs of TTS1 and TTS2, for which their capacity to be in equilibrium between their sp2/sp3 forms is also crucial to maximize their affinity during both the binding and inhibition processes (Figure 2). Thus, in the Michaelis complex formation step the sp2 form, which usually predominates at neutral pH,[112] can mimic the carbonyl group of 𝛽-lactam antibiotics as they also have an sp2 geometry.[33] Once this complex is established, the ability of the boronic acid to tune to the sp3 form provides a good mimetic of the high-energy tetrahedral transition states TTS1 and TTS2 in the serine-𝛽-lactamase mechanism, as well as TTS3 in the conversion catalyzed by the metallo-𝛽-lactamase enzymes (Figure 2). This effective “sp2/sp3 equilibrium process” is consistent with the high kon and low koff values experimentally observed in compounds of these types.[114,115] Initially, the main bottleneck for the use of boronic acids in this field was their potential side effects due to the additional inhibition of mammalian serine proteases.[116] However, the introduction of cyclic boronates (1,2-oxaborinan-2-ol and 3,4-dihydro2H-benzo[e][1,2]oxaborinin-2-ol) was a turning point in the field due to their greater selectivity against 𝛽-lactamase enzymes (Figure 6). The latter characteristic is mainly caused by the restricted conformation of this chemical scaffold, which would not fit in the smaller active sites of the serine proteases as they have been exquisitely designed for transforming more flexible substrates. As for the penicillin-based sulfone inhibitors [sulbactam (6), tazobactam (7), enmetazobactam (8)], the presence in the scaffold of a carboxylate group in the 𝛼-position to the cyclic oxygen atom proved to be pivotal for anchoring to the active site of the serine-𝛽-lactamase enzymes, either through electrostatic and/or hydrogen bonding interactions with key polar residues, as well as for coordination to the Zn2+ion of the metallo-𝛽lactamases. 5.1. Taniborbactam (VNRX-5133) The inspiration for the design of inhibitors based on cyclic boronates arose from the X-ray structure of TEM-1 𝛽-lactamase from Escherichia coli covalently modified by (1R)-1-acetamido2-(3-carboxy-2-hydroxyphenyl)ethylboronic acid (PDB 1ERQ, 1.9 Å) reported by Ness et al. (Figure 6A).[117] In the structure, the strong hydrogen bonding interaction observed between the phenol group in the modified ligand and the oxygen atom of the catalytic serine seems to induce a suitable arrangement of the ligand for coordination to the catalytic Zn2+ion through its phenol and carboxylate moieties. This hypothesis led Burns et al.[118] in 2010 at Protez pharmaceuticals, a subsidiary of Novartis, to develop the first boron-based compounds, specifically the bicyclic boronates, that were able to inhibit both serineand metallo-𝛽-lactamase enzyme. Further development at Venatorx Pharmaceuticals, with particular attention paid to the optimization of the flexible side chain of the ligand, led to the discovery of taniborbactam (1,formerly VNRX-5133), an injectable pan-𝛽-lactamase inhibitor (Figure 1C).[119–122] In parallel, Rempex Pharmaceuticals developed diverse monocyclic boronates that allowed the identification of vaborbactam (4, formerly RPX7009), which is the first boronbased 𝛽-lactamase inhibitor approved by the FDA in combination with meropenem (2017, vabomere) for the treatment of complicated urinary tract infections (Figure 1D).[123,124] The European Medicines Agency also approved the vaborbactam/meropenem combination for the treatment of intra-abdominal infections and hospital-acquired pneumonia.[125] The most remarkable feature of varborbactam is its strong capacity in restoring the activity of carbapenems against strains that produce ESBLs, especially KPC-producing Enterobacteriaceae, with Kivalues in the nanomolar range (Table 2).[123] This compound provides highly stable adducts, the tridimensional structure of which was solved by X-ray crystallography (PDB 6TD0,[126] 0.99 Å). Vaborbactam is a reversible inhibitor with a remarkable very slow off-rate constant (≈40 µs−1)andacorresponding residence time of ≈7 h against KPC-enzymes.[127] A substantially faster release of the vaborbactam bound form occurs against other serine-𝛽-lactamases. In vitro studies with KPC-producing strains showed that vaborbactam reduces the minimum inhibitory concentration (MIC) values of meropenem by ≥64-fold. This compound also efficiently inhibits other 𝛽lactamases of classes A (CTX-M, SHV, TEM-10) and C (Enterobacter cloacae cephalosporinase P99, K. pneumoniae CMY-2) in the 10 ×10−9–100 ×10−9m range but it does not have bactericidal activity (Table 2).[128] Unfortunately, vaborbactam lacks Adv. Therap. 2021,4, 2000246 2000246 (7 of 21) © 2021 The Authors. Advanced Therapeutics published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com Figure 6. The sp3 form of cyclic boronates is key for the inhibition of both serine and metallo-𝛽-lactamases. A) sp2/sp3 equilibrium forms of 3,4-dihydro2H-benzo[e][1,2]oxaborinin-2-ol derivatives and schematic representation of the resulting serine adduct I and di-zinc-complex II. B) Detailed view of the active site of 𝛽-lactamase TEM-1 from E. coli covalently modified by (1R)-1-acetamido-2-(3-carboxy-2-hydroxyphenyl)ethylboronic acid (PDB 1ERQ,[117] 1.9 Å). Note how the phenol group is in close contact with the catalytic Ser70 (2.3 Å). C) Tridimensional structure of I and II observed in PDB 6RTN[130] (OXA-10 from P. aeruginosa)and6SP7 [131] (VIM-2 from P. aeruginosa). Only the bicyclic boronate core of the ligand (cyan) is shown. clinical utility for the treatment of infections caused by bacterial pathogens that produce carbapenemases of classes B and D, as it proved to be a weak inhibitor of these enzymes (µM range). Among the B1 subclass, its inhibitory activity against the IMP-1 enzyme stands out when compared to VIM-1/VIM-2 and NMD1, which are lower by a factor of around 5. The bicyclic architecture of taniborbactam (1) has proven to be key in achieving nanomolar activity against class B enzymes because of the conformational restraints induced by the fused aromatic ring that preorganizes the ligand for binding. This bicyclic boronate efficiently inhibits the B1 subclass enzymes VIM and NDM, with IC50 values in the nanomolar range, as well as serine𝛽-lactamases of ESBLs, OXA-type, and KPC-type (Table 2).