Maria-Agustina Rossi

Bio: Maria-Agustina Rossi is an academic researcher from National Scientific and Technical Research Council. The author has contributed to research in topics: Thiazolidine & Active site. The author has an hindex of 1, co-authored 3 publications receiving 22 citations.

Metallo-β-Lactamase Inhibitors Inspired on Snapshots from the Catalytic Mechanism

Abstract: β-Lactam antibiotics are the most widely prescribed antibacterial drugs due to their low toxicity and broad spectrum. Their action is counteracted by different resistance mechanisms developed by bacteria. Among them, the most common strategy is the expression of β-lactamases, enzymes that hydrolyze the amide bond present in all β-lactam compounds. There are several inhibitors against serine-β-lactamases (SBLs). Metallo-β-lactamases (MBLs) are Zn(II)-dependent enzymes able to hydrolyze most β-lactam antibiotics, and no clinically useful inhibitors against them have yet been approved. Despite their large structural diversity, MBLs have a common catalytic mechanism with similar reaction species. Here, we describe a number of MBL inhibitors that mimic different species formed during the hydrolysis process: substrate, transition state, intermediate, or product. Recent advances in the development of boron-based and thiol-based inhibitors are discussed in the light of the mechanism of MBLs. We also discuss the use of chelators as a possible strategy, since Zn(II) ions are essential for substrate binding and catalysis.

The urgent need for metallo-β-lactamase inhibitors: an unattended global threat

Abstract: Due to their superior tolerability and efficacy, β-lactams are the most potent and prescribed class of antibiotics in the clinic. The emergence of resistance to those antibiotics, mainly due to the production of bacterial enzymes called β-lactamases, has been partially solved by the introduction of β-lactamase inhibitors, which restore the activity of otherwise obsolete molecules. This solution is limited because currently available β-lactamase inhibitors only work against serine β-lactamases, whereas metallo-β-lactamases continue to spread, evolve, and confer resistance to all β-lactams, including carbapenems. Furthermore, the increased use of antibiotics to treat secondary bacterial pneumonia in severely sick patients with COVID-19 might exacerbate the problem of antimicrobial resistance. In this Personal View, we summarise the main advances accomplished in this area of research, emphasise the main challenges that need to be solved, and the importance of research on inhibitors for metallo-B-lactamases amidst the current pandemic.

2-Mercaptomethyl Thiazolidines (MMTZs) Inhibit All Metallo-β-Lactamase Classes by Maintaining a Conserved Binding Mode.

Abstract: Metallo-β-lactamase (MBL) production in Gram-negative bacteria is an important contributor to β-lactam antibiotic resistance. Combining β-lactams with β-lactamase inhibitors (BLIs) is a validated route to overcoming resistance, but MBL inhibitors are not available in the clinic. On the basis of zinc utilization and sequence, MBLs are divided into three subclasses, B1, B2, and B3, whose differing active-site architectures hinder development of BLIs capable of "cross-class" MBL inhibition. We previously described 2-mercaptomethyl thiazolidines (MMTZs) as B1 MBL inhibitors (e.g., NDM-1) and here show that inhibition extends to the clinically relevant B2 (Sfh-I) and B3 (L1) enzymes. MMTZs inhibit purified MBLs in vitro (e.g., Sfh-I, Ki 0.16 μM) and potentiate β-lactam activity against producer strains. X-ray crystallography reveals that inhibition involves direct interaction of the MMTZ thiol with the mono- or dizinc centers of Sfh-I/L1, respectively. This is further enhanced by sulfur-π interactions with a conserved active site tryptophan. Computational studies reveal that the stereochemistry at chiral centers is critical, showing less potent MMTZ stereoisomers (up to 800-fold) as unable to replicate sulfur-π interactions in Sfh-I, largely through steric constraints in a compact active site. Furthermore, in silico replacement of the thiazolidine sulfur with oxygen (forming an oxazolidine) resulted in less favorable aromatic interactions with B2 MBLs, though the effect is less than that previously observed for the subclass B1 enzyme NDM-1. In the B3 enzyme L1, these effects are offset by additional MMTZ interactions with the protein main chain. MMTZs can therefore inhibit all MBL classes by maintaining conserved binding modes through different routes.

Metallo-β-lactamases in the Age of Multidrug Resistance: From Structure and Mechanism to Evolution, Dissemination, and Inhibitor Design.

Abstract: Antimicrobial resistance is one of the major problems in current practical medicine. The spread of genes coding for resistance determinants among bacteria challenges the use of approved antibiotics, narrowing the options for treatment. Resistance to carbapenems, last resort antibiotics, is a major concern. Metallo-β-lactamases (MBLs) hydrolyze carbapenems, penicillins, and cephalosporins, becoming central to this problem. These enzymes diverge with respect to serine-β-lactamases by exhibiting a different fold, active site, and catalytic features. Elucidating their catalytic mechanism has been a big challenge in the field that has limited the development of useful inhibitors. This review covers exhaustively the details of the active-site chemistries, the diversity of MBL alleles, the catalytic mechanism against different substrates, and how this information has helped developing inhibitors. We also discuss here different aspects critical to understand the success of MBLs in conferring resistance: the molecular determinants of their dissemination, their cell physiology, from the biogenesis to the processing involved in the transit to the periplasm, and the uptake of the Zn(II) ions upon metal starvation conditions, such as those encountered during an infection. In this regard, the chemical, biochemical and microbiological aspects provide an integrative view of the current knowledge of MBLs.

