Sweet Spot of a Killer

ANTIBACTERIAL WARFARE REQUIRES WITS, CREATIVITY AND TIME. In a post-antibiotic era, unicellular organisms are winning an arms race with sheer numbers, high reproductive rates and an acute response to strong selective pressure created by our indiscriminate use of antibacterial drugs. Where to now?

PHARMA & LIFE SCIENCES

WHITEPAPER

A POST-ANTIBIOTIC ERA 3

One view of the future of antibiotics

THE ANTIBACTERIAL BATTLEGROUND 4

The obstacles antibacterial drugs must overcome

TWO-FOR-ONE TARGET 6

A close look at DNA gyrase and topoisomerase IV

OF RINGS, BRIDGES AND POCKETS 7

Antibiotic compound structures and properties

A STRONG PUNCH CAN LEAD ASTRAY 9

Balancing strength and solubility

AN EASY FIT CAN GO A LONG WAY 11

The potential of improving binding interactions DECOY TO FREE A MOLECULE 13

The role of protein binding

THE FEAR OF WATER 16

Hydrophobicity and penetration

TIME TO DO THE WORK 17

Finding the sweet spot of effective killers

REFERENCES 18

CONTENTS

3

“ Finding the balance or ‘sweet spot’ that maximizes the amount of potent antibacterial agent that reaches and impacts its target will be the maker or breaker of future antibiotic development programs.”

A POST-ANTIBIOTIC ERA

Since the introduction of the first antibiotics at the beginning of the 20th century, we have blasted bacteria infecting our bodies with potent chemicals, only to discover that they have become resistant to our weapons. We adapt or combine antibiotics to circumvent this resistance, but the result is often multidrug unresponsiveness. These unicellular organisms are winning an arms race with sheer numbers, high reproductive rates and an acute response to strong selective pressure created by our indiscriminate use of antibacterial drugs.

Now we are running out of defensive options. A combination of financial and political factors has made the development of novel antibiotics a risky investment. Since the beginning of the 21st century, the FDA has approved only seven new chemical entities as systemic antibacterials. Of these, only two have truly novel mechanisms of action that can delay the development of resistance. Acknowledging the dwindling antibiotic arsenal, the World Health Organization has warned of a potential post-antibiotic era “in which common infections and minor injuries can kill”1.

Prevailing financial skepticism and an uncertain regulatory environment have clearly contributed to the decreasing number of novel antibiotics introduced into the clinical setting, but the scientific challenge of developing an effective antibacterial is an equally important factor. In a review article on antibacterial discovery, Dr. Lynn Silver points to a discovery void that has seen “no successful discoveries of novel agents since 1987”2. This void is not due to any lack of innovation or effort. Developing novel antibacterials is difficult because potential drug compounds must meet a number of criteria that are not always aligned.

The World Health Organization warned of a potential post-antibiotic era “in which common infections and minor injuries can kill”.

4

THE ANTIBACTERIAL BATTLEGROUND

Think about the last time you took an antibiotic. If it was oral, the pills were probably large and you were asked to take them two or more times a day over the course of several days.

This type of extended, high-dose regimen is essential to achieve the therapeutic goal of killing an evasive foreign cell inside of a host made up of drug-susceptible cells. Maintaining high drug levels in the host over an extended period of time to reach and wipe out the culprit bacterial population is the medical equivalent of ‘shock and awe’ warfare. Achieving the desired outcome is not a trivial task. Pelting bacteria with insufficient treatment dose or for an insufficient period increases the likelihood that an oddball bacterium with a mutation that makes it resistant to the antibiotic survives the chemical storm and lives to produce other bacteria with the same mutation. Voila! You have a drug-resistant strain of the pathogen.

The development of the antibiotic is also not a trivial matter. An antibiotic encounters a number of hurdles on the path from the point of administration to its target (Figure 1). All the tricks to overcome these obstacles must be incorporated into the chemical structure and formulation of the antibiotic.

Generally, the antibiotic compound must travel from the site of administration to the site of infection via the blood stream or tissue fluid. Blood and its derivatives are to a large extent water, so the compound travels best if it is water-soluble.

Blood also contains a repertoire of proteins, such as albumin and globulins, that have the ability to bind chemicals. This means that the chances of the antibiotic compound reaching the site of infection and being available to act on bacterial cells are significantly reduced if it does not dissolve well in water and if it tends to bind to proteins. A brute force solution to these bioavailability issues is to increase the drug dose – the more compound that enters the host, the more that is freely available. However, the host consists of cells that may also be susceptible to the compound, so the higher the dose, the greater the chance of adverse effects.

Once the compound arrives at the site of infection, it must reach its molecular target within the bacterium. Unlike the cells of the host, bacteria have a cell wall that poses a formidable barrier. Gram-positive bacteria have a cell wall consisting of a thick, extensively cross-linked peptidoglycan layer

Figure 1. Antibiotics (blue dots) face a number of potential obstacles on their way from the site of administration to their target within the infecting bacteria.

IV administration

Ingestion Dissolution& absorbtion

Direct entry

Must enterthe body

Must enter the bloodstream

Should not enterand adversely a�ecthost cells

Must travel easily in thebloodstream and avoid binding to proteins

Site ofinfection

Must crossoutermembrane,Must avoid

e�ux pumps

Must avoide�ux pumps

Must crossthe thickpeptidoglycanlayer

Must hit target

peptidoglycanlayerand plasmamembrane

and plasmamembrane

Potential obstaclesfor antibiotic

treatments

Gram-negative bacteria

Gram-positive bacteria

5

that surrounds the inner cell membrane and is porous to small substances. The underlying plasma membrane can be penetrated via passive diffusion of lipophilic molecules. Unfortunately, lipophilic molecules are not very water-soluble, which brings us back to the hurdle of bioavailability. Additionally, membrane-embedded proteins called efflux pumps are known to thwart antibiotics by removing them from the cell.