[129–131] This outstanding ultrabroad-spectrum activity makes taniborbactam (1) an excellent antibiotic partner for the treatment of infections due to carbapenem-resistant pathogens with distinct mechanisms of action, including carbapenem-resistant Enterobacterales and carbapenem-resistant P. aeruginosa.[132] Taniborbactam (1) is the first pan-𝛽-lactamase inhibitor to enter clinical trials that is able to inhibit both metalloand serine-𝛽-lactamases. Specifically, in combination with cefepime, a fourth-generation cephalosporin, it is currently in phase III studies. Venatorx Pharmaceuticals Inc. developed an orally bioavailable boron-based 𝛽-lactamase inhibitor, VNRX-7145 (2), which is a proform of VNRX-5236, a bicyclic boronate derivative structurally related to taniborbactam but with a short side chain (propionamido).[133,134] In combination with ceftibuten, a third generation cephalosporine, VNRX-5236 restores the antibiotic efficacy against ESBLs and carbapenem-resistant Enterobacterales, including strains that harbor KPC-type and OXA-type carbapenemases. The combination ceftibuten/VNRX-7145 is currently in phase I clinical development. Taniborbactam (1) is synthesized in six steps from commercially available boronic acid 16 as shown in Scheme 1.[118–120] As for the synthesis of other inhibitors based on cyclic boronates such as vaniborbactam (4), Matteson’s reaction is employed for the double homologation of the boronic acid pinanediol ester intermediate 17, which is readily prepared in two steps by double esterification of 16 to give chloride 19. SN2 reaction of chloride 19 with lithium bis(trimethylsilyl)amide and subsequent coupling with carboxylic acid 23 using HATU and NMO provide amide 20. The required acid 23 is prepared in five steps from commercially available carboxylic acid 21. Finally, treatment of 20 with BCl3leads to the deprotection of all functional groups and the formation of the bicyclic core in taniborbactam (1). Adv. Therap. 2021,4, 2000246 2000246 (8 of 21) © 2021 The Authors. Advanced Therapeutics published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com Table 2. Inhibitory activity [IC50 (µm)] of taniborbactam (1), vaborbactam (4), avibactam (9), clavulanic acid (5), and tazobactam (7) against relevant serineand metallo-𝛽-lactamase enzymes. Class Enzyme Taniborbactam (1) Vaborbactam (4) Avibactam (9) Clavulanic acid (5) Tazobactam (7) A TEM-116 0.12[128] 6[128] NAa)NA NA KPC-2 0.03[131] 0.09[128] 0.06[131] 1.8[131] 1.7[131] SHV-5 0.0004[131] 0.44[131] 0.013[131] 0.012[131] 0.015[131] CTX-M-15 0.01[131] 0.42[131] 0.003[131] 0.04[131] 0.001[131] B1 IMP-1 39.8[131] 126[128] >100[131] >100[131] >100[131] 2.51[130] NDM-1 0.19[131] 631[128] >100[131] >100[131] >100[131] 0.01[130] VIM-1 0.0079[130] 398[128] NA NA NA VIM-2 0.026[131] 316[128] >100[131] >100[131] >100[131] 0.0005[130] B2 CphA 2.51[130] 631[128] NA NA NA B3 L1 >10[130] 336[128] NA NA NA C AmpC 0.301[130] 5[128] NA NA NA P99 0.03[131] 0.09[131] 0.016[131] >100[131] 0.73[131] CMY-2 0.007[131] 0.22[131] 0.007[131] >100[131] 0.41[131] D OXA-1 0.16[131] 7.9[131] 0.04[131] 0.12[131] 0.43[131] OXA-48 0.42[131] 25[128] 0.55[131] 30[131] 0.55[131] 0.54[130] 38.8[131] 0.55[131] 30[131] 0.55[131] a) NA =Not available. Scheme 1. Synthesis of taniborbactam (1). Reagents, and conditions: (a) Isobutene, H2SO4(c), dioxane, RT. (b) (+)-pinanediol, THF, RT. (c) CH2ClI, nBuLi, THF, −100 °C to RT., CH2Cl2,−78 °C. (d) 1. CH2Cl2,nBuLi, THF, −100 °C. 2. ZnCl2,−100 °C to −10 °C. (e) 1. LHMDS, THF, −20 °C to RT. 2. 23,HATU,NMO,N,N-dimethylacetamide, RT. (f) BCl3,CH 2Cl2,−78 °C. (g) BnBr, K2CO3,DMF,RT.(h)CH 2Cl2, HCl (4 m in dioxane), RT. (i) 1. Et3N, (CH2Cl)2,RT.2.HOAc,N-Boc-aminoacetaldehyde, NaBH(OAc)3,RT.(j)1.Boc 2O, Et3N, CH2Cl2,RT.2.H 2(g), Pd/C (10%), EtOAc, RT. 5.1.1. Molecular Basis of Taniborbactam’s Efficacy against Class B1 Metallo-𝛽-Lactamases A differentiating feature between taniborbactam (1)andvaborbactam (4), beyond the markedly superior inhibitory capacity of the former against class B enzymes (nanomolar vs micromolar, respectively), is their distinct selectivity between B1 enzymes (Table 2). Thus, while vaborbactam has a modest and similar inhibition of the main B1 enzymes (VIM-1/2, NDM-1, IMP-1), taniborbactam is notably more effective against VIM-1/2 and NDM-1 Adv. Therap. 2021,4, 2000246 2000246 (9 of 21) © 2021 The Authors. Advanced Therapeutics published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com cefepime, ceftolozane, meropenem), or orally bioavailable (ceftibuten, cefpodoxime, tebipenem).[153] Specifically, these studies were performed with strains that express ESBLs (CTXM, SHV, TEM, VEB, PER), both plasmidic (CMY, FOX, MIR, DHA) and chromosomally encoded (P99, PDC, ADC) class C 𝛽-lactamases, class A carbapenemases (KPC, SME, NMC-A, BKC-1), class D carbapenemases (OXA-48, OXA-23, OXA-24/40, OXA-58), and class B carbapenemases (NDM, VIM, CcrA, IMP, and GIM). Moreover, susceptibility studies carried out on a large collection of carbapenem-resistant Enterobacterales revealed that the combination meropenem/QPX7728 has a very attractive microbiological profile for infections caused by pathogens with multiple resistance mechanisms.