The urgent need for metallo-β-lactamase inhibitors: an unattended global threat

Abstract: Due to their superior tolerability and efficacy, β-lactams are the most potent and prescribed class of antibiotics in the clinic. The emergence of resistance to those antibiotics, mainly due to the production of bacterial enzymes called β-lactamases, has been partially solved by the introduction of β-lactamase inhibitors, which restore the activity of otherwise obsolete molecules. This solution is limited because currently available β-lactamase inhibitors only work against serine β-lactamases, whereas metallo-β-lactamases continue to spread, evolve, and confer resistance to all β-lactams, including carbapenems. Furthermore, the increased use of antibiotics to treat secondary bacterial pneumonia in severely sick patients with COVID-19 might exacerbate the problem of antimicrobial resistance. In this Personal View, we summarise the main advances accomplished in this area of research, emphasise the main challenges that need to be solved, and the importance of research on inhibitors for metallo-B-lactamases amidst the current pandemic. Due to their superior tolerability and efficacy, β-lactams are the most potent and prescribed class of antibiotics in the clinic. The emergence of resistance to those antibiotics, mainly due to the production of bacterial enzymes called β-lactamases, has been partially solved by the introduction of β-lactamase inhibitors, which restore the activity of otherwise obsolete molecules. This solution is limited because currently available β-lactamase inhibitors only work against serine β-lactamases, whereas metallo-β-lactamases continue to spread, evolve, and confer resistance to all β-lactams, including carbapenems. Furthermore, the increased use of antibiotics to treat secondary bacterial pneumonia in severely sick patients with COVID-19 might exacerbate the problem of antimicrobial resistance. In this Personal View, we summarise the main advances accomplished in this area of research, emphasise the main challenges that need to be solved, and the importance of research on inhibitors for metallo-B-lactamases amidst the current pandemic. β-lactam antibiotics are the cornerstones of antimicrobial chemotherapy. However, antimicrobial resistance against these life-saving drugs is a major public health problem worldwide. The most concerning problem in antimicrobial resistance involves carbapenem-resistant Gram-negative bacteria. In the past decade, the so-called superbugs (eg, multidrug-resistant Klebsiella pneumoniae, carbapenem-resistant Acinetobacter baumannii, and multidrug-resistant Pseudomonas aeruginosa) have gained even more notoriety due to their intrinsic abilities to cause life-threatening disease in older people (older than 60 years) and immunocompromised hosts. These pathogens are responsible for more than 540 000 infections and nearly 14 000 deaths annually in the USA alone.1CDCAntibiotic resistance threats in the United States. Centers for Disease Control and Prevention, Atlanta, GA2019Google Scholar Regrettably, the situation is not expected to improve, since current estimates predict that antimicrobial resistance will be the main cause of death in 2050 (10 million deaths per year),2O'Neill J Antimicrobial resistance: tackling a crisis for the health and wealth of nations.http://www.jpiamr.eu/wp-content/uploads/2014/12/AMR-Review-Paper-Tackling-a-crisis-for-the-health-and-wealth-of-nations_1-2.pdfDate: 2014Date accessed: June 22, 2021Google Scholar threatening even the simplest medical procedure. The current COVID-19 pandemic might be aggravating this scenario in the future. Most deaths associated with the influenza pandemic of 1918 were caused by subsequent bacterial pneumonia,3Morens DM Taubenberger JK Fauci AS Predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness.J Infect Dis. 2008; 198: 962-970Crossref PubMed Scopus (1149) Google Scholar and secondary bacterial infections were also reported in the 2009 swine influenza pandemic,4Morris DE Cleary DW Clarke SC Secondary bacterial infections associated with influenza pandemics.Front Microbiol. 2017; 81041Crossref PubMed Scopus (267) Google Scholar during the 2003 severe acute respiratory syndrome outbreak,5Wilder-Smith A Green JA Paton NI Hospitalized patients with bacterial infections: a potential focus of SARS transmission during an outbreak.Epidemiol Infect. 2004; 132: 407-408Crossref PubMed Scopus (13) Google Scholar and during the 2012 Middle East respiratory syndrome outbreak.6Memish ZA Perlman S Van Kerkhove MD Zumla A Middle East respiratory syndrome.Lancet. 2020; 386: 995-1007Google Scholar In the current COVID-19 pandemic, sentinel reports showed that secondary infections were present in up to 30 % of critically ill patients7Dudoignon E Caméléna F Deniau B et al.Bacterial pneumonia in COVID-19 critically ill patients: a case series.Clin Infect Dis. 2020; 72: 905-906Crossref Scopus (57) Google Scholar and these infections were shown to markedly decrease the survival of patients with COVID-19.8Fattorini L Creti R Palma C Pantosti A Bacterial coinfections in COVID-19: an underestimated adversary.Ann Ist Super Sanita. 2020; 56: 359-364PubMed Google Scholar The bacterial species more frequently isolated are Mycoplasma pneumoniae, Staphylococcus aureus, Legionella pneumophila, Haemophilus spp, Klebsiella spp, P aeruginosa, Chlamydia spp, Streptococcus pneumoniae, and A baumannii. Notably, infections with antibiotic-resistant S aureus, K pneumoniae, P aeruginosa, or A baumannii have been reported in patients with COVID-19 in intensive care units.8Fattorini L Creti R Palma C Pantosti A Bacterial coinfections in COVID-19: an underestimated adversary.Ann Ist Super Sanita. 2020; 56: 359-364PubMed Google Scholar Understandably, as inpatients with SARS-CoV-2 infection often exhibit symptoms undistinguishable from hospital-acquired and ventilator-associated pneumonia, empirical treatments with broad-spectrum antibiotics are administered in many cases.9Rawson TM Moore LSP Zhu N et al.Bacterial and fungal coinfection in individuals with coronavirus: a rapid review to support COVID-19 antimicrobial prescribing.Clin Infect Dis Clin Infect Dis. 2020; 71: 2459-2468PubMed Google Scholar A 2020 literature review by Fattorini and colleagues8Fattorini L Creti R Palma C Pantosti A Bacterial coinfections in COVID-19: an underestimated adversary.Ann Ist Super Sanita. 