Gram-negative bacteria are even trickier. Their thin peptidoglycan layer is surrounded by a second outer membrane that works as an effective barrier between the environment and the periplasm, the space between the outer and inner cell membranes. The outer membrane is dotted with porins that regulate the traffic of molecules into and out of the periplasm and can act to exclude many antibiotics. Compared to Gram-positive bacteria, Gram-negative bacteria also have a much higher number and diversity of membrane-embedded efflux pumps. Finally,

Gram-negative bacteria produce a greater variety of enzymes that metabolize antibiotics, such as several ß-lactamases, that render penicillins, cephalosporins, monobactams and carbapenems inactive.

Since the type of proteins regulating molecular traffic is species-specific and the antibiotic must cross two chemically distinct cellular membranes, few antibiotics work against Gram-negative bacteria. One antibiotic class that is effective against Gram-negative bacteria, the fluoroquinolones, passes through the porins of the outer membrane as a charged, water-soluble molecule and then becomes uncharged and hydrophobic in the periplasm, which allows it to diffuse across the inner membrane. This environment-triggered change in molecular charge illustrates the complexity of developing effective broad-spectrum antibiotics3.

The final hurdle is hitting the target. Antibiotics work by binding to essential molecular machinery

of bacteria and preventing these targets from doing their job. Some antibiotics punch holes into the cell wall of bacteria by inhibiting the enzymes that make peptidoglycans. Others inhibit enzymes to prevent bacteria from synthesizing proteins or DNA.

New antibiotic targets are needed to circumvent target-specific resistance, which has emerged against every antibiotic class. These new targets can be bacterial enyzmes or pathways that have never been exploited before, or novel sites of action on currently exploited targets. Curbing resistance development will require a very careful selection that invokes not only new mechanisms of action but also enables the development of multi-target monotherapeutic drugs: single compounds that simultaneously impact the function of two or more targets3. In this way, if resistance develops along one line of fire, activity at the other target maintains the efficacy of the antibiotic.

30S

50S

Folate

Inhibit Cell Wall Sythesis or FunctionBeta-Lactams

PenicillinsCephalosporins

CarbapenemsMonobactams

VancomycinDaptomycinPolypeptides

Inhibit Nucleic Acid Sythesis or Function

Inhibit DNA Gyrase +/- Topoisomerase IVQuinolones

Inhibit Folate SynthesisTrimethoprim / Sulfamethoxazole

Create Free RadicalsMetronidazole, Nitrofurantoin

Inhibit Protein SythesisInhibit 50S subunitMacrolidesClindamycinLinezolidStreptograminsChloramphenicol

Inhibit 30S subunitAminoglycosidesTetracyclinesTigecycline

Target/ActionAntibiotic Class

Figure 2. Approved antibiotics work by inhibiting a limited repertoire of bacterial targets, disrupting essential functions such as cell wall synthesis, protein production, and DNA replication.

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A good target must have the ability to bind to a producible drug compound, be essential in many bacterial species and not present in the host, and have low potential for cross-resistance.

TWO-FOR-ONE TARGET

Current antibiotics hit a very narrow repertoire of targets2 and novel ones are hard to find (Figure 2). A good target must have the ability to bind to a producible drug compound, be essential in many bacterial species and not present in the host, and have low potential for cross-resistance, i.e., the mechanism of action must be something bacteria have not yet seen.

During the 1940s and 50s, the golden era of antibiotic discovery, natural products were screened for their ability to prevent the growth of pathogens of interest without much thought about how they worked. Successful candidates were then tested for toxicity in animals. By the late 1970s, the “low-hanging fruit” had been discovered and the need to come up with new compounds using limited resources gave rise to target-oriented discovery methods. Target selection came to have a critical role in the allocation of resources to the screening and development of compounds with antibacterial activity.

In the 1990s, the structure of a truncated subunit of the bacterial enzyme DNA gyrase was determined using x-ray crystallography4. This subunit was an ATPase, which binds adenosine triphosphate (ATP) and splits it into adenosine diphosphate (ADP) and phosphate. The energy released in this reaction powers the catalytic activity of gyrase, which prepares DNA for copying

by relieving the strain that results from unwinding the double-stranded molecule. Interestingly, the structure was published with a currently unused antibiotic molecule, novobiocin, bound to the ATP binding site. A number of pharmaceutical companies recognized the value of this information: this was a characterized binding site of an essential enzyme, validated to work as a target for an antibiotic. With time and a lot of research, they would expand their sights to include the ATP binding site of a related enzyme, topoisomerase IV5.

DNA gyrase and topoisomerase IV are present in a broad range of Gram-positive and Gram-negative bacteria, and both have key sequence differences at important sites compared to the human version, topoisomerase II6. Both bacterial enzymes are essential to DNA replication, but are involved at different points in the process. Finally, both have the same three-dimensional arrangement, with ATPase subunits containing very similar ATP binding sites, called GyrB in gyrase and ParE in topoisomerase IV (Figure 3). This similarity lends itself to the development of a single compound capable of concurrently binding and inhibiting the ATP binding sites of both enzymes: a multi-target monotherapeutic antibacterial.

