Computational analysis of the interactions of a novel cephalosporin derivative with β-lactamases

The problem of the antibiotic resistance is as old as the discovery of antibiotics themselves. Indeed, already in 1940, only about a decade after the discovery of penicillin and even before its introduction in clinics, two researchers found a bacterial enzyme able to inactivate it [1]. When the use of antibiotics was diffused worldwide, the presence of resistant strains of bacteria able to inactivate these drugs became more and more evident. Nevertheless, the impact of this phenomenon in clinics was probably underestimated, at least until the “golden age of antibiotics” has continued. However, since 1970s, the decline in the discovery of novel antimicrobial molecules and the irresponsible misuse/overuse of traditional antibiotics has led to the selection of multidrug resistant bacteria (the so-called “superbugs”) that are now considered as a main threat for global public health [2]. Despite many initiatives to address this issue, new resistance mechanisms are emerging and spreading globally. Therefore, on one side, there is an absolute need for coordinated action of all countries to avoid the spread of antibiotic resistance; on the other hand, there is the urgency to focus the research either on the development of new antimicrobial molecules able to escape to the mechanisms of antibiotic resistance in bacteria, or to counteract the resistance against the most commonly used antibiotics.

β-lactams are still the most widely used class of antibiotics, because of their pharmacological advantages such as potent activity combined with low toxicity, and also for their ease of delivery and low production costs [3]. The most important mechanism of resistance against this class of molecules is the activity of β-lactamases, a family of hydrolytic enzymes whose genes are mainly located on easily transferable plasmids and, as a consequence, have rapidly disseminated through the different bacteria species [4]. These enzymes catalyze the cleavage of the β-lactam ring responsible for the bactericidal activity of these antibiotics. Based on their mechanism of action, they can be divided in two principal groups: serine β-lactamases and metallo β-lactamases. This last group hosts a Zn ion in the active site, which is coordinated to metal ligating amino acids, and the cleavage of the β-lactam ring proceeds with the aid of polarized water molecules [5]. The first group evolved from the penicillin-binding proteins (PBPs), the targets of β-lactam antibiotics, and their mechanism of action proceeds via the same acylation/deacylation mechanism involving a Ser residue in the active site. Serine β-lactamases can be divided in three classes based on sequence identity. All of them share three sequence motifs common also to PBPs, but the class A enzymes are characterized by the presence of a conserved Glu residue in the so-called Ω-loop involved in the activation of the catalytic water molecule during deacylation, whereas class C enzymes are characterized by a conserved Tyr residue replacing the corresponding Ser residue of class A in motif II, and class D enzymes have a unique N-carboxylated Lys residue in motif I [3].

In order to overcome the serine β-lactamases-mediated resistance, several inhibitors have been introduced in the clinic for a long time. These compounds act as suicide inhibitors, covalently bonding the Ser residue in the active site of the β-lactamase and permanently inactivating the enzyme [6]. Many combinations of β-lactam antibiotics/β-lactamase inhibitors are currently approved for clinical use, and many others are in late stage clinical development [7]. Most of them pair a β-lactamase inhibitor with a cephalosporin derivative for their broad-spectrum activity and less susceptivity to hydrolysis compared with the penicillin counterparts [3].

In the last years, we have synthesized new 6-aminopenicillanic acid (6-APA) and 7-aminocephalosporanic acid (7-ACA) derivatives containing an additional β-lactam ring bound to the nitrogen of 6-APA/7-ACA scaffold via an amide bond. These compounds are active against Gram-positive bacteria at a concentration comparable with that of the reference antibiotic ceftriaxone (a third-generation cephalosporin widely used in clinics), with minimal in vitro cytotoxicity at a concentration even 10 times higher than that required for their antimicrobial effect. Moreover, the 7-ACA derivative (Fig. 1) showed better antimicrobial activity against S. aureus, one of the main pathogens responsible for nosocomial infections [8, 9]. An extended computational analysis of this compound allowed to study the interactions between this molecule and some candidate target PBPs. The results showed that this compound is potentially able to interact both with Gram-positive and Gram-negative enzymes, and that the 7-ACA nucleus is the preferred moiety targeting these enzymes, but also the isolated 2-azetidinone ring can interact with the active site of all PBPs. Moreover, a more favorable binding energy towards PBP2a, isolated from a multi-resistant S. aureus strain, was predicted for 7-ACA with respect to ceftriaxone [9]. All these data suggest that the 7-ACA derivative is a promising lead compound to develop a new class of antibiotics.

In our previous paper [9] we have tested this new 7-ACA derivative for its antibiotic activity and we have performed a computational study to dissect its interactions with its specific biological targets PBPs. In this study, we have extended the computational approach to serine β-lactamases in order to gain insight for the future development of antibiotics resistant to these enzymes. We have included in our study at least one representative for each Ambler class of serine β-lactamases [22], according also to the quality criteria that we have previously identified [9]. Moreover, most of the β-lactamases included in the current study are expressed by bacteria belonging to the group of the so-called “ESKAPE” pathogens, i.e. those bacteria belonging both to Gram-positive and Gram-negative species (the acronym “ESKAPE” has been created from the initial letters of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Enterobacter species), which are leading cause of nosocomial infections throughout the world. These pathogens can be commonly found in the human microbiota and in the environment, and under the selective pressure provoked by the use (and misuse/abuse) of antibiotics, they were particularly able to accumulate multiple different antimicrobial resistance mechanisms carried by bacterial chromosomes, plasmids or genetic transposable elements. In this way, they are now able to escape the most common antimicrobial treatments and, particularly in healthcare settings due to the high concentration of infected people, they can be transmitted among individuals or they can colonize immunocompromised patients, becoming one of the principal causes of death of hospitalised patients [25].

