Sweet Spot of a Killer

PHARMA & LIFE SCIENCES

WHITEPAPER 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? CONTENTS

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

“Finding the balance or ‘sweet A POST-ANTIBIOTIC ERA spot’ that maximizes the amount Since the introduction of the first antibiotics at the beginning of the 20th century, of potent antibacterial agent that we have blasted bacteria infecting our bodies with potent chemicals, only to discover reaches and impacts its target will that they have become resistant to our weapons. We adapt or combine antibiotics to be the maker or breaker of future circumvent this resistance, but the result is often multidrug unresponsiveness. These antibiotic development programs.” 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.

3 THE ANTIBACTERIAL BATTLEGROUND Think about the last time you took an antibiotic. If it Generally, the antibiotic compound must travel from was oral, the pills were probably large and you were the site of administration to the site of infection asked to take them two or more times a day over the via the blood stream or tissue fluid. Blood and course of several days. its derivatives are to a large extent water, so the compound travels best if it is water-soluble. This type of extended, high-dose regimen is essential to achieve the therapeutic goal of killing an evasive Blood also contains a repertoire of proteins, such foreign cell inside of a host made up of drug- as albumin and globulins, that have the ability to susceptible cells. Maintaining high drug levels in bind chemicals. This means that the chances of the the host over an extended period of time to reach antibiotic compound reaching the site of infection and wipe out the culprit bacterial population is and being available to act on bacterial cells are the medical equivalent of ‘shock and awe’ warfare. significantly reduced if it does not dissolve well in Achieving the desired outcome is not a trivial task. water and if it tends to bind to proteins. A brute force Pelting bacteria with insufficient treatment dose or solution to these bioavailability issues is to increase for an insufficient period increases the likelihood that the drug dose – the more compound that enters the an oddball bacterium with a mutation that makes it host, the more that is freely available. However, the resistant to the antibiotic survives the chemical storm host consists of cells that may also be susceptible to and lives to produce other bacteria with the same the compound, so the higher the dose, the greater mutation. Voila! You have a drug-resistant strain of the chance of adverse effects. the pathogen. Once the compound arrives at the site of infection, The development of the antibiotic is also not a trivial it must reach its molecular target within the matter. An antibiotic encounters a number of hurdles bacterium. Unlike the cells of the host, bacteria have on the path from the point of administration to its a cell wall that poses a formidable barrier. Gram- target (Figure 1). All the tricks to overcome these positive bacteria have a cell wall consisting of a obstacles must be incorporated into the chemical thick, extensively cross-linked peptidoglycan layer structure and formulation of the antibiotic.

Must enter the bloodstream Potential obstacles Dissolution Ingestion & absorbtion Must travel easily in the for antibiotic bloodstream and avoid binding to proteins treatments IV administration Direct entry Should not enter and adversely aect Site of host cells Must enter infection the body

Must cross Gram-negative bacteria outer Must avoid membrane, eux pumps peptidoglycan layer and plasma membrane Must hit target Must cross the thick peptidoglycan Must avoid layer eux pumps and plasma membrane Figure 1. Antibiotics (blue dots) face a number Gram-positive bacteria of potential obstacles on their way from the site of administration to their target within the infecting bacteria.