To sum up, in these times of high rates of antibiotic resistance development, an effective new antibacterial will need to be potent against a range of bacteria, especially the difficult Gram-negative species, hit more than one essential bacterial target, avoid hitting essential host targets, and exhibit effective bioavailability through good solubility and low binding to plasma proteins. Optimizing any one of these factors — antibacterial potency, solubility, or protein binding — often means sacrificing strength in another. Finding the balance or “sweet spot” that maximizes the amount of potent antibacterial agent that reaches and impacts its target will be the maker or breaker of future antibiotic development programs.

7Elsevier R&D Solutions

DNA gyrase and topoisomerase IV are not new targets, just underexploited ones. The fluoroquinolones represent the only currently marketed class of antibiotics that inhibit one or both of these enzymes. The fluoroquinolones inhibit the DNA cleavage and resealing activity of both enzymes by binding to the enzyme activity subunits rather than to the ATPase subunits GyrB and ParE. An older antibiotic class, the aminocoumarins, which includes novobiocin and clorobiocin, bind and inhibit the GyrB subunit of DNA gyrase and (to a lesser extent) the ParE subunit of topoisomerase IV. Clorobiocin was never clinically developed and novobiocin had only limited clinical use after the late 1950s due to rapid resistance development and alleged safety issues. Novobiocin was subsequently withdrawn from the market. Because of its limited use, widespread clinical resistance to antibiotics that target the ATPase subunits of DNA gyrase and topoisomerase IV never had time to develop7.

According to Dr. Greg Bisacchi, Associate Director and Principle Scientist for Infection Chemistry at AstraZeneca, multiple pharmaceutical companies initiated development programs that screened for compounds or compound fragments that bind to both targets. Taking advantage of the homology between the two ATP binding sites, the low potential for cross-resistance, and the essential nature of these enzymes, these programs aimed to design novel antibiotics with potent activity against Gram-positive and Gram-negative bacteria. As we will see, these programs encountered challenges in balancing antibacterial potency, solubility, and free-fraction (the amount of compound not bound to plasma proteins). Nonetheless, each program has also contributed to an increased understanding of target and compound interaction that in the end has made “a new class of effective and safe antibiotic drugs targeting gyrase and topoisomerase IV […] highly achievable”.

2

3

B subunit: Hydrolyzes ATPA subunit: Interacts with DNAATP binding site

2 DNA gate

3 Exit gate G segment

T segment

Figure 3. DNA gyrase and topoisomerase IV have very similar structures, consisting of two subunits that cut and rejoin DNA and two subunits that power that function through the hydrolysis (splitting) of ATP. DNA gyrase introduces negative supercoils into the DNA while topoisomerase IV decatenates daughter DNA strands. The homology of the ATP binding sites, the essential function of the two enzymes, and the low potential for cross-resistance development make the ATP binding sites of these two enzymes excellent targets for an antibiotic.

OF RINGS, BRIDGES AND POCKETS

Several compound series have been investigated to target DNA gyrase and topoisomerase IV. A closer look at five such compound series — the pyrrolamides8-10, azaindoles11 and pyridylureas7 from AstraZeneca, the benzimidazole ureas6,12 from Vertex Pharmaceuticals, and the pyrrolopyrimidines13,14 from Trius Therapeutics, now owned by Cubist Pharmaceuticals — provides interesting insights into the challenges of developing novel antibiotics. The initial molecular scaffolds of the pyrrolamides

8Elsevier R&D Solutions

and pyrrolopyrimidines were found by screening compound fragments that bind to GyrB8. The pyrrolamide team used nuclear magnetic resonance to detect binding and the pyrrolopyrimidine team used crystallographic screening13. The pyridylureas were designed to combine desirable features of the pyrrolamides and a published analog of the benzimidazole ureas7, and the azaindoles came from a virtual library of compound fragments docked in silico into GyrB11.

Despite different approaches to drug lead discovery, the development of all five compounds converged on structural features that interact at two conserved sites in GyrB and ParE (Figure 4). Compounds from all five series form hydrogen bonds with aspartic acid and a conserved water molecule in the pocket where the adenine of ATP binds to GyrB and ParE (the “adenine pocket”). All five series also form π-stacking, hydrogen bond, and electrostatic interactions with two arginines conserved outside of the ATP binding site (the “salt bridge pocket”). Two other interaction sites play an important role in the antibacterial activity of one or the other series. The pyrrolamides, azaindoles and pyridylureas all have hydrophobic groups (trifluoromethyl groups, for example) which project towards the hydrophobic floor of the ATP binding site. The development team of the pyrrolopyrimidine scaffold examined a fourth interaction site at the rim of the adenine pocket, where they situated a basic amine to hydrogen bond with a conserved asparagine6-9,11,13. Although not emphasized, this interaction was also noted for the azaindoles11 and pyridylureas7.

The development teams working on these antibacterial compounds explored different molecular features and functional groups to adjust the strength of interactions at these sites and thus maximize the potency with which the compounds shut down the bacterial enzyme machinery. At the same time, changes were made to affect solubility, protein binding, and other properties that impact the path of the antibacterial to its target, thereby altering therapeutic efficacy.

Knowledge acquired from modifications and testing streamlined the design of future versions or analogs of compounds within and across programs. The azaindole program exemplifies this information carryover. It took two years to take the pyrrolamides

from identification of the first lead compound to testing in vivo efficacy in a mouse study model. Using the same assays developed to test the pyrrolamides and leveraging what was learned about the behavior of ligands in GyrB and ParE, the subsequent azaindole program arrived at in vivo efficacy tests in less than a year. Furthermore, by using in silico modeling to predict the consequences of adding or removing different functional groups to or from the scaffold, the team only had to synthesize about a dozen analogs for testing11. While each modification revealed information that could improve a property or give a better understanding of tradeoffs between properties, the unique features of each compound series very often made balancing these attributes — finding that sweet spot — a process of educated trial and error.