[155] From the structural point of view, the main differences between taniborbactam (1) and QPX7728 (3) are i) the higher overall rigidity of the latter due to the additional restraint caused by the introduction of a cyclopropane ring in the 3,4-dihydro2H-benzo[e][1,2]oxaborinin-2-ol scaffold, and ii) the extra and/or more potent contacts resulting from (a) the substitution with an electron-withdrawing group (fluoro), and (b) the incorporation of the cyclopropane moiety. Analysis of the available crystal structures of KPC-2 from K. pneumoniae (PDB 6V1J,[147] 1.3 Å), OXA-48 from K. pneumoniae (PDB 6V1O,[147] 1.8 Å), VIM-2 from P. aeruginosa (PDB 6V1P,[147] 1.2 Å), and NDM-1 from K. pneumoniae (PDB 6V1M,[147] 1.05 Å) in complex with QPX7728, revealed that option (ii) is more likely (Figure 13). Thus, in all of the structures the cyclopropane ring is located in an apolar pocket of the enzyme active site, and the aromatic ring is exquisitely flanked by residues that can establish CH–𝜋interactions with this aromatic ring. The latter type of interaction is particularly relevant considering the electron-deficiency of this aromatic ring, which contains three electron-withdrawing substituents [F, CO2, OB(OH)2].[156] For instance, for the OXA-48/QPX7728 complex, the cyclopropyl moiety is surrounded by residues Trp105 and Val120, which are located close to the methylenic group, and the aromatic ring establishes CH–𝜋interactions with the side chains of residues Tyr211, Ile102, and Leu247, which are flanking both faces of the ring. This arrangement explains the much weaker inhibitory potency of its enantiomer (3S,4R)(≈10-fold against OXA-type enzymes), particularly for those enzymes that have large apolar subpockets in the active site, such as OXA-48.[147] 6. Conclusion The outstanding and ultrabroad-spectrum inhibitory capacity of taniborbactam, VNRX-7145, and QPX7728 against challenging 𝛽-lactamase enzymes, which have been summarized here, pinpoint bicyclic boronate inhibitors as the present hope against multidrug-resistant bacteria. With these recently discovered boron-based inhibitors a long-pursued unmet goal has been achieved, namely to restore the efficacy of 𝛽-lactam antibiotics against bacterial strains that produce metallo-𝛽-lactamases, for which inhibitors in clinical use are not currently available. Even more relevant is the fact that these inhibitors open the door to the use of the same compound for therapies that involve bacterial strains that coproduce both serineand metallo-𝛽-lactamase enzymes. Although the great structural diversity of these types enzymes makes it challenging to find an excellent inhibitor for all of them, QPX7728 seems to be the most universal of the inhibitors produced to date. The future of this bicyclic boronate inhibitor, which is currently under phase I clinical studies in combination with QPX2014, looks exciting. It is important to ascertain whether future combinations of QPX7728 with 𝛽-lactam antibiotics in clinical use could enter clinical development. In any case, the excellent structural information available and the results already achieved augur well for a boom in research around the bicyclic boronate scaffold in the near future. One can envisage that this will eventually bring more effective therapeutic solutions for dealing with one of the most serious challenges in global health. 7. Computational Methods Molecular Modeling Studies:All calculations were performed by using the Gaussian 09W[140] program package at DFT level by means of the B3LYP functional.[141,142] The standard 6–31+G(d,p)[143,144] basis set was used for C, H, O, S, B, and N, and the LANL2DZ relativistic pseudopotential was used for Zn.[145] The starting point of these calculations was taken from crystallographic structures PDB 6SP7 (chain E) including the following residues: Phe61, Trp87, His116, His118, Asp120, His196, CysS221, Gly232, Asn233, His263, both Zn atoms (labeled as residues 301 and 302), Zn-bonded taniborbactam (residue 306 named K9B) and the water molecule 441. All amino acids were truncated at C𝛼positions with a methyl group, except for Asn233, whose C𝛼was transformed into a methylene group keeping the nitrogen atom of the main chain and carbonyl group of Gly232 (transformed into an acetyl group). Hydrogen atoms were added to complete valences, considering all the histidine residues in the neutral form, and protonated in 𝜖position except His118 protonated in 𝛿position. Asp120 and Cys221 were used in the anionic form. The side chain of taniborbactam was truncated into an acetyl amide group, the oxygen atom O07 and the carboxylate group in the ligand were considered as hydroxyl and carboxylate groups, respectively. Vaborbactam was manually modeled from taniborbactam coordinates in a similar fashion. To represent better the geometry in the enzyme active site, minimization of both systems was performed while fixing the Cartesian coordinates for the C𝛼and C𝛽atoms of all residues. Molecular Dynamics Simulations Studies–Protein Preparation:The protein coordinates found in the crystal structure of IMP-1 from S. marcescens (PDB 5EV6, 1.98 Å)[138] and VIM-2 from P. aeruginosa (PDB 5ACU, 2.10 Å),[146] both in the wild-type form, were used. For the IMP-1 enzyme, coordinates from chain D were selected. Computation of the protonation state of all titratable groups at pH 7.0 was carried out using the H++ Web server.[157,158] As a result of this analysis His19 and His34 for IMP-1 and His55, His170, His252, His285, and His293 for VIM-2 were protonated in 𝜖 position. Parameterization of the Zn-contained active site was carried out as using MCPB module of AMBER Tools 17,[159,160] following the method described in the AMBER tutorial 2.4.