4 that surrounds the inner cell Gram-negative bacteria produce of bacteria and preventing these membrane and is porous to small a greater variety of enzymes that targets from doing their job. substances. The underlying plasma metabolize antibiotics, such as Some antibiotics punch holes membrane can be penetrated several ß-lactamases, that render into the cell wall of bacteria by via passive diffusion of lipophilic penicillins, cephalosporins, inhibiting the enzymes that make molecules. Unfortunately, monobactams and peptidoglycans. Others inhibit lipophilic molecules are not carbapenems inactive. enzymes to prevent bacteria from very water-soluble, which synthesizing proteins or DNA. brings us back to the hurdle Since the type of proteins of bioavailability. Additionally, regulating molecular traffic is New antibiotic targets are needed membrane-embedded proteins species-specific and the antibiotic to circumvent target-specific called efflux pumps are known to must cross two chemically resistance, which has emerged thwart antibiotics by removing distinct cellular membranes, few against every antibiotic class. them from the cell. antibiotics work against Gram- These new targets can be bacterial negative bacteria. One antibiotic enyzmes or pathways that have Gram-negative bacteria are even class that is effective against never been exploited before, or trickier. Their thin peptidoglycan Gram-negative bacteria, the novel sites of action on currently layer is surrounded by a second fluoroquinolones, passes through exploited targets. Curbing outer membrane that works as the porins of the outer membrane resistance development will an effective barrier between the as a charged, water-soluble require a very careful selection that environment and the periplasm, molecule and then becomes invokes not only new mechanisms the space between the outer uncharged and hydrophobic of action but also enables the and inner cell membranes. in the periplasm, which allows development of multi-target The outer membrane is dotted it to diffuse across the inner monotherapeutic drugs: single with porins that regulate the membrane. This environment- compounds that simultaneously traffic of molecules into and triggered change in molecular impact the function of two or out of the periplasm and can charge illustrates the complexity more targets3. In this way, if act to exclude many antibiotics. of developing effective broad- resistance develops along one line Compared to Gram-positive spectrum antibiotics3. of fire, activity at the other target bacteria, Gram-negative bacteria maintains the efficacy also have a much higher number The final hurdle is hitting the of the antibiotic. and diversity of membrane- target. Antibiotics work by binding embedded efflux pumps. Finally, to essential molecular machinery

Inhibit Nucleic Acid Sythesis or Function Folate Inhibit Folate Synthesis Trimethoprim / Sulfamethoxazole Inhibit DNA Gyrase +/- Topoisomerase IV Quinolones Create Free Radicals Metronidazole, Nitrofurantoin

Inhibit Cell Wall Sythesis or Function Inhibit Protein Sythesis Beta-Lactams Vancomycin Inhibit 50S subunit Penicillins Daptomycin Macrolides Polypeptides Cephalosporins Clindamycin Carbapenems Linezolid Monobactams Streptogramins 50S Chloramphenicol Inhibit 30S subunit 30S Aminoglycosides Tetracyclines Target/Action Tigecycline Antibiotic 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.

5 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.

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.

TWO-FOR-ONE TARGET Current antibiotics hit a very narrow by relieving the strain that results from repertoire of targets2 and novel ones are unwinding the double-stranded molecule. hard to find Figure( 2). A good target must Interestingly, the structure was published have the ability to bind to a producible with a currently unused antibiotic drug compound, be essential in many molecule, novobiocin, bound to the ATP bacterial species and not present in the binding site. A number of pharmaceutical host, and have low potential for cross- companies recognized the value of this resistance, i.e., the mechanism of action information: this was a characterized must be something bacteria have not binding site of an essential enzyme, yet seen. validated to work as a target for an antibiotic. With time and a lot of research, During the 1940s and 50s, the golden era they would expand their sights to include of antibiotic discovery, natural products the ATP binding site of a related enzyme, were screened for their ability to prevent topoisomerase IV5. the growth of pathogens of interest without much thought about how they worked. Successful candidates were then DNA gyrase and topoisomerase IV are tested for toxicity in animals. By the late present in a broad range of Gram- 1970s, the “low-hanging fruit” had been positive and Gram-negative bacteria, and discovered and the need to come up with both have key sequence differences at new compounds using limited resources important sites compared to the human gave rise to target-oriented discovery version, topoisomerase II6. Both bacterial methods. Target selection came to have a enzymes are essential to DNA replication, critical role in the allocation of resources but are involved at different points to the screening and development of in the process. Finally, both have the compounds with antibacterial activity. same three-dimensional arrangement, In the 1990s, the structure of a truncated with ATPase subunits containing very subunit of the bacterial enzyme DNA similar ATP binding sites, called GyrB gyrase was determined using x-ray in gyrase and ParE in topoisomerase IV crystallography4. This subunit was (Figure 3). This similarity lends itself to an ATPase, which binds adenosine the development of a single compound triphosphate (ATP) and splits it into capable of concurrently binding and adenosine diphosphate (ADP) and inhibiting the ATP binding sites of both phosphate. The energy released in this enzymes: a multi-target monotherapeutic reaction powers the catalytic activity of antibacterial. gyrase, which prepares DNA for copying

6 B subunit: Hydrolyzes ATP A subunit: Interacts with DNA ATP binding site 2 DNA gate 3 Exit gate 2 G segment T segment 3

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.

