Cyclobutanones and Antibiotic Resistance

Antibiotic resistance is a growing problem.  One way in which bacterial are able to resist antibiotics like penicillin is to use an enzyme called a beta-lactamase to react with the antibiotic and convert it into a form that does not work.

One strategy to overcome this is to inhibit the beta-lactamase before it gets a chance to deactivate the antibiotic molecule.  Compounds such as clavulanic acid have been used for just this purpose, but the bugs are starting to become resistant to this approach.  Beta-lactamases are not going away, so new compounds will need to be developed that inhibit them.

The center of activity in an enzyme is its active site - an openning in the enzyme where the substrate that is modified by the enzyme gets bound.  Typically the active site has a shape that complements the substrate, and has a number of catalytic groups inside which account for the chemical reaction that it catalyzes.  An inhibitor often binds to the active site of the enzyme and prevents the enzyme from binding to any of its "real" substrate.

When designing an inhibitor for an enzyme there are two things the molecule has to do.  First it has to have a shape that matches the active site.  Often enzymes are described as having a lock-and-key relationship with the substrate (or with an inhibitor).  Only a key with the right size and shape will fit into the keyhole of the lock.  Likewise, only a compound with the right shape will fit into the active site of the enzyme.  Secondly, the inhibitor should interact strongly with the catalytic groups inside the enzyme to help keep it lodged in the active site.  If the inhibitor comes out of the active site, the enzyme will no longer be inhibited.

A recent paper looks at a series of cyclobutanone-containing compounds as potential beta-lactamase inhibitors.  Beta-lactam antibiotics like penicillin , as well as the beta-lacatamse inhibitors clavulanic acid, sulbactam and tazobactam all contain the amide functionality.  Replacing the beta-lactam with a cyclobutanone is really interesting - it preserves the carbonyl and four-membered ring of the usual beta-lactamase substrates, and at the same time a ketone is very different from an amide.

When the beta-lactamase binds to penicillin it hydrolyzes the amide bond which leaves penicillin unable to act as an antibiotic.  Most beta-lactamases have a serine at the active site wich reacts with the beta lactam carbonyl.  After the serine OH attaches to the carbonyl carbon, the amide bond is broken to produce an acyl intermediate in which the penicillin molecule is covalently bound to the enzyme.  This is followed by a hydrolysis 

step which releases the resulting penicilloic acid.  

Currently used beta-lactam inhibitors do the same thing, except they get stuck at the acyl intermediate step.  With the inhibitors, the final hydrolysis step does not happen so the inhibitor remains covalently attached to the enzyme in its active site.  This prevents the enzyme from binding to penicillin and inactivating it.

The cyclobutanone analogs should also able to bind in the active site of the beta-lactamase enzymes.  Initially, the serine at the active site can attack the cyclobutanone carbonyl, which forms a hemi-ketal.  However, no further reaction can take place - there is amide group which can be broken to form the acyl intermediate.

Some beta-lactamases work through a different mechanism which involves two zinc ions at the active site.  Instead of a nucleophilic attack by serine, these metallo-enzymes use a hydroxide ion bound to one of the zinc ions as a nucleophile.  The substrate is never bound covalently to the enzyme and the acyl intermediate does not form.  Currently used beta-lactamase inhibitors are inactive against these metallo-enzymes.  The cyclobutanone analog can still react at the active site, forming a hydrate.  The hydrate can't react further, since there is no amide bond to be hydrolized.  In principle, the hydrate could remain bound to the zinc ions in the active site, and so inhibit the enzyme.

Since bacteria are developing resistance to beta-lactamase inhibitors, new strategies to counteract them are necessary.  Cyclobutanone-containing analogs show promise - since  they do not form a covalently bound acyl intermediate, they could inhibit the beta-lacatamase by a different mechanism than compounds like clavulanic acid and could be effective even in bacteria with resistance to clavulanic acid.  Furthermore, they could also be effective against metallo-enzymes which are not inhibited by the current crop of beta-lacatamase inhibitors.