Pyrrolamide series

Azaindole series

Pyridylurea series

Benzimidazole urea series

Pyrrolopyrimidine series

ADENINE POCKET

SALT BRIDGE POCKET

HYDROPHOBIC FLOOR

RIM OF ADENINE POCKET

Figure 4. Example compounds from each series targeting DNA gyrase and topoisomerase IV. All interact at 2 common sites — at the adenine pocket deep in the ATP binding site and at the salt bridge pocket near the solvent interface. Additional sites of interaction were a conserved hydrophobic surface of the binding site floor and interactions with a

conserved asparagine at the rim of the adenine pocket.

9

Problems arise when the drive to create a molecule that binds strongly to the target leads to reduced solubility or increased binding to plasma proteins.

A STRONG PUNCH CAN LEAD ASTRAY

The strength with which a compound inhibits a target enzyme is related to how well the former interacts with the latter to prevent function. Barring interfering factors, which will be explained in more detail below, antibacterial activity can be usually demonstrated to correlate with enzyme inhibition. The binding sites of enzymes are shaped to allow binding only with molecules of a specific configuration and charge distribution, so most antibiotic development strategies aim to optimize the shape and charge of a compound to best fit in its target. Then they test the designed compound to see how well it inhibits the target enzyme (inhibitory potency) and how well it kills or inhibits the growth of one or more bacterial cultures (minimum inhibitory concentration or MIC).

Problems arise when the drive to create a molecule that binds strongly to the target leads to the incorporation of functional groups that either reduce the solubility of the compound or increase the likelihood that it will bind to plasma proteins. A strong punch is meaningless if the compound cannot reach its target. This was an issue with the pyrrolamides.

The pyrrole in clorobiocin, one of the aminocoumarins known to bind GyrB and inhibit gyrase, mimics the portion of ATP that interacts at the adenine pocket. Thus, it is not surprising that pyrrole surfaced as a fragment hit in a discovery program that led to the screen for a lead in the pyrrolamide series. In a second screen using GyrB with an occupied adenine pocket, another compound fragment surfaced that weakly interacted at a distal region of the binding site, where it opens to the surrounding solvent (the salt bridge pocket). The team set out to find compounds that interacted with GyrB at both sites simultaneously8,9.

They first anchored the compound in the adenine pocket by maximizing the strength of hydrogen bonds at that site. Testing different substituents on the pyrrole ring, they positioned two chlorine atoms opposite to the nitrogen in the pyrrole ring and achieved an over 150-fold improvement in inhibitory potency and antibacterial activity (MIC Figure 5). The chlorines increased hydrophobic interactions in the adenine pocket and, as atoms that draw away electrons, made the NH group on the pyrrole a better hydrogen donor in its interaction with aspartic acid8.

Figure 5. Positioning two chloride atoms on the pyrrole of pyrrolamide generated an early lead with significantly improved antibacterial activity. Potency against Gram-negative bacteria (Haemophilus influenzae, Escherichia coli, and Moraxella catarrhalis) was still limited (extracted from Sherer et al. 2011).

6.9

>64

>64

>64

>64

>64

>64

R1 = R2 = H R1 = R2 = Cl

>64

>64

0.03

1

4

4

2

IC50 (µM)

GyrB S. aureus

MIC (µg/ml)

S. pneumoniae

S. aureus

M. catarrhalis

E. faecium

H. inuenzae

E. coli

10

Then the team grew the molecule to reach out toward the salt bridge pocket. Exploring different functional groups for this extension, the team concluded that

aromatic heterocycles π-stacking with one arginine (aligning parallel one on top of the other) and carboxlyate or carboxamide groups forming hydrogen bonds with the other arginine achieved the strongest interactions in this pocket9 (Figure 6).

A pitfall of this “anchor and link” approach was that it emphasized the interaction at the hydrophobic adenine pocket. The resulting compounds were therefore all molecules with a large hydrophobic head at one end, which greatly restricted solubility, and potentially increased protein binding, or had both effects. Due to unoptimized physical properties and potency, relatively high doses of an early analog in the pyrrolamide series were needed to achieve a maximum response

against Streptococcus pneumoniae in mice. The effective dose (ED50) was estimated to be 54 mg/kg/day. By comparison, other antibacterials like ciprofloxacin and vancomycin achieve ED50 in the range of 2–15 mg/kg/day in other mouse models9.

Aiming to improve the balance between antibacterial activity and solubility, development teams of other compounds turned their focus to other interaction sites. By strengthening interactions there, they could de-emphasize the adenine pocket and use a functional group that was less hydrophobic. The azaindoles and pyridylureas were developed under this strategy. Both programs sought leads with the same hydrogen donor/acceptor binding motif seen in the adenine of ATP, the pyrrolamides, and in arylureas like benzimidazole urea: a hydrogen donor separated by one carbon from a hydrogen acceptor (Figure 7). They found that motif in pyridylurea and azaindole. Then, both programs worked toward improving interactions at sites outside of the adenine pocket.

Figure 8. X-ray structure of the lead azaindole analog bound to ParE of the Gram-positive bacterium S. pneumoniae. The trifluoromethyl group abuts the hydrophobic floor of the binding site, created by the conserved enzymatic residues isoleucine, proline, and methionine (behind the group). Reproduced with permission from Manchester et al. 2012.

Figure 7. A donor for aspartic acid and an acceptor for water. Pyridylureas and azaindoles match the interaction pattern of other compounds known to bind the adenine pocket.