TEM-1 was the first β-lactamase identified in E. coli [26]. The proteins of this family are encoded generally by plasmidic genes and are commonly detected in E. coli based infections. TEM-1 belongs to Ambler class A and to group 2b according to Bush-Jacoby-Medeiros classification [23, 24] and is active against both penicillins and cephalosporins. The crystal structure of wild type TEM-1 β-lactamase selected for the present work has been determined at 1.9 Å resolution in 1995 [11]. Similarly to other known β-lactamases of the same class, it consists of two domains, with the active site located at the interface between them. In addition to the wild type, in order to improve our knowledge about the binding of 7-ACA derivative with this family of enzymes, we also simulated the interaction of our cephalosporin derivative with some mutants whose 3D structure is available. In particular, we selected the structure of the double mutant p.Val84Ile + p.Ala184Val, considered as a high resolution “wild type” enzyme [12]; of TEM-1 p.Asn132Ala, a mutant that shows a dramatic change in stability when bound to inhibitors such as moxalactam and imipenem [13]; of p.Met182Thr, which is isofunctional but more stable and diffracts to a higher resolution with respect to the wild type TEM-1 [14]; finally, of p.Glu166Asn, a deacylation-defective mutant of this β-lactamase [15].

The PC-1 β-lactamase used in this study derives from the Gram positive organism S. aureus, belonging to the ESKAPE group [25]. It is classified by Ambler as a class A serine penicillinase and is more specific for penicillins, being classified in the group 2a according to Bush, Jacoby and Medeiros. The structure selected for the present work has been determined at 2.0 Å [10] and, similarly to TEM-1, it consists of two closely associated domains with the active cleft located between them.

SHV-1 is another β-lactamase belonging to class A and group 2b, sharing approximately 68% sequence identity with TEM-1 [27]. It can be considered another common β-lactamase, found primarily in K. pneumoniae (another bacterial species belonging to the ESKAPE group) [25]. We have selected two structures for our study: the wild type protein, solved at a resolution of 1.33 Å in a complex with a boronic acid transition state inhibitor [16], and that of the mutant p.Glu166Ala complexed to a penam sulfone, solved at high (1.22 Å) resolution [17]. This last mutant, similarly to the mutant p.Glu166Asn of TEM-1, is a deacylation deficient variant.

AmpC from E. coli has been selected as a representative enzyme for class C β-lactamases (cephalosporinases, i.e. specific for cephalosporins). More than 100 structures are present in PDB for this protein, and we have selected the one of the wild type protein determined at 1.43 Å [18] and the structure of the mutant p.Asn152His, involving one of the catalytic residues, whose mutation determines the impairment of the enzymatic activity and a concomitant enhancement of the stability of the enzyme [19].

Finally, we have selected two representative structures of β-lactamases belonging to class D of Ambler classification (the so-called oxacillinases): OXA-10 from P. aeruginosa and OXA-23 from A. baumanii [20, 21]. Both organisms belong to the ESKAPE group, and in particular, the latter one, regarded as of little clinical importance just a decade ago, has been recently re-classified as a major opportunistic pathogen [25]. The rapid spread of the OXA-23-producing pathogens in the clinics worldwide constitutes a serious threat to human health [21]. The structure of OXA-10 has been determined at 2 Å in complex with a cyclobutanone analogue of β-lactams [20]. The structure of OXA-23 has been determined in complex with meropenem, a clinically important carbapenem antibiotic [21].

The interactions predicted for the 7-ACA derivative show several features in common within all these different β-lactamases and with the PBPs previously tested [9]. The predicted binding scores are in the same range of amplitude for the two families of enzymes, but the two β-lactam rings show different reactivity towards acylation, being the isolated 2-azetidinone ring the less reactive one in both cases. This would therefore increase the ability of this compound to escape from the inactivation of β-lactamases, conserving a reactive moiety for the interaction with PBPs. We are investigating if the different reactivities of these two rings could be modulated by their reciprocal distance in the molecule, by synthesizing and testing derivatives with a longer spacer. The polar interactions with the carboxylic moiety of 7-ACA and with the amide moiety in the chain connecting the two halves of this compound appear to be well conserved, although in the case of class D β-lactamases they involve hydrophobic residues. The two phenyl rings seems to be important for the interaction with different residues of the active site, and we think that the introduction of substituents could modulate the affinity of the 7-ACA derivative for the different enzymes.

By using an innovative computational approach, we have performed a deep characterization of the interaction of a new cephalosporin derivative bearing an additional 2-azetidinone ring bound to the 7-ACA nucleus, with serine β-lactamases belonging to different classes, in order to collect information about the potential ability of this compound to be more resistant to their inactivation. The results show that the interaction between this class of enzymes and the isolated β-lactam ring is less favored with respect to the interaction with the “canonical” 7-ACA moiety, with no significant differences between the two diastereoisomeric forms obtained from the chemical synthesis of this compound [9]. The isolated ring seems therefore less prone to the catalytic cleavage of these enzymes, and this would improve the resistance of this class of compounds towards inactivation. The detailed analysis of the interactions between the 7-ACA derivatives and the residues of the active site of the selected β-lactamases suggests that the two phenyl rings bound to the isolated 2-azetidinone moiety and the amidic group that connects this moiety to the 7-ACA nucleus could be modified in order to modulate the affinity of this compound for these enzymes.

This study represents an important step to develop a new class of antibiotics hopefully able to overcome the bacterial resistance, thus becoming a new, effective weapon in the battle for survival against microbes that we cannot absolutely risk to loose.