[161] Active site of both enzymes consisted of the following residues: i) for coordination to Zn1 ion (residue number 301/1296): His77/His116, His79/His118, and His139/His196 (in IMP-1/VIM-2, respectively); ii) for coordination to Zn2 ion (residue number 302/1297): His197/His263, Asp81/Asp120, and Cys158/Cys221; and a water molecule (residue number 400/1001) situated between both Zn2+ cations that was modeled as hydroxide. All histidine residues were considered in their neutral form and coordinated to the Zn atoms through the nitrogen atom in 𝜖position (NE2) except for His79/His118 that is coordinated through the nitrogen atom in 𝛿position (ND1). Residues Asp81/Asp120 and Cys158/Cys221 both are bound in the anionic form through the atoms OD2 and SG, respectively. The bond and angle force constants for the active site residues were determined from the sub matrices of the Cartesian Hessian matrix calculated using Gaussian 09 using the Seminario Method.[162] Partial charges for the active site residues were derived by quantum mechanical calculations using Gaussian 09, according to the RESP[163] model. Addition of hydrogen atoms and molecular mechanics parameters from the ff14SB[163] force field were assigned to the proteins using the LEaP Adv. Therap. 2021,4, 2000246 2000246 (16 of 21) © 2021 The Authors. Advanced Therapeutics published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com module of AMBER Tools 17. IMP-1 protein was immersed in a truncated octahedron of ≈9100 TIP3P water molecules and neutralized by addition of chloride ions. VIM-2 protein was immersed in a truncated octahedron of ≈6400 TIP3P water molecules and neutralized by addition of sodium ions. Minimization of the Unbound Forms:The system was minimized in four stages: a) initial minimization of the active site residues, hydroxide and Zn ions (1000 steps, first half using steepest descent and the rest using conjugate gradient); b) minimization of the solvent and ions (5000 steps, first half using steepest descent and the rest using conjugate gradient); c) minimization of the side chain residues, waters, and ions (5000 steps, first half using steepest descent and the rest using conjugate gradient); d) final minimization of the whole system (5000 steps, first half using steepest descent, and the rest using conjugate gradient). A positional restraint force of 50 kcal mol−1Å−2was applied to the whole system except the active site, the whole protein and 𝛼carbons during the first three stages (a–c), respectively. Simulations of the Unbound Forms:MD simulations were performed using the pmemd.cuda_SPFP[165–167] module from the AMBER 16 suite of programs. Periodic boundary conditions were applied, and electrostatic interactions were treated using the smooth particle mesh Ewald method[168] with a grid spacing of 1 Å. The cutoff distance for the nonbonded interactions was 9 Å. The SHAKE algorithm[169] was applied to all bonds containing hydrogen using a tolerance of 10−5Å and an integration step of 2.0 fs. The minimized system was then heated at 300 K at 1 atm by increasing the temperature from 0 to 300 K over 100 ps and by keeping the system at 300 K another 100 ps. A positional restraint force of 50 kcal mol−1Å−2 was applied to all 𝛼carbons during the heating stage. Finally, an equilibration of the system at constant volume (200 ps with positional restraints of 5 kcal mol−1Å−2to 𝛼carbons) and constant pressure (another 100 ps with positional restraints of 5 kcal mol−1Å−2to 𝛼carbons) was performed. The positional restraints were gradually reduced from 5 to 1 mol−1Å−2 (5 steps, 100 ps each), and the resulting systems were allowed to equilibrate further (100 ps) without restraints. Unrestrained MD simulations were carried out for 100 ns. System coordinates were collected every 10 ps for further analysis. The molecular graphics program PyMOL[170] and CHIMERA[171] was employed for visualization and depicting enzyme structures. The cpptraj module in AMBER Tools 17 was used to analyze the trajectories and to calculate the rmsd of the protein during the simulation.[172] The vibrational modes for both metallo-𝛽-lactamases were calculated by principal component analysis with the cpptraj module from the corresponding MD trajectories.[173] Supporting Information Supporting Information is available from the Wiley Online Library or from the author. Acknowledgements Financial support from the Spanish Ministry of Economy and Competiveness (SAF2016-75638-R, PID2019-105512RB-I00), the Xunta de Galicia [ED431B 2018/04 and Centro singular de investigación de Galicia accreditation 2019–2022 (ED431G 2019/03)], and the European Regional Development Fund (ERDF) is gratefully acknowledged. The authors also thank the Centro de Supercomputación de Galicia (CESGA) for use of the Finis Terrae computer. Conflict of Interest The authors declare no conflict of interest. Keywords antibiotic adjuvants, boron-based inhibitors, metallo-𝛽-lactamases, resistance breakers, serine-𝛽-lactamases Received: October 30, 2020 Revised: December 6, 2020 Published online: February 15, 2021 [1] A. M. Thayer, Chem. Eng. News 2016,94, 36. [2] E.D.Brown,G.D.Wright,Nature 2016,529, 336. [3] C. L. Ventola, Pharm. Ther. 2015,40, 277. [4] I. Levin-Reisman, I. Ronin, O. Gefen, I. Braniss, N. Shoresh, N. Q. Balaban, Science 2017,355, 826. [5] M.E.A.deKraker,A.J.Stewardson,S.Harbarth,PLoS Med. 2016, 13, 1002184. [6] N. Gilbert, Nature 2018,555,S5. [7] C. Lepore, L. Silver, U. Theuretzbacher, J. Thomas, D. Visi, Nat. Rev. Drug Discovery 2019,18, 739. [8] S. Chakradhar, Nat. Med. 2016,22, 1197. [9] C. González-Bello, Bioorg. Med. Chem. Lett. 2017,27, 4221. [10] E. E. Gill, O. L. Franco, R. E. W. Hancock, Chem. Biol. Drug Des. 2015, 85, 56. [11] P. Bernal, C. Molina-Santiago, A. Daddaoua, M. A. Llamas, Microb. Biotechnol. 