DNA gyrase and topoisomerase IV are According to Dr. Greg Bisacchi, Associate not new targets, just underexploited ones. Director and Principle Scientist for The fluoroquinolones represent the only Infection Chemistry at AstraZeneca, currently marketed class of antibiotics multiple pharmaceutical companies that inhibit one or both of these enzymes. initiated development programs that The fluoroquinolones inhibit the DNA screened for compounds or compound cleavage and resealing activity of both fragments that bind to both targets. enzymes by binding to the enzyme Taking advantage of the homology activity subunits rather than to the between the two ATP binding sites, the ATPase subunits GyrB and ParE. An older low potential for cross-resistance, and antibiotic class, the aminocoumarins, the essential nature of these enzymes, which includes novobiocin and these programs aimed to design novel clorobiocin, bind and inhibit the GyrB antibiotics with potent activity against subunit of DNA gyrase and (to a lesser Gram-positive and Gram-negative extent) the ParE subunit of topoisomerase bacteria. As we will see, these programs IV. Clorobiocin was never clinically encountered challenges in balancing developed and novobiocin had only antibacterial potency, solubility, and free- limited clinical use after the late 1950s fraction (the amount of compound not due to rapid resistance development and bound to plasma proteins). Nonetheless, alleged safety issues. Novobiocin was each program has also contributed to an subsequently withdrawn from the market. increased understanding of target and Because of its limited use, widespread compound interaction that in the end clinical resistance to antibiotics that target has made “a new class of effective and the ATPase subunits of DNA gyrase and safe antibiotic drugs targeting gyrase and topoisomerase IV never had time to develop7. topoisomerase IV […] highly achievable”.

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

Elsevier R&D Solutions 7 and pyrrolopyrimidines were found by screening from identification of the first lead compound to compound fragments that bind to GyrB8. The testing in vivo efficacy in a mouse study model. pyrrolamide team used nuclear magnetic resonance Using the same assays developed to test the to detect binding and the pyrrolopyrimidine team pyrrolamides and leveraging what was learned used crystallographic screening13. The pyridylureas about the behavior of ligands in GyrB and ParE, were designed to combine desirable features of the subsequent azaindole program arrived at in vivo the pyrrolamides and a published analog of the efficacy tests in less than a year. Furthermore, by benzimidazole ureas7, and the azaindoles came from using in silico modeling to predict the consequences a virtual library of compound fragments docked in of adding or removing different functional groups to 11 silico into GyrB . or from the scaffold, the team only had to synthesize about a dozen analogs for testing11. While each Despite different approaches to drug lead discovery, modification revealed information that could improve the development of all five compounds converged a property or give a better understanding of tradeoffs on structural features that interact at two conserved between properties, the unique features of each sites in GyrB and ParE (Figure 4). Compounds compound series very often made balancing these from all five series form hydrogen bonds with attributes — finding that sweet spot — a process of aspartic acid and a conserved water molecule in educated trial and error. 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 Pyrrolamide series 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 Azaindole series 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 Pyridylurea series 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 Benzimidazole urea series 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. Pyrrolopyrimidine series Knowledge acquired from modifications and testing streamlined the design of future versions or analogs of compounds within and across programs. The ADENINE POCKET SALT BRIDGE POCKET azaindole program exemplifies this information HYDROPHOBIC FLOOR carryover. It took two years to take the pyrrolamides 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.