2020; 56: 359-364PubMed Google Scholar reported that 476 (88·3%) of 539 patients with COVID-19 were treated with broad-spectrum antibiotics, including expanded-spectrum cephalosporins (eg, ceftriaxone, ceftazidime, and cefepime), quinolones, and carbapenems. Consequently, antibiotic use has substantially increased in many settings.9Rawson TM Moore LSP Zhu N et al.Bacterial and fungal coinfection in individuals with coronavirus: a rapid review to support COVID-19 antimicrobial prescribing.Clin Infect Dis Clin Infect Dis. 2020; 71: 2459-2468PubMed Google Scholar With the spread of the pandemic, hospitals around the world have seen an increase of patients infected with COVID-19. This situation has demanded major adjustments to health-care systems and infrastructure, and especially to the infection control and antimicrobial stewardship programmes.10Davis MW McManus D Koff A et al.Re-purposing antimicrobial stewardship tools in the electronic medical record for the management of COVID-19 patients.Infect Control Hosp Epidemiol. 2020; 41: 1335-1337Crossref PubMed Scopus (10) Google Scholar Unfortunately, less robust health-care systems, such as those in many Latin American and Asian countries where antimicrobial resistance rates are already perilously high11García-Betancur JC Appel TM Esparza G et al.Update on the epidemiology of carbapenemases in Latin America and the Caribbean.Expert Rev Anti Infect Ther. 2021; 19: 197-213Crossref PubMed Scopus (29) Google Scholar, 12Sader HS Castanheira M Arends SJR Goossens H Flamm RK Geographical and temporal variation in the frequency and antimicrobial susceptibility of bacteria isolated from patients hospitalized with bacterial pneumonia: results from 20 years of the SENTRY Antimicrobial Surveillance Program (1997–2016).J Antimicrob Chemother. 2019; 74: 1595-1606Crossref PubMed Scopus (41) Google Scholar and antimicrobial stewardship programmes are just beginning to be implemented,13Cox JA Vlieghe E Mendelson M et al.Antibiotic stewardship in low- and middle-income countries: the same but different?.Clin Microbiol Infect. 2017; 23: 812-818Summary Full Text Full Text PDF PubMed Scopus (231) Google Scholar are adjusting their response to the pandemic at varying degrees.14Acosta LD Capacidad de respuesta frente a la pandemia de COVID-19 en América Latina y el Caribe.Rev Panam Salud Publica. 2020; 44: 1Crossref Scopus (23) Google Scholar, 15Garcia PJ Alarcón A Bayer A et al.COVID-19 response in Latin America.Am J Trop Med Hyg. 2020; 103: 1765-1772Crossref PubMed Scopus (83) Google Scholar, 16Ezequiel GE Jafet A Hugo A et al.The COVID-19 pandemic: a call to action for health systems in Latin America to strengthen quality of care.Int J Qual Heal Care. 2020; 2020: 1-2Google Scholar Regrettably, these circumstances create a so-called perfect storm for an accelerated evolution of antimicrobial resistance.17Reardon S Antibiotic treatment for COVID-19 complications could fuel resistant bacteria.https://www.sciencemag.org/news/2020/04/antibiotic-treatment-covid-19-complications-could-fuel-resistant-bacteriaDate: 2020Date accessed: June 22, 2021Google Scholar, 18Antimicrobial resistance in the age of COVID-19.Nat Microbiol. 2020; 5: 779Crossref PubMed Scopus (54) Google Scholar Resistance to carbapenems among Gram-negative bacteria is primarily due to the production of carbapenemases, which inactivate these life-saving drugs. Bacteria achieve these challenging chemical reactions with two types of enzymes: one based on a serine residue (serine β-lactamases) or another using one or two zinc ions (metallo-β-lactamases). Some serine β-lactamases (such as K pneumoniae carbapenemases [KPC] and oxacillinase-48 [OXA-48-like] carbapenemases) can hydrolyse carbapenems, whereas all metallo-β-lactamases are carbapenemases. Metallo-β-lactamases are of particular interest and concern given several factors: (1) their ability to hydrolyse and provide resistance to virtually all β-lactam antibiotics; (2) the unavailability of clinically useful metallo-β-lactamase inhibitors; (3) the rapid pace at which new variants are isolated (figure 1A); (4) the transferability of their encoding genes; and (5) their ubiquity, because they are isolated from nosocomial and environmental sources.19Tooke CL Hinchliffe P Bragginton EC et al.β-lactamases and β-lactamase inhibitors in the 21st century.J Mol Biol. 2019; 431: 3472-3500Crossref PubMed Scopus (351) Google Scholar, 20Bush K Bradford PA Epidemiology of β-lactamase-producing pathogens.Clin Microbiol Rev. 2020; 33: e00047-e00119Crossref PubMed Scopus (291) Google Scholar Metallo-β-lactamases are further divided into three subclasses (B1, B2, and B3), based primarily on their metal content and different active site features. Most metallo-β-lactamases that have been identified so far belong to subclass B1, among which imipenemase (IMP), Verona imipenemase (VIM), and New Delhi metallo-β-lactamase (NDM) families are the three most common metallo-β-lactamases found in clinical isolates.21Bush K The ABCD's of β-lactamase nomenclature.J Infect Chemother. 2013; 19: 549-559Summary Full Text PDF PubMed Scopus (171) Google Scholar Subclass B2 has the fewest members and includes enzymes produced by different species of Aeromonas, such as A hydrophila, A veronii, and Serratia fonticola (the enzyme are commonly named in the literature as CphA, ImiS, and Sfh-I, respectively). Subclass B3 includes L1 of Stenotrophomonas maltophilia, an emerging multidrug-resistant bacterium in patients who are severely immunocompromised. This microorganism is associated with respiratory tract infections in patients who are chronically ill, who have cancer, or who have cystic fibrosis, and for whom antibiotic treatment options are limited.22Chang YT Lin CY Chen YH Hsueh PR Update on infections caused by Stenotrophomonas maltophilia with particular attention to resistance mechanisms and therapeutic options.Front Microbiol. 2015; 6: 893Crossref PubMed Scopus (224) Google Scholar The β-lactamase genes (bla genes) encoding subclass B1 metallo-β-lactamases are largely plasmid-borne and are of greater clinical relevance compared with metallo-β-lactamase subclasses B2 and B3, and other B1 enzymes that are not plasmid borne. This characteristic means subclass B1 metallo-β-lactamases can be transferred between bacterial strains via these mobile genetic elements. IMP-type β-lactamases were identified in 1991 in Japan23Watanabe M Iyobe S Inoue M Mitsuhashi S Transferable imipenem resistance in Pseudomonas aeruginosa.Antimicrob Agents Chemother. 