2013,6, 445. [12] H. Douafer, V. Andrieu, O. Phanstiel, J. M. Brunel, J. Med. Chem. 2019,62, 8665. [13] M. A. Farha, E. D. Brown, Nat. Biotechol. 2013,31, 120. [14] D. Brown, Nat. Rev. Drug Discovery 2015,14, 821. [15] R. J. Worthington, C. Melander, Trends Biotechnol. 2013,31,177. [16] G. D. Wright, Trends Microb. 2016,24, 862. [17] K. Bush, ACS Infect. Dis. 2015,13, 509. [18] R. I. Melander, C. Melander, ACS Infect. Dis. 2017,3, 559. [19] L. Kalan, G. D. Wright, Expert Rev. Mol. Med. 2011,13,5. [20] J. A. Hill, L. E. Cowen, Future Microbiol. 2015,10, 1719. [21] K. Bush, P. A. Bradford, Cold Spring Harb. Perspect. Med. 2016,6, 025247. [22] M. Toth M, N. T. Antunes, N. K. Stewart, H. Frase, M. Bhattacharya, C. A. Smith, S. B. Vakulenko, Nat. Chem. Biol. 2016,12,9. [23] K. Poole, Cell Mol. Life Sci. 2004,61, 2200. [24] R. P. Elander, Appl. Microbiol. Biotechnol. 2003,61, 385. [25] C. Walsh, Nature 2000,406, 775. [26] C. L. Tooke, P. Hinchi, E. C. Bragginton, C. K. Colenso, V. H. A. Hirvonen, Y. Takebayashi, J. Spencer, J. Mol. Biol. 2019,431, 3472. [27] K. Baren, P. A. Bradford, Clin. Microbiol. Rev. 2020,33, e00047-19. [28] WHO, Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics, http://www.who.int/medicines/publications/WHO-PPL-Short_ Summary_25Feb-ET_NM_WHO.pdf?ua=1 (accessed: July 2020). [29] T. Naas, S. Oueslati, R. A. Bonnin, M. L. Dabos, A. Zavala, L. Dortet, P. Retailleau, B. I. Iorga, J. Enzyme Inhib. Med. Chem. 2017,32, 917. [30] K. Bush, P. A. Bradford, Nat. Rev. Microbiol. 2019,17, 295. [31] K. Bush, Antimicrob. Agents Chemother. 2018,62, e01076-18. [32] K. Bush, Ann. N. Y. Acad. Sci. 2013,1277, 84. [33] C. González-Bello, D. Rodríguez, M. Pernas, A. Rodríguez, E. Colchón, J. Med. Chem. 2020,63, 1859. [34] A. Krajnc, P. A. Lang, T. D. Panduwawala, J. Brem, C. J. Schofield, Curr. Opin. Chem. Biol. 2019,50, 101. [35] A. Philippon, P. Slama, P. Dény, R. Labia, Clin. Microbiol. Rev. 2016, 29, 29. [36] J. Walther-Rasmussen, N. Høiby, J. Antimicrob. Chemother. 2007,60, 470. [37] G. A. Jacoby, Clin. Microbiol. Rev. 2009,22, 161. [38] C. Juan, G. Torrens, M. González-Nicolau, A. Oliver, FEMS Microbiol. Rev. 2017,41, 781. [39] D. A. Leonard, R. A. Bonomo, R. A. Powers, Acc. Chem. Res. 2013, 46, 2407. Adv. Therap. 2021,4, 2000246 2000246 (17 of 21) © 2021 The Authors. Advanced Therapeutics published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com [40] K.-C. J. Kaitany,N. V. Klinger, C. M. June, M. E. Ramey, R. A. Bonomo, R. A. Powers, D. A. Leonard, Antimicrob. Agents Chemother. 2013,57, 4848. [41] L. Poirel, T. Naas, P. Nordmann, Antimicrob. Agents Chemother. 2010,54, 24. [42] B. A. Evans, S. G. Amyes, Clin. Microbiol. Rev. 2014,27, 241. [43] M. Afzal-Shah, N. Woodford, D. M. Livermore, Antimicrob. Agents Chemother. 2001,45, 583. [44] A. M. Queenan, K. Bush, Clin. Microbiol. Rev. 2007,20, 440. [45] G. Bou, A. Oliver, J. Martínez-Beltrán, Antimicrob. Agents Chemother. 2000,44, 1556. [46] S. Corvec, L. Poirel, T. Naas, H. Drugeon, P. Nordmann, Antimicrob. Agents Chemother. 2007,51, 1530. [47] L. Poirel, S. Marqué, C. Héritier, C. Segonds, G. Chabanon, P. Nordmann, Antimicrob. Agents Chemother. 2005,49, 202. [48] T. Palzkill, Ann. N. Y. Acad. Sci. 2013,1277, 91. [49] P. Linciano, L. Cendron, E. Gianquinto, F. Spyrakis, D. Tondi, ACS Infect. Dis. 2019,5,9. [50] P. Salahuddin, A. Kumar, A. U. Khan, Curr. Protein Pept. Sci. 2018, 19, 130. [51] I. Garcia-Saez, J. D. Docquier, G. M. Rossolini, O. Dideberg, J. Mol. Biol. 2008,375, 604. [52] H. Park, E. N. Brothers, K. M. Merz, J. Am. Chem. Soc. 2005,127, 4232. [53] A. I. Karsisiotis, C. F. Damblon, G. C. K. Roberts, Metallomics 2014, 6, 1181. [54] H. Zhang, Q. Hao, FASEB J. 2011,25, 2574. [55] D. T. King, L. J. Worrall, R. Gruninger, N. C. Strynadka, J. Am. Chem. Soc. 2012,134, 11362. [56] M. R. Meini, L. I. Llarrull, A. J. Vila, FEBS Lett. 2015,589, 3419. [57] M.-N. Lisa, A. R. Palacios, M. Aitha, M. M. González, D. M. Moreno, M. W. Crowder, R. A. Bonomo, J. Spencer, D. L. Tierney, L. I. Llarrull, A. J. Vila, Nat. Commun. 2017,8, 538. [58] H. Feng, X. Liu, S. Wang, J. Fleming, D.-C. Wang, W. Liu, Nat. Commun. 2017,8, 2242. [59] A. U. Khan, L. Maryam, R. Zarrilli, BCM Microbiol. 2017,17, 101. [60] P. Nordmann, L. Dortet, L. Poirel, Trends Mol. Med. 2012,18, 263. [61] C. Bebrone, Biochem. Pharmacol. 2007,74, 1686. [62] G. Garau, C. Bebrone, C. Anne, M. Galleni, J. M. Frère, O. Dideberg, J. Mol. Biol. 2005,345, 785. [63] C. Bebrone, H. Delbrück, M. B. Kupper, P. Schlömer, C. Willmann, J. M. Frère, R. Fischer, M. Galleni, K. M. Hoffmann, Antimicrob. Agents Chemother. 2009,53, 4464. [64] R. P. McGeary, D. T. Tan, G. Schenk, Future Med. Chem. 2017,9, 673. [65] M. P. Pedroso, D. W. Waite, O. Melse, L. Wilson, N. Mitic, R. P. McGeary, I. Antes, L. W. Guddat, P. Hugenholtz, G. Schenk, Protein Cell 2020,11, 613. [66] Z. Cheng, C. A. Thomas, A. R. Joyner, R. L. Kimble, A. M. Sturgill, N. Y. Tran, M. R. Vulcan, S. A. Klinsky, D. J. Orea, C. R. Platt, F. Cao, B. Li, Q. Yang, C. J. Yurkiewicz, W. Fast, M. W. Crowder, Biomolecules 2020,10, 459. [67] H. J. Morrill, J. M. Pogue, K. S. Kaye, K. L. LaPlante, Open Forum Infect. Dis. 2015,2, 050. [68] R. F. Potter, A. W. D’Souza, G. Dantas, Drug Resist. Updat. 2016,29, 30. [69] D. van Duin, Y. Doi, Virulence 2017,8, 460. [70] P. Nordmann, L. Poirel, Clin. Infect. Dis. 2019,69, S521. [71] M. I. Abboud, C. Damblon, J. Brem, N. Smargiasso, P. Mercuri, B. Gilbert, A. M. Rydzik, T. D. W. Claridge, C. J. Schofield, J.