Figure 6. X-ray structure of an early pyrrolamide lead bound to GyrB of the Gram-positive bacterium Staphylococcus aureus. Reproduced with permission from Sherer et al. 2011.

ADENINE PYRROLAMIDES ARYLUREAS AZAINDOLES PYRIDYLUREAS

11

They did not approach this task blindly; a lot had been learned from the pyrrolamides. They had learned, for example, that a single fluorine atom incorporated on the piperidine ring of the pyrrolamide created shape-specific versions of the compound (stereoisomers) that exhibited a 10- to 20-fold improvement in inhibitory potency and antibacterial activity compared to other isomers9. In this conformation, the fluorine pointed to the hydrophobic floor of the GryB binding site, which may have contributed to the improved potency. Both the azaindoles and the pyridylureas included a trifluoromethyl group that reached further than

a single fluoride to abut the hydrophobic floor in both GyrB and ParE (Figure 8). Compounds with this lipophilic group exhibited significantly improved inhibitory potency over other analogs in the respective series (Figure 9) and measures of their hydrophobicity (LogD; the larger the number, the more hydrophobic the molecule) were in the acceptable range7,11. It is worth noting that improvements in antibacterial activity required the presence of the right functional group interacting at the salt bridge pocket, emphasizing the importance of that site.

R

0.058

4.3

0.022

-1.1 -0.62

0.13 <0.01

<0.01

<0.01

0.51

IC50 (µM)

GyrB S. aureus

GyrB E. coli

ParE E. coli

ParE S. pneumoniaeLogD

Cl

Figure 9. The halogenic group extending from the scaffold of the pyridylureas the hydrophobic floor of GyrB and ParE improved inhibitory potency of early analogs (extracted from Basarab et al. 2013).

Figure 10. X-ray structure of an early pyrrolopyrimidine analog bound to GyrB of the Gram-positive bacterium Enterococcus faecalis. The amino-azetidine group interacts with a conserved asparagine (N48) at the rim of the adenine pocket. Reproduced with permission from Trzoss et al. 2013.

AN EASY FIT CAN GO A LONG WAY

Improving the fit of a compound in the binding site of its target and its alignment with protein residues to form binding interactions can also lead to increased potency without necessarily altering the charge or hydrophobicity of the molecule. Simply minimizing strain on the molecule or increasing the likelihood of interaction reduces the energetic and entropic cost of binding.

While development efforts for both the pyridylureas and the azaindoles emphasized the interaction at the hydrophobic floor, they noted the existence of another interaction site at

the opening of the adenine pocket. At this site in ParE, the urea carbonyl of the pyridylureas interacted electrostatically with a conserved asparagine across a water molecule. The carbonyl on the azaindoles formed a hydrogen bond directly with the same asparagine (visible in the x-ray structure).

This interaction site was explored extensively in the development program of the pyrrolopyrimidines. Early analogs featured a 1-aminopropan-2-ol that formed a hydrogen bond with the conserved asparagine at

12

this site in GyrB and ParE, but the rotational freedom of this functional group made the interaction unfavorable. The development team replaced it with an amino-azetidine group (Figure 10). This functional group presented an amine to asparagine on a more rigid extension, which afforded a 4 to 6-fold improvement in antibacterial activity (Figure 11). Further improvements were achieved by presenting the amine on an azabicyclohexane14.

More than improving inhibitory potency, i.e., inhibition of the enzymes, the modifications made at this interaction site also dramatically improved antibacterial activity against the Gram-negative bacterium Escherichia coli. This activity was exhibited by all analogs that achieved balanced inhibition of both GyrB and ParE14. Inhibitory potencies in GyrB and ParE were similar in Gram positive bacteria. However, in E. coli, potencies were 10- to 100-fold lower against ParE than GyrB8. While selected pyrrolamides exhibited good activity against the Gram-negative bacteria Moraxella catarrhalis and Haemophilus influenzae, none worked against wild-type E. coli9. The team developing the pyrrolopyrimidine series concluded that a potent and balanced dual inhibition of GyrB and ParE, in addition to positioning a moderately basic amine to interact with the asparagine at the adenine pocket rim, was an important factor to achieve broad Gram-negative antibacterial activity14.

Taking a more granular look at differences between the GryB and ParE interior surfaces, the pyrrolopyrimidine team carefully analyzed the crystal structures of several GyrB and ParE from different Gram-positive and Gram-negative bacteria. The objective was to encompass as much of the diversity at the binding site as possible to accommodate these differences in the design of the compound series. A comparison of the two binding sites showed that, due to a shift in the position of a conserved proline in the nearby hydrophobic floor, the salt bridge pocket of Gram-negative ParE is wider than that of GyrB and that of Gram-positive ParE13.

Functional groups of early analogs in this series that reached into the salt bridge pocket were connected to the pyrrolopyrimidine via a thioether linker. Upon binding GyrB and ParE, these compounds adopted a strained conformation with the thioether bond opening 10 degrees more than its ideal value. The S-linker was replaced with a more flexible O-linker, which accommodates a wider bond angle and thus allows the molecule to adapt to differences in pocket morphology without altering the interactions at the salt bridge pocket13

(Figure 12). The O-linked pyrrolopyrimidines showed balanced inhibition of GyrB and ParE of Enterococcus faecalis, Francisella tularensis and E. coli and proved potent against H. influenzae, E. coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa, all Gram-negative bacteria14.

Figure 11. Using a rigid structure to present an amine to interact at the rim of the adenine pocket dramatically improved the antibacterial activity of early pyrrolopyrimidine analogs. Interestingly, strengthened interactions at this site brought about potency against the Gram-negative bacterium E. coli (extracted from Trzoss et al. 2013).