1991; 35: 147-151Crossref PubMed Scopus (488) Google Scholar and are still the predominant metallo-β-lactamases in southeast Asia, where they can be found among P aeruginosa, A baumannii, and different species of Enterobacterales.20Bush K Bradford PA Epidemiology of β-lactamase-producing pathogens.Clin Microbiol Rev. 2020; 33: e00047-e00119Crossref PubMed Scopus (291) Google Scholar However, VIM-type β-lactamases were discovered in 1997 in Italy24Lauretti L Riccio ML Mazzariol A et al.Cloning and characterization of blaVIM, a new integron-borne metallo-beta-lactamase gene from a Pseudomonas aeruginosa clinical isolate.Antimicrob Agents Chemother. 1999; 43: 1584-1590Crossref PubMed Google Scholar and, until 2017, were the predominant metallo-β-lactamases in Europe, especially in Mediterranean countries.25Mojica MF Bonomo RA Fast W B1-Metallo-β-lactamases: where do we stand?.Curr Drug Targets. 2016; 17: 1029-1050Crossref PubMed Scopus (135) Google Scholar VIM-2-like β-lactamases are associated mostly with P aeruginosa, whereas VIM-1-like β-lactamases (eg, VIM-4) are frequently reported in strains of Enterobacterales.26López C Ayala JA Bonomo RA González LJ Vila AJ Protein determinants of dissemination and host specificity of metallo-β-lactamases.Nat Commun. 2019; 103617Crossref PubMed Scopus (37) Google Scholar Lastly, the first documented case of infection caused by NDM-producing bacteria occurred in 2008, when the blaNDM gene was detected in K pneumoniae and Escherichia coli strains from a patient returning to Sweden from India.27Yong D Toleman MA Giske CG et al.Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India.Antimicrob Agents Chemother. 2009; 53: 5046-5054Crossref PubMed Scopus (1906) Google Scholar Since then, blaNDM has disseminated globally, and it is considered endemic in the Indian subcontinent and the Middle East (figure 1B). Notably, NDM is currently the predominant metallo-β-lactamase in Europe.28Nordmann P Poirel L Epidemiology and diagnostics of carbapenem resistance in Gram-negative bacteria.Clin Infect Dis. 2019; 69: S521-S528Crossref PubMed Scopus (270) Google Scholar The blaNDM gene has been reported in several families of Enterobacterales as well as in other Gram-negative bacteria, such as Vibrio cholerae, Pseudomonas spp, and A baumannii.20Bush K Bradford PA Epidemiology of β-lactamase-producing pathogens.Clin Microbiol Rev. 2020; 33: e00047-e00119Crossref PubMed Scopus (291) Google Scholar, 25Mojica MF Bonomo RA Fast W B1-Metallo-β-lactamases: where do we stand?.Curr Drug Targets. 2016; 17: 1029-1050Crossref PubMed Scopus (135) Google Scholar, 29Wu W Feng Y Tang G Qiao F McNally A Zong Z NDM metallo-β-lactamases and their bacterial producers in health care settings.Clin Microbiol Rev. 2019; 32: e00115-e00118Crossref PubMed Scopus (299) Google Scholar NDM-producing microorganisms can cause life-threatening infections; therefore, that fact that these microorganisms are actively disseminating outside the health-care system is a matter of concern.30Mills MC Lee J The threat of carbapenem-resistant bacteria in the environment: Evidence of widespread contamination of reservoirs at a global.Environ Pollut. 2019; 255113143Crossref PubMed Scopus (77) Google Scholar, 31Köck R Daniels-Haardt I Becker K et al.Carbapenem-resistant Enterobacteriaceae in wildlife, food-producing, and companion animals: a systematic review.Clin Microbiol Infect. 2018; 24: 1241-1250Summary Full Text Full Text PDF PubMed Scopus (176) Google Scholar NDM-1 is unique in being a membrane-bound protein, by contrast with other metallo-β-lactamases. This cellular localisation favours its secretion and dissemination in outer membrane vesicles.32González LJ Bahr G Nakashige TG Nolan EM Bonomo RA Vila AJ Membrane anchoring stabilizes and favors secretion of New Delhi metallo-β-lactamase.Nat Chem Biol. 2016; 12: 516-522Crossref PubMed Scopus (102) Google Scholar Outer membrane vesicles are spherical portions of the outer membrane that protrude and detach from growing cells in response to a wide variety of environments.33Schwechheimer C Kuehn MJ Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions.Nat Rev Microbiol. 2015; 13: 605-619Crossref PubMed Scopus (897) Google Scholar, 34Schwechheimer C Kulp A Kuehn MJ Modulation of bacterial outer membrane vesicle production by envelope structure and content.BMC Microbiol. 2014; 14: 324Crossref PubMed Scopus (104) Google Scholar NDM-1 is selectively secreted in an active form into outer membrane vesicles by different bacteria, therefore being protected against the action of extracellular proteases.32González LJ Bahr G Nakashige TG Nolan EM Bonomo RA Vila AJ Membrane anchoring stabilizes and favors secretion of New Delhi metallo-β-lactamase.Nat Chem Biol. 2016; 12: 516-522Crossref PubMed Scopus (102) Google Scholar These outer membrane vesicles possess potent carbapenemase activity that helps with titrating the amount of available antibiotic at the infection site and can protect neighbouring bacteria otherwise susceptible to antibiotics.35Rumbo C Fernández-Moreira E Merino M et al.Horizontal transfer of the OXA-24 carbapenemase gene via outer membrane vesicles: a new mechanism of dissemination of carbapenem resistance genes in Acinetobacter baumannii.Antimicrob Agents Chemother. 2011; 55: 3084-3090Crossref PubMed Scopus (224) Google Scholar, 36Chatterjee S Mondal A Mitra S Basu S Acinetobacter baumannii transfers the blaNDM-1 gene via outer membrane vesicles.J Antimicrob Chemother. 2017; 72: 2201-2207Crossref PubMed Scopus (78) Google Scholar Furthermore, the finding of a plasmid containing the blaNDM-1 gene in outer membrane vesicles from A baumannii37Kim SW Lee JS Bin PS et al.The importance of porins and β-lactamase in outer membrane vesicles on the hydrolysis of β-lactam antibiotics.Int J Mol Sci. 2020; 212822Crossref Scopus (25) Google Scholar has also suggested an active role of vesicles in direct gene transfer.36Chatterjee S Mondal A Mitra S Basu S Acinetobacter baumannii transfers the blaNDM-1 gene via outer membrane vesicles.J Antimicrob Chemother. 2017; 72: 2201-2207Crossref PubMed Scopus (78) Google Scholar Despite that, the effects of outer membrane vesicles in the dissemination of resistance is controversial, and this area deserves attention. Notably, NDM-1 is also tailored to be expressed by all bacterial hosts highlighted as Priority One by WHO,26López C Ayala JA Bonomo RA González LJ Vila AJ Protein determinants of dissemination and host specificity of metallo-β-lactamases.Nat Commun. 