Elsevier R&D Solutions 8 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 The pyrrole in clorobiocin, one of the inhibits a target enzyme is related to how aminocoumarins known to bind GyrB and well the former interacts with the latter inhibit gyrase, mimics the portion of ATP to prevent function. Barring interfering that interacts at the adenine pocket. Thus, factors, which will be explained in more it is not surprising that pyrrole surfaced detail below, antibacterial activity can as a fragment hit in a discovery program be usually demonstrated to correlate that led to the screen for a lead in the with enzyme inhibition. The binding pyrrolamide series. In a second screen sites of enzymes are shaped to allow using GyrB with an occupied adenine binding only with molecules of a specific pocket, another compound fragment configuration and charge distribution, so surfaced that weakly interacted at a distal most antibiotic development strategies region of the binding site, where it opens aim to optimize the shape and charge of to the surrounding solvent (the salt a compound to best fit in its target. Then bridge pocket). The team set out to find they test the designed compound to see compounds that interacted with GyrB at how well it inhibits the target enzyme both sites simultaneously8,9. (inhibitory potency) and how well it kills They first anchored the compound in or inhibits the growth of one or more the adenine pocket by maximizing the bacterial cultures (minimum inhibitory strength of hydrogen bonds at that site. concentration or MIC). Testing different substituents on the Problems arise when the drive to create a pyrrole ring, they positioned two chlorine molecule that binds strongly to the target atoms opposite to the nitrogen in the leads to the incorporation of functional pyrrole ring and achieved an over 150-fold groups that either reduce the solubility of improvement in inhibitory potency and the compound or increase the likelihood antibacterial activity (MIC that it will bind to plasma proteins. Figure 5). The chlorines increased A strong punch is meaningless if the hydrophobic interactions in the adenine compound cannot reach its target. This pocket and, as atoms that draw away was an issue with the pyrrolamides. electrons, made the NH group on the pyrrole a better hydrogen donor in its interaction with aspartic acid8.

R1 = R2 = H R1 = R2 = Cl IC50 (µM) GyrB S. aureus 6.9 0.03 MIC (µg/ml) S. pneumoniae >64 1 Figure 5. Positioning two chloride atoms on S. aureus >64 4 the pyrrole of pyrrolamide generated an early M. catarrhalis >64 2 lead with significantly improved antibacterial activity. Potency against Gram-negative E. faecium >64 4 bacteria (Haemophilus influenzae, Escherichia H. in uenzae >64 >64 coli, and Moraxella catarrhalis) was still limited E. coli >64 (extracted from Sherer et al. 2011). >64

9 Then the team grew against Streptococcus pneumoniae

the molecule to in mice. The effective dose (ED50) was reach out toward estimated to be 54 mg/kg/day. By the salt bridge comparison, other antibacterials like

pocket. Exploring ciprofloxacin and vancomycin achieve ED50 different functional in the range of 2–15 mg/kg/day in other groups for this mouse models9. extension, the team Aiming to improve the balance between concluded that Figure 6. X-ray structure of an early antibacterial activity and solubility, aromatic heterocycles π-stacking with one pyrrolamide lead bound to GyrB of the development teams of other compounds Gram-positive bacterium Staphylococcus arginine (aligning parallel one on top of turned their focus to other interaction aureus. Reproduced with permission from the other) and carboxlyate or carboxamide sites. By strengthening interactions there, Sherer et al. 2011. groups forming hydrogen bonds with they could de-emphasize the adenine the other arginine achieved the strongest pocket and use a functional group that interactions in this pocket9 (Figure 6). was less hydrophobic. The azaindoles and A pitfall of this “anchor and link” approach pyridylureas were developed under this was that it emphasized the interaction strategy. Both programs sought leads at the hydrophobic adenine pocket. The with the same hydrogen donor/acceptor resulting compounds were therefore binding motif seen in the adenine of ATP, all molecules with a large hydrophobic the pyrrolamides, and in arylureas like head at one end, which greatly restricted benzimidazole urea: a hydrogen donor solubility, and potentially increased separated by one carbon from a hydrogen protein binding, or had both effects. Due acceptor (Figure 7). They found that motif Figure 7. A donor for aspartic acid and to unoptimized physical properties and in pyridylurea and azaindole. Then, both an acceptor for water. Pyridylureas and potency, relatively high doses of an early programs worked toward improving azaindoles match the interaction pattern of other compounds known to bind the adenine analog in the pyrrolamide series were interactions at sites outside of the pocket. needed to achieve a maximum response adenine pocket.