-M. Frère, Antimicrob. Agents Chemother. 2016,60, 5655. [72] C. Lascols, M. Hackel, S. H. Marshall, A. M. Hujer, S. Bouchillon, R. Badal, D. Hoban, R. A. Bonomo, J. Antimicrob. Chemother. 2011, 66, 1992. [73] F.Al-Marzooq,Y.F.Ngeow,S.T.Tay,Int. J. Antimicrob. Agents 2015, 45, 445. [74] S. N. Seiffert, J. Marschall, V. Perreten, A. Carattoli, H. Furrer, A. Endimiani, Int. J. Antimicrob. Agents 2014,44, 260. [75] M. Avolio, C. Vignaroli, M. Crapis, A. Camporese, Future Microbiol. 2017,12, 1119. [76] T. Kwon, J. W. Yang, S. Lee, M. R. Yun, W. G. Yoo, H. S. Kim, J. O. Cha, D. W. Kim, Genome Announc. 2016,4, e01550-15. [77] C. A. Moubareck, S. F. Mouftah, T. Pal, A. Ghazawi, D. H. Halat, A. Nabi, M. A. AlSharhan, Z. O. AlDeesi, C. C. Peters, H. Celiloglu, M. Sannegowda, D. K. Sarkis, A. Sonnevend, Int. J. Antimicrob. Agents 2018,52, 90. [78] T. Tada, T. Miyoshi-Akiyama, R. K. Dahal, S. K. Mishra, H. Ohara, K. Shimada, T. Kirikae, B. M. Pokhrel, Int. J. Antimicrob. Agents 2013, 42, 372. [79] D. A. Contreras, S. P. Fitzwater, D. D. Nanayakkara, J. Schaenman, G. M. Aldrovandi, O. B. Garner, S. Yang, Antimicrob. Agents Chemother. 2020,64, e00948-19. [80] D. R. Giacobbe, M. Bassetti, F. G. De Rosa, V. del Bono, P. A. Grossi, F. Menichetti, F. Pea, G. M. Rossolini, M. Tumbarello, P. Viale, C. Viscoli, ISGRI-SITA (Italian Study Group on Resistant Infections of the Società Italiana Terapia Antinfettiva), Expert Rev. Anti-Infect. Ther. 2018,16, 307. [81] B. D. VanScoy, D. Tenero, S. Turner, D. M. Livermore, J. McCauley, H. Conde, S. M. Bhavnani, C. M. Rubino, P. G. Ambrose, Antimicrob. Agents Chemother. 2017,61, e01052-17. [82] L. Tselepis, G. W. Langley, A. F. Aboklaish, E. Widlake, D. E. Jackson, T. R. Walsh, C. J. Schofield, J. Brem, J. M. Tyrrell, Int. J. Antimicrob. Agents 2020,56, 105925. [83] A. Bonnefoy, C. Dupuis-Hamelin, V. Steier, C. Delachaume, C. Seys, T. Stachyra, M. Fairley, M. Guitton, M. Lampilas, J. Antimicrob. Chemother. 2004,54, 410. [84] K. Garber, Nat. Rev. Drug Discovery 2015,14, 445. [85] A. Vena, N. Castaldo, M. Bassetti, Curr. Opin. Infect. Dis. 2019,32, 638. [86]K.L.Chew,M.K.L.Tay,B.Cheng,R.T.P.Lin,S.Octavia, J. W. P. Teo, Antimicrob. Agents Chemother. 2018,62, e00414-18. [87] J. A. Karlowsky, K. M. Kazmierczak, B. L. M. de Jonge, M. A. Hackel, D. F. Sahm, P. A. Bradford, Antimicrob. Agents Chemother. 2017,61, e00472-17. [88] C. Ramsey, A. P. MacGowan, J. Antimicrob. Chemother. 2016,71, 2704. [89] M. A. Powles, A. Galgoci, A. Misura, L. Colwell, K. H. Dingley, W. Tang, J. Wu, T. Blizzard, M. Motyl, K. Young, Antimicrob. Agents Chemother. 2018,62, e02577-17. [90] J. Wu, F. Racine, M. K. Wismer, K. Young, D. M. Carr, J. C. Xiao, R. Katwaru, Q. Si, P. Harradine, M. Motyl, P. R. Bhagunde, M. L. Rizk, Antimicrob. Agents Chemother. 2018,62, e02323-17. [91] D. M. Livermore, S. Mushtaq, M. Warner, A. Vickers, N. Woodford, J. Antimicrob. Chemother. 2017,72, 1373. [92] Z. Khan, A. Iregui, D. Landman, J. Quale, J. Antimicrob. Chemother. 2019,74, 2938. [93] A. J. Lepak, M. Zhao, D. R. Andes, Antimicrob. Agents Chemother. 2019,63, e01648-19. [94] S. Mushtaq, A. Vickers, N. Woodford, A. Haldimann, D. M. Livermore, J. Antimicrob. Chemother. 2019,74, 953. [95] T. E. Asempa, A. Motos, K. Abdelraouf, C. Bissantz, C. Zampaloni, D. P. Nicolau, Int. J. Antimicrob. Chemother. 2020,55, 105838. [96] M. D. Barnes, M. A. Taracila, C. E. Good, S. Bajaksouzian, R. J. Rojas, D. van Duin, B. N. Kreiswirth, M. R. Jacobs, A. Haldimann, K. M. Papp-Wallace, R. A. Bonomo, Antimicrob. Agents Chemother. 2019, 63, e00432-19. [97] B.Moya,I.M.Barcelo,S.Bhagwat,M.Patel,G.Bou,K.M.PappWallace, R. A. Bonomo, A. Oliver, Antimicrob. Agents Chemother. 2017,61, e01238-17. Adv. Therap. 2021,4, 2000246 2000246 (18 of 21) © 2021 The Authors. Advanced Therapeutics published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com [98] N. L. Mallalieu, E. Winter, S. Fettner, K. Patel, E. Zwanziger, G. Attley, I. Rodriguez, A. Kano, S. M. Salama, D. Bentley, A. M. Geretti, Antimicrob. Agents Chemother. 2020,64, e02229-19. [99] S. D. Lahiri, S. Mangani,H. Jahic, M. Benvenuti, T. F. Durand-Reville, F. De Luca, D. E. Ehmann, G. M. Rossolini, R. A. Alm, J. D. Docquier, ACS Chem. Biol. 2015,10, 591. [100] T. F. Durand-Réville, S. Gluler, J. Comita-Prevoir, B. Chen, N. Bifulco, H. Huynh, S. Lahiri, A. B. Shapiro, S. M. McLeod, N. M. Carter, S. H. Moussa, C. Velez-Vega, N. B. Olivier, R. McLaughlin, N. Gao, J. Thresher, T. Palmer, B. Andrews, R. A. Giacobbe, J. V. Newman, D. E. Ehmann, B. de Jonge, J. O´Donnell, J. P. Mueller, R. A. Tommasi, A. A. Miller, Nat. Microbiol. 2017,2, 17104. [101] O. Sagan, R. Yakubsevitch, K. Yanev, R. Fomkin, E. Stone, D. Hines, J. O’Donnell, A. Miller, R. Isaacs, S. Srinivasan, Antimicrob. Agents Chemother. 2020,64, e01506-19. [102] M. D. Barnes, V. Kumar, C. R. Bethel, S. H. Moussa, J. O’Donnell, J. D. Rutter, C. E. Good, K. M. Hujer, A. M. Hujer, S. H. Marshall, B. N. Kreiswirth, S. S. Richter, P. N. Rather, M. R. Jacobs, K. M. PappWallace, F. van den Akker, R. A. Bonomo, mBio 2019,10, e00159-19. [103] T. F. Durand-Reville, J. Comita-Prevoir, J. Zhang, X. Wu, T. MayDracka, J. Romero, F. Wu, A. Chen, A. B. Shapiro, N. Carter, S. McLeod, R. Giacobbe, J. Verheijen, S. Lahiri, M. Sacco, Y. Chen, J. O’Donnell, A. A. Miller, J. P. Mueller, R. Tommasi, J. Med. Chem. 2020,63, 12511. [104] J. O’Donnell, A. Tanudra, A. Chen, D. Hines, R. Tommasi, J. Mueller, ACS Infect. Dis. 2020,6, 1378. [105] A. A. Miller, A. B. Shapiro, S. M. McLeod, N. M. Carter, S. H. Moussa,R.Tommasi,J.P.Mueller,ACS Infect. Dis. 2020,6, 1389. [106] A. G. Stewart, P. N. A. Harris, A. Henderson, M. A. Schembri, D. L. Paterson, J. Antimicrob. Chemother. 