2019; 103617Crossref PubMed Scopus (37) Google Scholar, 38WHOWHO publishes list of bacteria for which new antibiotics are urgently needed.https://www.who.int/news/item/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-neededDate: 2017Date accessed: June 22, 2021Google Scholar by contrast with other host-specific metallo-β-lactamases. The rapid evolution (figure 1A) and spread (figure 1B) of NDM presents a threat that requires urgent action. Two main strategies are currently used to counteract resistance due to β-lactamases. The first involves the synthesis of new β-lactam compounds refractory to hydrolysis by these enzymes (eg, cefiderocol). The second strategy is the development of β-lactamase inhibitors, which can be paired with the antibiotics and formulated as a single product.39Bush K Bradford PA Interplay between β-lactamases and new β-lactamase inhibitors.Nat Rev Microbiol. 2019; 17: 295-306Crossref PubMed Scopus (250) Google Scholar In this way, β-lactamase inhibitors prolong the usefulness of otherwise obsolete β-lactam antibiotics. Currently, clinical inhibitors approved for metallo-β-lactamases are not available. Metallo-β-lactamases do not share an evolutionary relationship with serine β-lactamases, because these β-lactamases possess different structures, active sites, and catalytic mechanisms. The first generation of β-lactamase inhibitors introduced in the clinic includes mechanistic-based inhibitors—ie, so-called suicide inactivators with a β-lactam structure (eg, clavulanic acid, tazobactam, and sulbactam; table). Suicide inactivators are β-lactam compounds that are hydrolysed by the serine β-lactamases but remain bound to the active site serine residue, thereby inactivating the enzyme. None of these compounds are markedly active against the current carbapenemases. The second generation of β-lactamase inhibitors corresponds to non-β-lactam-based compounds—the diazabicyclooctanones—such as avibactam40Coleman K Diazabicyclooctanes (DBOs): a potent new class of non-β-lactam β-lactamase inhibitors.Curr Opin Microbiol. 2011; 14: 550-555Crossref PubMed Scopus (185) Google Scholar, 41Ehmann DE Jahić H Ross PL et al.Avibactam is a covalent, reversible, non-β-lactam β-lactamase inhibitor.Proc Natl Acad Sci USA. 2012; 109: 11663-11668Crossref PubMed Scopus (385) Google Scholar and relebactam.42Blizzard TA Chen H Kim S et al.Discovery of MK-7655, a β-lactamase inhibitor for combination with Primaxin®.Bioorganic Med Chem Lett. 2014; 24: 780-785Crossref PubMed Scopus (129) Google Scholar These compounds act as reversible inhibitors of class A carbapenemases (eg, KPC-2) and extended-spectrum β-lactamases (eg, CTX-M), class C cephalosporinases (eg, AmpC, as commonly referred to in the literature, from Enterobacter cloacae complex and P aeruginosa), and some class D, notably the OXA-48 carbapenemase. However, none of these compounds effectively inactivate metallo-β-lactamases in a clinically meaningful way. In 2017, the approval of vaborbactam in combination with meropenem defined a third generation of β-lactamase inhibitors: boronate compounds, designed as transition state analogues. Vaborbactam is a potent inhibitor of serine β-lactamases, especially KPC-2, but is not active against metallo-β-lactamases.43Hecker SJ Reddy KR Totrov M et al.Discovery of a cyclic boronic acid β-lactamase inhibitor (RPX7009) with utility vs class A serine carbapenemases.J Med Chem. 2015; 58: 3682-3692Crossref PubMed Scopus (300) Google Scholar Overall, there are six approved serine β-lactamase inhibitors in different combinations with antibiotics, but none of these inhibitors work against metallo-β-lactamases (table).TableClinically available serine β-lactamase inhibitors and metallo-β-lactamase inhibitors in clinical trialsYear of FDA approvalClinical trial phaseβ-lactam partnerFormulationUsageInhibitor typeInhibition mechanismInhibition profile of the inhibitorSerine β-lactamasesMetallo-β-lactamasesClass A (extended-spectrum β-lactamase)Class A (KPC)Class CClass DSubclass B1Subclass B3Clavulanic acid1984..AmoxicillinAugmentinWide*Clavulanic acid is used to treat a wide variety of bacterial infections, among them: lower respiratory tract infections, acute bacterial otitis media, sinusitis, skin and skin structure infections, and cUTIs.First generationSuicide inhibitor, β-lactam analogueYesNoNoNoNoNoSulbactam1987..AmpicillinUnasynWide†Sulbactam is used to treat a wide variety of bacterial infections, among them: gynaecological infections, bone and joint infections, cIAIs, and skin and skin structure infections.First generationSuicide inhibitor, β-lactam analogueYesNoNoNoNoNoTazobactam1993..PiperacillinZosynWide‡Tazobactam with piperacillin is used to treat a wide variety of bacterial infections, among them: cIAIs, skin and skin structure infections, gynaecological infections, community-acquired pneumonia, and nosocomial pneumonia.First generationSuicide inhibitor, β-lactam analogueYesNoNoNoNoNoTazobactam2014..CeftolozaneZerbaxacIAIs and cUTIsFirst generationSuicide inhibitor, β-lactam analogueYesNoNoNoNoNoAvibactam2015..CeftazidimeAvycazcIAIs and cUTIsSecond generationReversible inhibitor, DBO typeYesYesYesYesNoNoAvibactam..Phase 3 (NCT03580044)Aztreonam..To be determined§The phase 3 study will determine the efficacy against cIAIs, nosocomial pneumonia (including hospital-acquired pneumonia and ventilator associated pneumonia), cUTIs, or bloodstream infections due to metallo-β-lactamase-producing Gram-negative bacteria.Second generationReversible inhibitor, DBO typeYesYesYesYesYesYesVaborbactam2017..MeropenemVabomerecUTIsThird generationβ-lactam transition state analogue, boronate typeYesYesYesYesNoNoRelebactam2019..Imipenem and cilastatin¶Relebactam is in combination with imipenem and cilastatin.RecarbriocUTIs and cIAIsSecond generationReversible inhibitor, DBO typeYesYesYesNoNoNoTaniborbactam..Phase 3 (NCT03840148)Cefepime..cUTIsThird generationβ-lactam transition state analogue, boronate typeYesYesYesYesYes‖IMP-type metallo-β-lactamases are weakly inhibited.NoQPX7728..Phase 1 (NCT04380207)......Third generationβ-lactam transition state analogue, boronate typeYesYesYesYesYes‖IMP-type metallo-β-lactamases are weakly inhibited.NoFDA=US Food and Drug Administration. cIAI=complicated intra-abdominal infection. cUTI=complicated urinary tract infection. DBO=diazabicyclooctanone.* Clavulanic acid is used to treat a wide variety of bacterial infections, among them: lower respiratory tract infections, acute bacterial otitis media, sinusitis, skin and skin structure infections, and cUTIs.