S. aureus E. coli

MIC (μg/ml)

4

1

0.13

>64

64

8

13

The role of optimizing the shape of a compound in achieving balanced dual targeting also surfaced in the development of the benzimidazole ureas. This compound series was modified during early exploration with aryl substituents adjacent to the benzimidazole NH group. Intramolecular hydrogen bonds between the aryl substituent and the NH group maintained planarity of the overall molecule, giving an improved and more balanced inhibition of gyrase and topoisomerase IV. Modeling showed that the planar

molecule optimized interactions in the binding site of ParE, which itself is tighter than GyrB. A selected analog resulting from this optimization exhibited effective antibacterial activity against six Gram-positive and six Gram-negative bacteria6. In the cases of both the pyrrolopyrimidines and the benzimidazole ureas, antibacterial activity was enhanced without altering the binding mode of the compound, thus reducing the risk of impairing solubility or other properties.

Figure 12. (A) Comparison of a pyrrolopyrimidine analog binding in the salt bridge pocket of ParE (red protein, gold ligand) and GyrB (gray protein, green ligand). The pocket in ParE is wider than in GyrB due to a different position of a conserved proline. (B) Using a more flexible O-linker to position the compound extension into the salt bridge pocket allows the molecule to adjust to differences in the shape of the pocket, which conversely enables more even dual targeting. Reproduced with permission from Tari et al. 2013.

A

B

14

Figure 13. A substituent on the thiazole ring reduces pKa value of the carboxylic acid and overall clearance rates. The effect may be due to impaired recognition by transporters. Note that the fluorine on the piperidine was substituted with a methoxyl group in the course of optimization work (extracted from Basarab et al. 2014).

DECOY TO FREE A MOLECULE

As mentioned previously, a strong inhibitory potency mostly translates to good antibacterial activity, unless something interferes with the molecule reaching and binding to the target. One form of interference is protein binding. An antibiotic molecule can be sequestered by proteins in blood plasma and tissues, severely limiting the concentration of drug actually available to interact with its target. Consequently, antibacterial activity of the compound may be low regardless of potent enzyme inhibition.

It is not uncommon for antibacterials to show reduced activity when tested against bacterial cultures containing proteins, like albumin, that are commonly found in blood serum. This change in antibacterial activity is called a “serum shift”12. Protein binding has rather complex mechanics. On the one hand, protein binding reduces the amount of compound available to affect the pathogen. On the other hand, protein binding extends the time over which the drug remains in the body because it cannot be eliminated until it is slowly released. Protein binding is also a consequence of several factors. For example, hydrophobic compounds tend to be more readily bound by proteins, but the presence of ionizable groups that interact with specific sites on a protein also contributes to binding12. The problem arises when modifications made to a compound to reduce its serum shift also reduce its potency.

Early analogs of the benzimidazole urea series showed strong inhibition of GyrB and ParE and excellent potency against a number of bacterial strains. However, this activity was weakened by protein binding of up to 95% for some of these analogs and serum shifts ranging from 15- to 128-fold12. It was imperative to reduce the serum shift of this compound series to improve therapeutic efficacy. The team performed a systematic analysis of the properties of the compound series and identified two criteria that should result in compounds with low serum shifts: medium hydrophobicity and high polar surface area12. This makes sense as these criteria match the properties of known polar, hydrophilic compounds with good solubility and reduced likelihood of binding to proteins. These criteria were, on the one hand, used to select one of the analogs that surfaced as potent dual target inhibitors from the incorporation of aryl substituents that maintained planarity of the molecule and, on the other, to design an analog with significantly reduced serum shift6.

R

H

IC50 (µM)

GyrB S. aureus

MIC (µg/ml)

Clearance (ml/min/kg)

S. pneumoniaeM. catarrhalisH. in�uenzaeE. coliS. aureus

0.016

0.031

0.18

64

0.32

66

26

99

0.240

0.016

0.036

17

0.64

10

<0.01 <0.01

0.073

<0.008

24

0.13

pKa 3.6 4.2

ParE E. coli

Rat

Dog

Mouse

15

Unexpectedly, the lead compound of the series also covalently bound liver proteins in vitro, which could reduce the amount of drug available to interact with the target and potentially lead to safety issues. Studies suggested that the compound was being metabolized via cytochrome P oxidation and the resulting modification of the urea that binds in the adenine pocket produced a reactive metabolite that bound to protein. In a classic example of the tradeoff between inhibitory potency and other properties, attempts to replace the urea diminished the antibacterial activity of the resulting analogs, in some instances several fold6.

To solve the problem, instead of removing the site of metabolism, the team decided to reduce the amount of compound being converted and bound to liver proteins. To do this, they incorporated a functional group on the compound that would redirect metabolic activity away from the urea — a veritable molecular decoy. Careful to ensure that the newly designed compound remained planar and had as low a hydrophobicity as possible, they achieved zero binding to liver proteins and the compound maintained potent

dual inhibition of GyrB and ParE, as well as excellent antibacterial activity against several Gram-positive and two Gram-negative bacteria6. By approaching the problem from a different perspective, the benzimidazole ureas drew closer to the right balance.

Along similar lines, the pyrrolamide team improved bioavailability of an analog that went into Phase I clinical trials by disguising the carboxylic acid bound to the thiazole ring interacting in the salt bridge pocket. They reasoned that anion transporters, membrane proteins in kidney and liver cells that remove negatively charged compounds from body fluids, recognized the carboxylic acid and thus cleared early pyrrolamide analogs at high rates. The team added a functional group adjacent to the carboxylic acid to form an intramolecular hydrogen bond, essentially reducing the likelihood that the carboxylic acid would dissociate and thus, become an anion (Figure 13). As a result, clearance rates in multiple study models were significantly lower for the new analog and its antibacterial activity remained strong15.