2020,75, 2384. [107] J. Plescia, N. Moitessie, Eur. J. Med. Chem. 2020,195, 112270. [108] R. C. Kane, A. T. Farrell, R. Sridhara, R. Pazdur, Clin. Cancer Res. 2006, 12, 2955. [109] M. Shirley, Drugs 2016,76, 405. [110] A. Markham, Drugs 2014,74, 1555. [111] O. Dobozy, I. Mile, I. Ferencz, V. Csányi, Acta Biochim. Biophys. Acad. Sci. Hung. 1971,6, 97. [112] R. Martin, J. B. Jones, Tetrahedron Lett. 1995,36, 8399. [113] D. Tondi, R. A. Powers, E. Caselli, M. C. Negri, J. Blázquez, M. P. Costi, B. K. Shoichet, Chem. Biol. 2001,8, 593. [114] C. T. Lohans, D. W. Wareham, H. P. Oswin, J. Mohammed, J. Spencer, C. W. Fishwick, M. A. McDonough, C. J. Schofield, J. Brem, Antimicrob. Agents Chemother. 2017,61, e02260-16. [115] S. T. Cahill, J. M. Tyrrell, I. H. Navratilova, K. Calvopiña, S. W. Robinson, C. T. Lohans, M. A. McDonough, R. Cain, C. W. G. Fishwick, M. B. Avison, T. R. Walsh, C. J. Schofield, J. Brem, Biochim. Biophys. Acta, Gen. Subj. 2019,1863, 742. [116] S. J. Baker, C. Z. Ding, T. Akama, Y.-K. Zhang, V. Hernandez, Y. Xia, Future Med. Chem. 2009,1, 1275. [117] S. Ness, R. Martin, A. M. Kindler, M. Paetzel, M. Gold, S. E. Jensen, J. B. Jones, N. C. Strynadka, Biochemistry 2000,39, 5312. [118] C. J. Burns, R. Goswami, R. W. Jackson, T. Lessen, W. Li, D. C. Pevear, P. K. Tirunahari, H. Xu, (Protez pharmaceuticals, Inc), WO 2010/130708A1,2010. [119] C. J. Burns, D. Daigle, B. Liu, D. McGrarry, D. C. Pevear, R. E. L. Trout, (Protez pharmaceuticals, Inc), WO 2014/089365A1,2014. [120] C. J. Burns, D. Daigle, B. Liu, R. W. Jackson, J. Hamrick, D. McGrarry, D. C. Pevear, R. E. L. Trout, (Protez pharmaceuticals, Inc), WO 2015/191907A1,2015. [121] C. J. Burns, D. Daigle, B. Liu, D. McGrarry, D. C. Pevear, E. L. Trout, (Venatorx pharmaceuticals, Inc), WO 2014/151958A1,2014. [122] C. J. Burns, D. Daigle, B. Liu, D. McGrarry, D. C. Pevear, E. L. Trout, R. W. Jackson, (Venatorx pharmaceuticals, Inc), US 2014/0194386, 2014. [123] S. J. Hecker, K. R. Reddy, M. Totrov, G. C. Hirst, O. Lomovskaya, D. C. Griffith, P. King, R. Tsivkovski, D. Sun, M. Sabet, Z. Tarazi, M. C. Clifton, K. Atkins, A. Raymond, K. T. Potts, J. Abendroth, S. H. Boyer, J. S. Loutit, E. E. Morgan, S. Durso, M. N. Dudley, J. Med. Chem. 2015,58, 3682. [124] G. Hirst, R. Reddy, S. Hecker, M. Totrov, D. C. Griffith, O. Rodny, M. N. Dudley, S. Boyer, (Rempex Pharmaceuticals, Inc), WO2012/021455A1,2012. [125] S. Dhillon, Drugs 2018,78, 1259. [126] C. L. Tooke, P. Hinchliffe, A. Krajnc, A. J. Mulholland, J. Brem, C. J. Schofield, J. Spencer, RSC Med. Chem. 2020,11, 491. [127] R. Tsivkovski, O. Lomovskaya, Antimicrob. Agents Chemother. 2020, 64, e01935-19. [128] G. W. Langley, R. Cain, J. M. Tyrrell, P. Hinchliffe, K. Calvopiña, C. L. Tooke, E. Widlake, C. G. Dowson, J. Spencer, T. R. Walsh, C. J. Schofield, J. Brem, Bioorg. Med. Chem. Lett. 2019,29, 1981. [129] J. C. Hamrick, J.-D. Docquier, T. Uehara, C. L. Myers, D. A. Six, C. L. Chatwin, K. J. John, S. F. Vernacchio, S. M. Cusick, R. E. L. Trout, C. Pozzi, F. De Luca, M. Benvenuti, S. Mangani, B. Liu, R. W. Jackson, G. Moeck, L. Xerri, C. J. Burns, D. C. Pevear, D. M. Daigle, Antimicrob. Agents Chemother. 2020,64, e01963-19. [130] A. Krajnc, J. Brem, P. Hinchliffe, K. Calvopina, T. D. Panduwawala, P. A. Lang, J. J. A. G. Kamps, J. M. Tyrrell, E. Widlake, B. G. Saward, T. R. Walsh, J. Spencer, C. J. Schofield, J. Med. Chem. 2019,62, 8544. [131] B. Liu, R. E. L. Trout, G.-H. Chu, D. McGarry, R. W. Jackson, J. C. Hamrick, D. M. Daigle, S. M. Cusick, C. Pozzi, F. De Luca, M. Benvenuti, S. Mangani, J.-D. Docquier, W. J. Weiss, D. C. Pevear, L. Xerri, C. J. Burns, J. Med. Chem. 2020,63, 2789. [132] S. Mushtaq, A. Vickers, M. Doumith, M. J. Ellington, N. Woodford, D. M. Livermore, J. Antimicrob. Chemother. 2021,76, 160. [133] C. J. Burns, R. Trout, A. Zulli, E. Mesaros, R. Jackson, S. Boyd, B. Liu, L. McLaughlin, C. Chatwin, J. Hamrick, D. Daigle, D. Pevear, 257th National Meet. Exposition of the American Chemical Society, Orlando, FL, U.S., March 31–April 4, 2019, American Chemical Society, Washington, DC 2019. [134] J. Hamrick, C. Chatwin, K. John, C. Burns, L. Xerri, G. Moeck, D. Pevear, 29th ECCMID, Amsterdam, The Netherlands, April 13–16, 2019, European Society of Clinical Microbiology and Infectious Diseases, Switzerland 2019. [135] L. M. Salonen, M. Ellermann, F. Diederich, Angew. Chem., Int. Ed. 2011,50, 4808. [136] A. Cardomí, J. C. Gómez-Tamayo, V. Gigoux, D. Fourmy, Trends Pharmacol. Sci. 2013,34, 320. [137] M. Maneiro, A. Peón, E. Lence, J. M. Otero, M. J. van Raaij, P. Thompson, A. R. Hawkins, C. González-Bello, Biochem. J. 2014,462, 415. [138] P. Hinchliffe, M. M. González, M. F. Mojica, J. M. González, V. Castillo, C. Saiz, M. Kosmopoulou, C. L. Tooke, L. I. Llarrull, G. Mahler, R. A. Bonomo, A. J. Vila, J. Spencer, Proc. Natl. Acad. Sci. USA 2016,113, E3745. [139] M. Aitha, A. R. Marts, A. Bergstrom, A. J. Møller, L. Moritz, L. Turner, J. C. Nix, R. A. Bonomo, R. C. Page, D. L. Tierney, M. W. Crowder, Biochemistry 2014,53, 7321. [140] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, G. A. Petersson, H. Nakatsuji, X. Li, M. Caricato, A. Marenich, J. Bloino, B. G. Janesko, R. Gomperts, B. Mennucci, H. P. Hratchian, J. V. Ortiz, A. F. Izmaylov, J. L. Sonnenberg, D. Williams-Young, F. Ding, F. Lipparini, F. Egidi, J. Goings, B. Peng, A. Petrone, T. Henderson, D. Ranasinghe, V. G. Zakrzewski, J. Gao, N. Rega, G. Zheng, W. Liang, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, K. Throssell, J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, T. Keith, R. Kobayashi, Adv. Therap. 