† Sulbactam is used to treat a wide variety of bacterial infections, among them: gynaecological infections, bone and joint infections, cIAIs, and skin and skin structure infections.‡ Tazobactam with piperacillin is used to treat a wide variety of bacterial infections, among them: cIAIs, skin and skin structure infections, gynaecological infections, community-acquired pneumonia, and nosocomial pneumonia.§ The phase 3 study will determine the efficacy against cIAIs, nosocomial pneumonia (including hospital-acquired pneumonia and ventilator associated pneumonia), cUTIs, or bloodstream infections due to metallo-β-lactamase-producing Gram-negative bacteria.¶ Relebactam is in combination with imipenem and cilastatin.‖ IMP-type metallo-β-lactamases are weakly inhibited. Open table in a new tab FDA=US Food and Drug Administration. cIAI=complicated intra-abdominal infection. cUTI=complicated urinary tract infection. DBO=diazabicyclooctanone. The development of an efficient metallo-β-lactamase inhibitor that is active against the three subclasses is a challenging task. The clinically available serine β-lactamase inhibitors target the catalytic serine residue that cleaves the β-lactam ring. Metallo-β-lactamases do not possess this serine residue in their active site. Instead, a water molecule activated by the zinc ions pursues β-lactam hydrolysis. As a result of this fundamental difference in the catalytic mechanism, none of the currently available serine β-lactamase inhibitors are active against metallo-β-lactamases. Furthermore, the active sites of serine β-lactamases and metallo-β-lactamases present different sizes and topologies. Serine β-lactamases have a narrow and deep catalytic site. By contrast, the active site in metallo-β-lactamases is in a shallow groove, with only a few contact points to bind the inhibitor or substrate. An additional challenge in achieving metallo-β-lactamase inhibition resides on their large structural diversity, which involves low homology among active site residues and different Zn2+ content. Lastly, we note that, unlike serine β-lactamases, which are exclusively bacterial enzymes, metallo-β-lactamases belong to a superfamily of metalloproteins with diverse biological functions beyond β-lactam hydrolysis. This superfamily, designated as the metallo-hydrolase/oxidoreductase superfamily, groups more than 30 000 genes distributed in all three domains of life, Archaea, Bacteria, and Eukarya.44Murzin AG Brenner SE Hubbard T Chothia C SCOP: a structural classification of proteins database for the investigation of sequences and structures.J Mol Biol. 1995; 247: 536-540Crossref PubMed Scopus (5610) Google Scholar, 45Daiyasu H Osaka K Ishino Y Toh H Expansion of the zinc metallo-hydrolase family of the beta-lactamase fold.FEBS Lett. 2001; 503: 1-6Crossref PubMed Scopus (276) Google Scholar Members of this superfamily share a common protein fold that results in all of them having similar active sites. This characteristic explains why L-captopril, an inhibitor of the angiotensin-converting enzyme, can inhibit metallo-β-lactamases. Therefore, another challenge is to achieve a fine balance to develop effective broad-spectrum metallo-β-lactamase inhibitors that are still selective enough to avoid toxicity due to inhibition of off-target enzymes. Taniborbactam46Liu B Trout REL Chu GH et al.Discovery of taniborbactam (VNRX-5133): a broad-spectrum serine- and metallo-β-lactamase inhibitor for carbapenem-resistant bacterial infections.J Med Chem. 2020; 63: 2789-2801Crossref PubMed Scopus (141) Google Scholar and QPX772847Tsivkovski R Totrov M Lomovskaya O Biochemical characterization of QPX7728, a new ultrabroad-spectrum beta-lactamase inhibitor of serine and metallo-beta-lactamases.Antimicrob Agents Chemother. 2020; 64: e00130-e00150Crossref PubMed Scopus (56) Google Scholar, 48Hecker SJ Reddy KR Lomovskaya O et al.Discovery of cyclic boronic acid QPX7728, an ultrabroad-spectrum inhibitor of serine and metallo-β-lactamases.J Med Chem. 2020; 63: 7491-7507Crossref PubMed Scopus (97) Google Scholar are novel boronate compounds that are effective against most of the B1 metallo-β-lactamases (figure 2A). Both compounds are currently in phase 3 and phase 1 clinical trials, respectively, giving some hope (table). The development of boronate-based inhibitors, although promising, is still in its infancy. There are limitations in the scope of currently available boronates, as well as an incomplete understanding of their mechanism of inhibition and spectrum of action. What makes some boronates potent metallo-β-lactamase inhibitors and others not? Why do boronates inhibit some B1 metallo-β-lactamases (eg, the IMP-type metallo-β-lactamases) less efficiently? Why are boronates not active against B3 metallo-β-lactamases, such as L1 metallo-β-lactamase? Expanding this knowledge will enable the design of potent boronate molecules able to be cross-class metallo-β-lactamase inhibitors. Metallo-β-lactamases can also be inactivated by metal chelators that remove the essential zinc ions, such as Aspergillomarasmine A (figure 2A),49King AM Reid-Yu SA Wang W et al.Aspergillomarasmine A overcomes metallo-β-lactamase antibiotic resistance.Nature. 2014; 510: 503-506Crossref PubMed Scopus (385) Google Scholar or metal-based compounds that replace the zinc ions with other metals that gives rise to an inactive variant. The latter situation is the case of colloidal bismuth subcitrate,50Wang R Lai TP Gao P et al.Bismuth antimicrobial drugs serve as broad-spectrum metallo-β-lactamase inhibitors.Nat Commun. 2018; 9: 439Crossref PubMed Scopus (140) Google Scholar a compound used for the treatment of Helicobacter pylori infections. The use of chelators is appealing because these agents mimics a natural defence mechanism in vertebrate hosts that is triggered by bacterial infections, which consists of a massive metal sequestration by metal-binding proteins, such as calprotectin.51Zygiel EM Nolan EM transition metal sequestration by the host-defense protein calprotectin.Annu Rev Biochem. 2018; 87: 621-643Crossref PubMed Scopus (109) Google Scholar, 52Kehl-Fie TE Skaar EP Nutritional immunity beyond iron: a role for manganese and zinc.Curr Opin Chem Biol. 2010; 14: 218-224Crossref PubMed Scopus (458) Google Scholar, 53Corbin BD Seeley EH Raab A et al.Metal chelation and inhibition of bacterial growth in tissue abscesses.Science. 