16

Figure 14. Carboxylic acids interacting in the salt bridge pocket made pyridylurea analogs more soluble and more effective at inhibiting GyrB and ParE, but their antibacterial activity was limited, especially compared to analogs with a carboxamide group. Higher acidity appeared to limit the ability of the compound to permeate bacterial membranes (extracted from Basarab et al. 2013).

2.0

64

<0.01

<0.01

<0.01

>64

>64

0.51

<0.01

>64

0.017

0.96

0.012

0.09

5.5

0.7

IC50 (µM)

GyrB S. aureus

MIC (µg/ml)

S. pneumoniaeM. catarrhalisH. in�uenzaeE. coli

GryB E. coliParE E. coliParE S. pneumoniae

-0.62 2.0Log D

R

The team realized that excessively polar compounds, though more soluble and less protein bound, could not penetrate bacterial cell membranes.

THE FEAR OF WATER

Hydrophobic features in the various series were needed to match hydrophobic interaction sites in GyrB and ParE to achieve good enzyme potency, and also to enable cell membrane permeation, but excessive initial hydrophobicity led to poor physical properties such as low solubility and/or high protein binding.

Case in point, analysis of the azaindole series revealed improvements in antibacterial activity that tracked with changes in hydrophobicity. The team realized that excessively polar compounds, though more soluble and less protein bound, could not penetrate bacterial cell membranes11.

This observation was corroborated by work with the pyridylureas. In the process of finding functional groups that would strengthen interactions at the salt bridge pocket, the team noticed that charged, polar carboxylic acids at this site generated more soluble compounds but analogs with a neutral, less polar carboxamide achieved higher antibacterial activity7 (Figure 14).

In fact a correlation emerged between the acidity of a compound and its antibacterial activity -- the higher the acidity, the weaker the antibacterial

activity of a compound due to poor membrane penetration, even if enzyme inhibitory potencies were equally high. LogD measurements, which reflect the extent of polarity (negative values indicate high polarity), were used to guide the optimal polarity needed to achieve both membrane penetration and good physical properties. High logD values (>3) indicate excessive hydrophobicity which could cause physical properties (solubility and/or protein binding) to suffer, while values too low (much below zero) could cause membrane permeability to suffer. Compounds having LogD values between 0 and 2 seemed like the best compromise. Based on this tradeoff, the team chose a compound for efficacy experiments that exhibited moderate solubility but strong antibacterial activity.

Selecting a compound having a low clearance rate from the body was another factor in the selection. Low clearance rates are often correlated with higher extents of protein binding, although if protein binding is too high, then antibacterial activity in the body suffers, so balancing clearance and extent of antibacterial activity in this regard is also a compromise.

17

A wealth of knowledge has accumulated about how to achieve balanced dual inhibition of GyrB and ParE, while maintaining adequate solubility and small serum shift.

TIME TO DO THE WORK

As with the pyridylureas, compromising on one or two important properties in order to maximize the therapeutic efficacy of a compound is necessary to accommodate development goals. For example, a top candidate of the pyrrolopyrimidine series featuring a single ring at the salt bridge site had lower inhibitory potential and antibacterial activity than analogs with two rings. However, testing the compounds against mutated E. coli with compromised cell membrane and efflux pumps showed that the activity of all but the single-ringed compound was impacted by efflux and limited ability to permeate the bacterial membrane14. The smaller candidate would be the right choice if a goal of the development program was to demonstrate effectiveness against Gram-negative bacteria.

Creativity is also essential for balancing all factors that make a successful antibacterial. The introduction of a metabolic decoy in the benzimidazole urea series is an example of an unconventional approach to a problem that could have severely compromised the potency and safety of the series. The same team was also creative about solving solubility issues. Their optimized compound with strong antibacterial attributes had very low solubility at physiological pH. Therefore, the team created a prodrug by adding a phosphate group to the compound. A prodrug is a version of a compound that includes a functional group used to transiently alter an undesirable property. Typically,

the functional group is removed after administration of the prodrug. In the case of the benzimidazole urea, the developed prodrug had good solubility from the added phosphate group. Once administered, the phosphate group was hydrolyzed via normal metabolic processes, leaving the active antibacterial to reach its target and achieve high exposure in animal models6.

What is clear from examining the work of these five antibacterial development programs is that a wealth of knowledge has accumulated about how to achieve balanced dual inhibition of GyrB and ParE, while maintaining adequate solubility and small serum shift. It is also clear that, although this knowledge improves the likelihood of developing a successful novel DNA gyrase and topoisomerase IV inhibitor, nothing can substitute the need to test and re-test promising compounds. Achieving an antibacterial that successfully hits these two targets and mitigates resistance development will require time — time to characterize the target, time to examine different mechanisms by which a compound can inhibit that target, and time to understand toxicity by thoroughly investigating development paths that failed.

We cannot afford to lose the arms race against pathogenic bacteria. Our best bet will be to recruit the best minds, implement the best technology, and above all, take the time to find the sweet spot of, hopefully, several effective killers.

18

REFERENCES

1. World Health Organization (2014) Antimicrobial resistance: global report on surveillance. WHO Press, France.

2. Silver, L. L. (2011) Challenges of Antibacterial Discovery. Clin. Microbiol. Rev. 24, 71

3. Friedman, D. and Alper, J. (2014) Technological challenges in antibiotic discovery and development. A workshop summary. The National Academies Press, Washington, D.C.