ADENINE PYRROLAMIDES ARYLUREAS AZAINDOLES PYRIDYLUREAS

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.

10 They did not approach this task blindly; a lot a single fluoride to abut the hydrophobic floor had been learned from the pyrrolamides. They in both GyrB and ParE (Figure 8). Compounds had learned, for example, that a single fluorine with this lipophilic group exhibited significantly atom incorporated on the piperidine ring of the improved inhibitory potency over other analogs pyrrolamide created shape-specific versions of the in the respective series (Figure 9) and measures compound (stereoisomers) that exhibited a 10- to of their hydrophobicity (LogD; the larger the 20-fold improvement in inhibitory potency and number, the more hydrophobic the molecule) were antibacterial activity compared to other isomers9. in the acceptable range7,11. It is worth noting that In this conformation, the fluorine pointed to the improvements in antibacterial activity required the hydrophobic floor of the GryB binding site, which presence of the right functional group interacting at may have contributed to the improved potency. the salt bridge pocket, emphasizing the importance Both the azaindoles and the pyridylureas included of that site. a trifluoromethyl group that reached further than R

IC50 (µM) GyrB S. aureus 0.13 <0.01

GyrB E. coli 0.058 <0.01

ParE E. coli 4.3 0.51

ParE S. pneumoniae 0.022 <0.01 LogD -1.1 -0.62 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).

AN EASY FIT CAN GO A LONG WAY Improving the fit of a the opening of the adenine compound in the binding site pocket. At this site in ParE, of its target and its alignment the urea carbonyl of the with protein residues to form pyridylureas interacted binding interactions can also electrostatically with a lead to increased potency conserved asparagine across a without necessarily altering water molecule. The carbonyl the charge or hydrophobicity on the azaindoles formed a of the molecule. Simply hydrogen bond directly with minimizing strain on the the same asparagine (visible in molecule or increasing the the x-ray structure). likelihood of interaction This interaction site was reduces the energetic and Figure 10. X-ray structure of an early pyrrolopyrimidine analog bound explored extensively in the entropic cost of binding. to GyrB of the Gram-positive bacterium Enterococcus faecalis. The development program of amino-azetidine group interacts with a conserved asparagine (N48) While development efforts for the pyrrolopyrimidines. at the rim of the adenine pocket. Reproduced with permission from both the pyridylureas and the Early analogs featured a Trzoss et al. 2013. azaindoles emphasized the 1-aminopropan-2-ol that interaction at the hydrophobic formed a hydrogen bond with floor, they noted the existence the conserved asparagine at of another interaction site at