2008; 319 (65): 96Crossref Scopus (671) Google Scholar However, exposure to this environment of metal starvation has led to the selection of metallo-β-lactamases that have developed a higher zinc binding affinity, thus being able to escape the action of chelators.54Bahr G Vitor-Horen L Bethel CR Bonomo RA González LJ Vila AJ Clinical evolution of New Delhi metallo-β-lactamase (NDM) optimizes resistance under Zn(II) deprivation.Antimicrob Agents Chemother. 2017; 62: e01849-e01917PubMed Google Scholar, 55Cheng Z Thomas PW Ju L et al.Evolution of New Delhi metallo-β-lactamase (NDM) in the clinic: effects of NDM mutations on stability, zinc affinity, and mono-zinc activity.J Biol Chem. 2018; 293: 12606-12618Summary Full Text Full Text PDF PubMed Scopus (63) Google Scholar Additionally, a major concern regarding the use of chelators is their low specificity because these agents can also target many other metalloproteins. In this regard, Aspergillomarasmine A seems to be more selective and less toxic in animal infections than other chelators. However, the clinical efficacy of Aspergillomarasmine A remains to be determined. A limitation of concern is its inactivity against some metallo-β-lactamases of the B1 subclass, such as São Paolo metallo-1 (SPM-1), Adelaide imipenemase (AIM), or IMP-1, with high affinity towards zinc.56Rotondo CM Sychantha D Koteva K Wright GD Suppression of β-lactam resistance by Aspergillomarasmine A is influenced by both the metallo-β-lactamase target and the antibiotic partner.Antimicrob Agents Chemother. 2020; 64: e01386-e01419Crossref PubMed Scopus (12) Google Scholar Remarkably, despite the different active site topology and Zn2+ content among the three metallo-β-lactamase subclasses, a shared mechanism of hydrolysis of carbapenems has been elucidated.57Lisa M-N Palacios AR Aitha M et al.A general reaction mechanism for carbapenem hydrolysis by mononuclear and binuclear metallo-β-lactamases.Nat Commun. 2017; 8: 538Crossref PubMed Scopus (83) Google Scholar The zinc ions in metallo-β-lactamases activate a water molecule, which is responsible for the cleavage of the β-lactam ring. After this step, a central reaction intermediate with a negative charge is formed. This intermediate is bound to the active site by interaction with the metal centre and some conserved residues, such as Asn233 and Lys224 (figure 2B). Finally, this intermediate species receives a proton from a water molecule before being released from the active site. Notably, this intermediate is not present in the reaction of carbapenem hydrolysis that is catalysed by serine β-lactamases. The elucidation of this common catalytic mechanism is of major relevance to advance in the metallo-β-lactamase inhibitor design, given that novel strategies inspired by the mechanism are possible.58Palacios AR Rossi M-A Mahler GS Vila AJ Metallo-β-lactamase inhibitors inspired on snapshots from the catalytic mechanism.Biomolecules. 2020; 10: 854Crossref Scopus (37) Google Scholar A representative example of this novel approach is the family of bisthiazolidines, which mimic the β-lactam antibiotics and are active against the three subclasses of metallo-β-lactamases (figure 2A).59Hinchliffe P González MM Mojica MF et al.Cross-class metallo-β-lactamase inhibition by bisthiazolidines reveals multiple binding modes.Proc Natl Acad Sci USA. 2016; 113: e3745-e3754Crossref PubMed Scopus (105) Google Scholar It is also necessary to elucidate which is the action of these new compounds on other cellular targets in bacteria. The present prospects offers hope, but further efforts are needed in understanding how metallo-β-lactamases can be inhibited and, also importantly, how metallo-β-lactamases can escape the action of novel inhibitors by means of new mutations. Future developments should also consider that tebipenem, the first oral carbapenem, is in phase 3 clinical trials.60Jain A Utley L Parr TR Zabawa T Pucci MJ Tebipenem, the first oral carbapenem antibiotic.Expert Rev Anti Infect Ther. 2018; 16: 513-522Crossref PubMed Scopus (45) Google Scholar, 61Kobayashi R Konomi M Hasegawa K Morozumi M Sunakawa K Ubukata K In vitro activity of tebipenem, a new oral carbapenem antibiotic, against penicillin-nonsusceptible Streptococcus pneumoniae.Antimicrob Agents Chemother. 2005; 49: 889-894Crossref PubMed Scopus (64) Google Scholar Therefore, the development of oral β-lactamase inhibitors to be paired with this antibiotic would be a major step forward in the fight against bacterial resistance. As made evident by the COVID-19 pandemic, densely populated urban settlements characterised by poor hygiene, contaminated water, and close proximity of domestic animals favours the dissemination of antimicrobial resistance.62Nadimpalli ML Marks SJ Montealegre MC et al.Urban informal settlements as hotspots of antimicrobial resistance and the need to curb environmental transmission.Nat Microbiol. 2020; 5: 787-795Crossref PubMed Scopus (65) Google Scholar This finding corresponds to cities, where 90% of the urban population growth is expected to take place. Global travel and food transportation will soon recover; however, the growing threat of antimicrobial resistance will not diminish. As bla genes become more and more widespread, vulnerable populations will probably bear the burden of untreatable infections. The wealth of information that has been gathered during the past 5 years in understanding metallo-β-lactamases (particularly NDM-1) should now be translated into practical solutions to counteract antimicrobial resistance. Contributors RAB and AJV conceived the idea for this Personal View and wrote the Personal View. MFM and M-AR did the literature search, analysed and discussed the data, made the figures, and also assisted in writing the Personal View. All authors discussed and approved the final version of the Personal View. We declare no competing interests. Acknowledgments This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health to RAB and AJV (award number R01AI100560). This work was also supported, in part, by funds and/or facilities provided by the Cleveland Department of Veterans Affairs (award number 1I01BX001974) to RAB from the Biomedical Laboratory Research & Development Service of the Veterans Affairs Office of Research and Development, and the Geriatric Research, Education, and Clinical Center VISN 10. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Department of Veterans Affairs. Editorial note: the Lancet Group takes a neutral position with respect to territorial claims in published maps and institutional affiliations.