4. Lewis, R. J., Singh, O. M., Smith, C. V., Skarzynski, T., Maxwell, A., Wonacott, A. J., and Wigley, D. B. (1996) The nature of inhibition of DNA gyrase by the coumarins and the cyclothialidines revealed by x-ray crystallography. EMBO J. 15, 1412.

5. Bellon, S., Parsons, J. D., Wei, Y., Hayakawa, K., Swenson, L. L., Charifson, P. S., Lippke, J. A., Aldape, R., and Gross, C. H. (2004) Crystal structures of Escherichia coli topoisomerase IV Par E subunit (24 and 43 kilodaltons): a single residue dictates differences in novobiocin potency against topoisomerase IV and DNA gyrase. Antimicrob. Agents Chemother. 48, 1856.

6. Grillot, A.-L. (2014) Design and evolution of a novel class of gyrase B and topoisomerase IV dual-targeting antibacterials. 15th Annual Drug Discovery Summit, Geneva, Switzerland.

7. Basarab, G. S., Manchester, J. I., Bist, S., Boriack-Sjodin, A., Dangel, B., Illingworth, R., Sherer, B. A., Sriram, S., Uria-Nickelsen, M., and Eakin, A. E. (2013) Fragment-to-hit-lead discovery of a novel pyridylurea scaffold of ATP competitive dual targeting type II topoisomerase inhibiting antibacterial agents. J. Med. Chem. 56, 8712.

8. Eakin, A. E., Green, O., Hales, N., Walkup, G. K., Bist, S., Singh, A., Mullen, G., Bryant, J., Embrey, K., Gao, N., Breeze, A., Timms, D., Andrews, B., Uria-Nickelsen, M., Demeritt, J., Loch III, J. T., Hull, K., Blodgett, A., Illingworth, R. N., Prince, B., Boriack-Sjodin, P. A., Hauck, S., MacPherson, L. J., Ni, H., Sherer, B., et al. (2012) Pyrrolamide DNA gyrase inhibitors: fragment-based nuclear magnetic resonance screening to identify antibacterial agents. Antimicrob. Agents Chemother. 56, 1240.

9. Sherer, B. A., Hull, K., Green, O., Basarab, G., Hauck, S., Hill, P., Loch III, J. T., Mullen, G., Bist, S., Bryant, J., Boriack-Sjodin, A., Read, J., Degrace, N., Uria-Nickelsen, M., Illingworth, R. N., Eakin, A. E., et al. (2011) Pyrrolamide DNA gyrase inhibitors: optimization of antibacterial activity and efficacy. Bioorg. Med. Chem. Lett. 21, 7416.

10. Uria-Nickelsen, M., Blodgett, A., Kamp, H., Eakin, A w., Sherer, B., and Green, O. (2013) Novel DNA gyrase inhibitors: microbiological characterisation of pyrrolamides. Int. J. Antimicrob. Agents 41, 28.

11. Manchester, J. I., Dussault, D. D., Rose, J. A., Boriack-Sjodin, A., Uria-Nickelsen, M., Ioannidis, G., Bist, S., Fleming, P., and Hull, K. G. (2012) Discovery of a novel azaindole class of antibacterial agents targeting the ATPase domains of DNA gyrase and topoisomerase IV. Bioorg. Med. Chem. Lett. 22, 5150.

12. Perola, E. A., Stamos, D., Grillot, A.-L., Ronkin, S., Wang, T., Letiran, A., Tang, Q., Deininger, D. D., Liao, Y., Tian, S.-K., Drumm, J. E., Nicolau, D.P., Tessier, P. R., Mani, N., Grossman, T. H., Charifson, P. S. et al. (2014) Successful application of serum shift prediction models to the design of dual targeting inhibitors of bacterial gyrase B and topoisomerase IV with improved in vivo efficacy. Bioorg. Med. Chem. Lett. 24, 2177.

13. Tari. L. W., Trzoss, M., Bensen, D. C., Li, X., Chen, Z., Lam, T., Zhang, J., Creighton, C. J., Cunningham, M. L., Kwan, B., Stidham, M., Shaw, K. J., Lightstone, F. C., Wong, S. E., Nguyen, T. B., Nix, J., Finn, J., et al. (2013) Pyrrolopyrimidine inhibitors of DNA gyrase B (GyrB) and topoisomerase IV (ParE). Part I: Structure guided discovery and optimization of dual targeting agents with potent, broad-spectrum enzymatic activity. Bioorg. Med. Chem. Lett. 23, 1529.

14. Trzoss, M., Bensen, D. C., Li, X., Chen, Z., Lam, T., Zhang, J., Creighton, C. J., Cunningham, M. L., Kwan, B., Stidham, M., Nelson, K., Brown-Driver, V., Castellano, A., Shaw, K. J., Lightstone, F. C., Wong, S. E., Nguyen, T. B., Finn, J., Tari, L. W., et al. (2013) Pyrrolopyrimidine inhibitors of DNA gyrase B (GyrB) and topoisomerase IV (ParE). Part II: Development of inhibitors with broad spectrum, Gram-negative antibacterial activity. Bioorg. Med. Chem. Lett. 23, 1537.

15. Basarab, G. S., Hill, P. J., Garner, C. E., Hull, K., Green, O., Sherer, B. A., P. Brian Dangel, B., Manchester, J. I., Bist, S., Hauck, S., Zhou, F., Uria-Nickelsen, M., Illingworth, R., Alm, R., Rooney, M., Eakin, A.E., et al. (2014) Optimization of pyrrolamide topoisomerase II inhibitors toward identification of an antibacterial clinical candidate (AZD5099). J. Med. Chem. Doi: 10.1021/jm500462x

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