2021,4, 2000246 2000246 (19 of 21) © 2021 The Authors. Advanced Therapeutics published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, J. M. Millam, M. Klene, C. Adamo, R. Cammi, J. W. Ochterski, R. L. Martin, K. Morokuma, O. Farkas, J. B. Foresman, D. J. Fox, Gaussian 09, Revision E.01, Wallingford, CT, 2016. [141] A. D. Becke, J. Chem. Phys. 1993,98, 5648. [142] P. J. Stephens, F. J. Devlin, C. F. Chabalowski, M. J. Frisch, J. Phys. Chem. 1994,98, 11623. [143] G. A. Petersson, A. Bennett, T. G. Tensfeldt, M. A. Al-Laham, W. A. Shirley, J. Mantzaris, J. Chem. Phys. 1988,89, 2193. [144] G. A. Petersson, M. A. Al-Laham, J. Chem. Phys. 1991,94, 6081. [145] G. A. Petersson, A. Bennett, T. G. Tensfeldt, M. A. Al-Laham, W. A. Shirley, J. Mantzaris, J. Chem. Phys. 1988,89, 2193. [146] T. Christopeit, T. J. O. Carlsen, R. Helland, H.-K. S. Leiros, J. Med. Chem. 2015,58, 8671. [147] S. J. Hecker, K. R. Reddy, O. Lomovskaya, D. C. Griffith, D. RubioAparicio, K. Nelson, R. Tsivkovski, D. Sun, M. Sabet, Z. Tarazi, J. Parkinson, M. Totrov, S. H. Boyer, T. W. Glinka, O. A. Pemberton, Y. Chen, M. N. Dudley, J. Med. Chem. 2020,63, 7491. [148] R. Reddy, T. Glinka, M. Totrov, S. Hecker, (Rempex Pharmaceuticals, Inc.), WO 2014/107535A1,2014. [149] R. Reddy, T. Glinka, S. Hecker, M. Totrov, O. Rodny, (Rempex pharmaceuticals, Inc), WO 2015/179308A1,2015. [150] R. K. Reddy, T. Glinka, M. Totrov, S. Hecker, O. Rodny, (Rempex pharmaceuticals, Inc), WO 2016/003929A1,2016. [151] O. Lomovskaya, K. Nelson, D. Rubio-Aparicio, R. Tsivkovski, D. Sun, M. N. Dudley, Antimicrob. Agents Chemother. 2020,64, e00552-20. [152] O. Lomovskaya, R. Tsivkovski, K. Nelson, D. Rubio-Aparicio, D. Sun, M. Totrov, M. N. Dudley, Antimicrob. Agents Chemother. 2020,64, e00212-20. [153] S. Hecker, R. K. Reddy, T. Glinka, O. Rodny, (Rempex pharmaceuticals, Inc), WO 2018/005662A1,2018. [154] R. Tsivkoski, M. Totrov, O. Lomovskaya, Antimicrob. Agents Chemother. 2020,64, e00130-20. [155] K. Nelson, D. Rubio-Aparicio, D. Sun, M. Dudley, O. Lomovskaya, Antimicrob. Agents Chemother. 2020,64, e00757-20. [156] E. A. Meyer, R. K. Castellano, F. Diederich, Angew. Chem., Int. Ed. 2003,42, 1210. [157] J. C. Gordon, J. B. Myers, T. Folta, V. Shoja, L. S. Heath, A. Onufriev, Nucleic Acids Res. 2005,33, W368. [158] http://biophysics.cs.vt.edu/H++ (acessed: November 2020). [159] M. B. Peters, Y. Yang, B. Wang, L. Füsti-Molnár, M. N. Weaver, K. M. Merz, J. Chem. Theory Comput. 2010,6, 2935. [160] D. A. Case, D. S. Cerutti, T. E. Cheatham, III, T. A. Darden, R. E. Duke, T. J. Giese, H. Gohlke, A. W. Goetz, D. Greene, N. Homeyer, S. Izadi, A. Kovalenko, T. S. Lee, S. LeGrand, P. Li, C. Lin, J. Liu, T. Luchko, R. Luo, D. Mermelstein, K. M. Merz, G. Monard, H. Nguyen, I. Omelyan, A. Onufriev, F. Pan, R. Qi, D. R. Roe, A. Roitberg, C. Sagui, C. L. Simmerling, W. M. Botello-Smith, J. Swails, R. C. Walker, J. Wang, R. M. Wolf, X. Wu, L. Xiao, D. M. York, P. A. Kollman, AMBER 2017, University of California, San Francisco 2017. [161] Building bonded model with MCPB: https://ambermd.org/ tutorials/advanced/tutorial20/mcpb.php (accessed: November 2020). [162] J. M. Seminario, Int. J. Quantum Chem. 1996,30, 1271. [163] W. D. Cornell, P. Cieplak, C. I. Bayly, I. R. Gould, K. M. Merz, Jr., D. M. Ferguson, D. C. Spellmeyer, T. Fox, J. W. Caldwell, P. A. Kollman, J. Am. Chem. Soc. 1995,117, 5179. [164] J. A. Maier, C. Martinez, K. Kasavajhala, L. Wickstrom, K. E. Hauser, C. Simmerling, J. Chem. Theory Comput. 2015,11, 3696. [165] A. W. Goetz, M. J. Williamson, D. Xu, D. Poole, S. Le Grand, R. C. Walker, J. Chem. Theory Comput. 2012,8, 1542. [166] R. Salomon-Ferrer, A. W. Goetz, D. Poole, S. Le Grand, R. C. Walker, J. Chem. Theory Comput. 2013,9, 3878. [167] S. Le Grand, A. W. Goetz, R. C. Walker, Comput. Phys. Commun. 2013,184, 374. [168] T. A. Darden, D. York, L. G. Pedersen, J. Chem. Phys. 1993,98, 10089. [169] J.-P. Ryckaert, G. Ciccotti, H. J. C. Berendsen, J. Comput. Phys. 1977, 23, 327. [170] W. L. DeLano, The PyMOL Molecular Graphics System, DeLano Scientific LLC, Palo Alto, CA, USA 2008. http://www.pymol.org. [171] E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch, D. M. Greenblatt, E. C. Meng, T. E. Ferrin, J. Comput. Chem. 2004,25, 1605. [172] D. R. Roe, T. E. Cheatham, J. Chem. Theory Comput. 2013,9, 3084. [173] AMBER 2015 London Workshop. Introduction to Principal Component Analysis: http://www.amber.utah.edu/AMBER-workshop/ London-2015/pca/ (accessed: November 2020). Emilio Lence obtainedhisPh.D.attheUniversityofSantiagodeCompostela(USC,Spain)in2009. HedidapredoctoralstayintheUniversityofCambridge(UK)withProfessorAbell.Afterconcluding hisdoctoralthesis,hejoinedtheDepartmentofChemistryattheUniversityofBristol(UK)asaXunta deGaliciapostdoctoralfellow inProfessorMulholland’sgroup.In2015hereturnedtotheUSCasa researchassociate,focusinghisresearchonusingcomputationaltoolstounderstandenzymerecognitionandmechanisticprocesseswithpotentialapplicationsforthediscoveryofnovelantibioticsand 𝛽-lactamaseinhibitors. Adv. Therap. 2021,4, 2000246 2000246 (20 of 21) © 2021 The Authors. Advanced Therapeutics published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com Concepción González-Bello obtainedherPh.D.attheUniversityofSantiagodeCompostela(USC, Spain)in1994.ShedidpredoctoralstaysattheUniversityofGhent(Belgium)withProfessorVandewalleandattheScrippsResearchInstitute(USA)withProfessorNicolaou.In1994shejoinedtheUniversityofCambridge(UK)asapostdocinProfessorAbell’sgroup.In1996shejoinedtheUSCasan assistantprofessorandwaspromotedtoassociateprofessorin2003.Since2011shehasbeengroup leaderattheCiQUS.Hermainresearchinterestisthestructure-baseddesignofnovelantibioticsand 𝛽-lactamaseinhibitorsinthefightagainstsuperbugs. Adv. Therap. 2021,4, 2000246 2000246 (21 of 21) © 2021 The Authors. Advanced Therapeutics published by Wiley-VCH GmbH