New Carbapenemase Inhibitors: Clearing the Way for the β-Lactams.

Abstract: Carbapenem resistance is a major global health problem that seriously compromises the treatment of infections caused by nosocomial pathogens. Resistance to carbapenems mainly occurs via the production of carbapenemases, such as VIM, IMP, NDM, KPC and OXA, among others. Preclinical and clinical trials are currently underway to test a new generation of promising inhibitors, together with the recently approved avibactam, relebactam and vaborbactam. This review summarizes the main, most promising carbapenemase inhibitors synthesized to date, as well as their spectrum of activity and current stage of development. We particularly focus on β-lactam/β-lactamase inhibitor combinations that could potentially be used to treat infections caused by carbapenemase-producer pathogens of critical priority. The emergence of these new combinations represents a step forward in the fight against antimicrobial resistance, especially in regard to metallo-β-lactamases and carbapenem-hydrolysing class D β-lactamases, not currently inhibited by any clinically approved inhibitor.

The past, present, and future of antibiotics

Abstract: Antibiotics have transformed modern medicine. They are essential for treating infectious diseases and enable vital therapies and procedures. However, despite this success, their continued use in the 21st century is imperiled by two orthogonal challenges. The first is that the microbes targeted by these drugs evolve resistance to them over time. The second is that antibiotic discovery and development are no longer cost-effective using traditional reimbursement models. Consequently, there are a dwindling number of companies and laboratories dedicated to delivering new antibiotics, resulting in an anemic pipeline that threatens our control of infections. The future of antibiotics requires innovation in a field that has relied on highly traditional methods of discovery and development. This will require substantial changes in policy, quantitative understanding of the societal value of these drugs, and investment in alternatives to traditional antibiotics. These include narrow-spectrum drugs, bacteriophage, monoclonal antibodies, and vaccines, coupled with highly effective diagnostics. Addressing the antibiotic crisis to meet our future needs requires considerable investment in both research and development, along with ensuring a viable marketplace that encourages innovation. This review explores the past, present, and future of antimicrobial therapy. Description Antibiotics are imperiled by antimicrobial resistance, but innovations in antimicrobial discovery hold promise for the future.

The urgent need for metallo-β-lactamase inhibitors: an unattended global threat

Abstract: Due to their superior tolerability and efficacy, β-lactams are the most potent and prescribed class of antibiotics in the clinic. The emergence of resistance to those antibiotics, mainly due to the production of bacterial enzymes called β-lactamases, has been partially solved by the introduction of β-lactamase inhibitors, which restore the activity of otherwise obsolete molecules. This solution is limited because currently available β-lactamase inhibitors only work against serine β-lactamases, whereas metallo-β-lactamases continue to spread, evolve, and confer resistance to all β-lactams, including carbapenems. Furthermore, the increased use of antibiotics to treat secondary bacterial pneumonia in severely sick patients with COVID-19 might exacerbate the problem of antimicrobial resistance. In this Personal View, we summarise the main advances accomplished in this area of research, emphasise the main challenges that need to be solved, and the importance of research on inhibitors for metallo-B-lactamases amidst the current pandemic.