Beta+Lactam+resistant+bacteria

=Disease/Drug of Interest=

New Delhi Metallo-Beta-Lactamase (NDM-1)/Clavulanic acid

Motivation and Background
Since their discovery, society has come to heavily rely on beta lactam drugs, including penicillin, to treat serious bacterial infections. Thus the rise of antibiotic resistance to beta lactams presents a troubling situation. One of these resistance mechanisms is the synthesis of beta-lactamases, which cleave beta-lactam ring structures via hydrolysis. Even more concerning, class B beta-lactamases do not utilize a covalent catalytic mechanism, leading to resistance to most lactam-based inhibitors. One recent beta-lactamase currently studied is the New Delhi metallo-beta-lactamase, otherwise known as NDM-1 [1].

The first case of NDM-1 positive bacterial infection was in 2008. The infection, caused by Klebsiella pneumonia, was found to contain this novel metallo-beta-lactamase and resistant to all beta-lactam antibiotics except aztreonam;. [2,3,4]. NDM-1 was also found in a E. coli strain derived from the patient’s feces, which suggested NDM-1 gene could be horizontally transferred.[2]. As beta-lactam antibiotics are used as a last line of defense against drug-resistant bacteria, with carbapenems and penicillins constituting half of all antibiotic prescriptions worldwide, NDM-1 spread has been a significant concern. Bacterial plasmids that harbor the NDM-1 gene facilitate transmission to other species [5]. Thus, NDM-1 producers are not limited to a single species. E. coli, K. pneumoniae, K. oxytoca, Enterobacter cloacae, Proteus spp., Citrobacter freundii, Morganella morganii, Providencia spp. Pseudomonas aeroginosa and Acinetobacter baumanni, have all been found to encode the NDM-1 gene. [7]



The spread of NDM-1 has been facilitated by poor conditions in India into a global problem. Its population consists of many ill individuals, with the most tuberculosis patients globally and Asia’s highest incidence of cholera. Testing and surveillance of NDM-1 has been inadequate in India. Poor sanitation, lack of control of prescription antibiotics, crowding and poverty has amounted to the selection of multi-drug resistant bacteria in India. Bacteria with the NDM-1 gene, //bla-NDM-1//, have spread into the water systems, food, as well as hospitals.[6,8] Patients especially vulnerable are newborns, transplant recipients and individuals with compromised immune systems. Medical tourists are also susceptible, with 850,000 people traveling to India in 2010 for treatment. Over 40 countries have identified NDM-1 positive cases. Furthermore, in Canada, Italy, Kosovo, France and South Africa, cases with no links to travel have been reported, suggesting NDM-1 has already taken hold in these countries. [6]

Target Information
A s with other metallo-beta-lactamases of B1, NDM-1 overall structure has the common characteristic folding of αβ/βα sandwich. [4] As a metallo-beta-lactamase, NDM-1 depends on zinc ions on its active site to catalyze the hydrolysis of beta-lactam rings. Monozinc and dizinc metalloforms of NDM-1 exist, with the dizinc form being more common . The two zincs contained in NDM-1 have different binding affinities, and when supplemented with zinc ions, both metalloforms had maximal activity for penem substrates, such as penicillin G, ampicillin, and carbenicillin [1]. Significant residues associated with NDM-1’s active site (see Figure 3) include His120, His122, His189, His250, Asp124, Cys208 (see Figure 1). NDM-1 contains two mobile loops in the active site important to recognize, bind and catalyze MBLs. Significant conformational changes in response to the presence of substrate and other unique structural characteristics probably contributes to NDM-1’s ability to hydrolyze a broader range of substrates, especially those not degraded by other MBLs. [4]  See external link for a video on Beta-lactams and Beta-lactamase.

Drug Information


One potential inhibitor for NDM-1 is Clavulanic acid.[9] Molecular formula is C8H9NO5 . Molecular weight is 199.16 g/mol. CAS number is 58001-44-8. [10]

GlaxoSmithKline (GSK), formerly Beecham,  held 3 patents for Augmentin, which contained clavulanic acid and amoxicillin, until 2002. [11,12] Martin Cole, Thomas T. Howarth; Christopher  Reading were the original discoverers of clavulanic acid were employed by Beecham.[13] <span style="font-family: Arial,sans-serif; font-size: 11pt;">Information about clinical trials regarding specific interactions clavulanic acid and NDM-1 not found. It is naturally derived from <span style="background-color: white; font-family: Arial,sans-serif; font-size: 11pt;">Streptomyces clavuligerus and synthetically derived from arginine and the sugar glyceraldehyde 3-phosphate. Clavulanic acid <span style="font-family: Arial,sans-serif; font-size: 11pt;">is currently given in combinations with other antibiotics in order to increase effectiveness as it is a competitive inhibitor, such as with <span style="background-color: white; font-family: Arial,sans-serif; font-size: 11pt;">ticarcillin or amoxicillin. [14]

<span style="background-color: white; font-family: Arial,sans-serif; font-size: 11pt;">The combination of amoxicillin and clavulanic acid is prescribed in tablet and suspension forms to be taken by mouth. Side effects include diarrhea, upset stomach, vomiting, rashes, hives, difficuly breathing or swallowing, wheezing, etc. Brand names include Amoclav, Augmentin, and Clavamox. [15] Amoxicillin is an effective antibiotic against many gram-positive and gram-negative organisms. Thus as clavulanic acid protects amoxicillin from beta-lactamase enzymes through competitive inhibition, drugs such as Augmentin can target a wide range of types of infections. [16]  <span style="background-color: white; font-family: Arial,sans-serif; font-size: 11pt;">There have been indications that clavulanic acid could be a viable inhibitor to the NDM-1 enzyme. As NDM-1 naturally accommodated all beta lactams, a beta-lactam that could inhibit the enzyme would be ideal, Results from docking indicate that potential inhibitor conformations would block or displace the bridging hydroxide ion. Clavulanic acid in the docking process showed an unanimous inhibitor conformation in 100 poses. <span class="apple-converted-space" style="color: #403838; font-family: Arial,sans-serif; font-size: 11pt;"> In addition, as large beta-lactams tend to interact with remaining pockets of NDM-1 to bind beta-lactams into substrate conformers, clavulanic acid as a small beta-lactam would be viable as it would bind into an inhibitor conformation. [9]

An Alternative to Clavulanic Acid
<span style="color: #403838; font-family: Arial,sans-serif; font-size: 11pt;">There is a discrepancy in scientific literature over the effectiveness of clavulanic acid toward NDM-1 as a beta lactamase enzyme. In the Charan et al. paper as well as Arpin et. al state that NDM-1 is not susceptible to clavulanic acid, [8,17] The Kim et. al paper also asserts that NDM-1 is resistant with the explanation that class B metallo-beta-lactamases, such as NDM-1, cannot not be inhibited by classic irreversible beta lactamase inhibitors such as clavulanic acid, as NDM-1 is not a serine-based beta-lactamase. [18]

Aztreonam, another beta-lactam, is also a potential inhibitor to NDM-1. <span style="font-family: Arial,sans-serif; font-size: 11pt; line-height: 1.5;"> [9,19] In docking, aztreonam was found to block or replace the bridging hydroxide/ water in all conformations via its sulfate group. No hydrolysis activity of NDM-1 was evident in enzymatic assay. [9] However aztreonam's major disadvantage is that many NDM-1 producers also encode other beta-lactamases that are able to hydrolyze aztreonam, such as AMPC cephalosporinases and ESBLs.[19,20]. An approved inhibitor to those beta-lactamases will thus be necessary for a proper drug to combat NDM-1.