11 this site in GyrB and ParE, but the rotational freedom Taking a more granular look at differences of this functional group made the interaction between the GryB and ParE interior surfaces, the unfavorable. The development team replaced it with pyrrolopyrimidine team carefully analyzed the crystal an amino-azetidine group (Figure 10). This functional structures of several GyrB and ParE from different group presented an amine to asparagine on a Gram-positive and Gram-negative bacteria. The more rigid extension, which afforded a 4 to 6-fold objective was to encompass as much of the diversity improvement in antibacterial activity (Figure 11). at the binding site as possible to accommodate these Further improvements were achieved by presenting differences in the design of the compound series. A the amine on an azabicyclohexane14. comparison of the two binding sites showed that, due to a shift in the position of a conserved proline in More than improving inhibitory potency, i.e., the nearby hydrophobic floor, the salt bridge pocket inhibition of the enzymes, the modifications made of Gram-negative ParE is wider than that of GyrB and at this interaction site also dramatically improved that of Gram-positive ParE13. antibacterial activity against the Gram-negative bacterium Escherichia Functional groups of coli. This activity was MIC (μg/ml) early analogs in this exhibited by all analogs S. aureus E. coli series that reached into that achieved balanced the salt bridge pocket inhibition of both GyrB were connected to and ParE14. Inhibitory 4 >64 the pyrrolopyrimidine potencies in GyrB and via a thioether linker. ParE were similar in Upon binding GyrB Gram positive bacteria. and ParE, these However, in E. coli, compounds adopted a potencies were 10- to strained conformation 1 64 100-fold lower against with the thioether ParE than GyrB8. While bond opening 10 selected pyrrolamides degrees more than exhibited good activity its ideal value. The against the Gram- S-linker was replaced negative bacteria with a more flexible Moraxella catarrhalis 0.13 8 O-linker, which and Haemophilus accommodates a wider influenzae, none bond angle and thus worked against wild- allows the molecule type E. coli9. The Figure 11. Using a rigid structure to present an amine to to adapt to differences team developing the interact at the rim of the adenine pocket dramatically improved in pocket morphology pyrrolopyrimidine the antibacterial activity of early pyrrolopyrimidine analogs. without altering the series concluded that Interestingly, strengthened interactions at this site brought about interactions at the a potent and balanced potency against the Gram-negative bacterium E. coli (extracted salt bridge pocket13 from Trzoss et al. 2013). dual inhibition of GyrB (Figure 12). The O-linked and ParE, in addition to positioning a moderately pyrrolopyrimidines showed balanced inhibition of basic amine to interact with the asparagine at the GyrB and ParE of Enterococcus faecalis, Francisella adenine pocket rim, was an important factor to tularensis and E. coli and proved potent against achieve broad Gram-negative antibacterial activity14. H. influenzae, E. coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa, all Gram-negative bacteria14.

12 The role of optimizing the shape of molecule optimized interactions in a compound in achieving balanced the binding site of ParE, which itself dual targeting also surfaced in the is tighter than GyrB. A selected analog development of the benzimidazole resulting from this optimization exhibited ureas. This compound series was effective antibacterial activity against six modified during early exploration Gram-positive and six Gram-negative with aryl substituents adjacent to the bacteria6. In the cases of both the benzimidazole NH group. Intramolecular pyrrolopyrimidines and the benzimidazole hydrogen bonds between the aryl ureas, antibacterial activity was enhanced substituent and the NH group maintained without altering the binding mode of planarity of the overall molecule, the compound, thus reducing the risk of giving an improved and more balanced impairing solubility or other properties. inhibition of gyrase and topoisomerase IV. Modeling showed that the planar

Figure 12. (A) Comparison of a pyrrolopyrimidine analog binding in the salt bridge pocket of ParE (red protein, gold ligand) and GyrB A (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.

13 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 Early analogs of the benzimidazole urea show reduced activity when tested against series showed strong inhibition of GyrB bacterial cultures containing proteins, and ParE and excellent potency against a like albumin, that are commonly found in number of bacterial strains. However, this blood serum. This change in antibacterial activity was weakened by protein binding activity is called a “serum shift”12. Protein of up to 95% for some of these analogs binding has rather complex mechanics. and serum shifts ranging from 15- to On the one hand, protein binding reduces 128-fold12. It was imperative to reduce the the amount of compound available to serum shift of this compound series to affect the pathogen. On the other hand, improve therapeutic efficacy. The team protein binding extends the time over performed a systematic analysis of the which the drug remains in the body properties of the compound series and because it cannot be eliminated until it identified two criteria that should result is slowly released. Protein binding is also in compounds with low serum shifts: a consequence of several factors. For medium hydrophobicity and high polar example, hydrophobic compounds tend surface area12. This makes sense as these to be more readily bound by proteins, criteria match the properties of known but the presence of ionizable groups that polar, hydrophilic compounds with good interact with specific sites on a protein solubility and reduced likelihood of also contributes to binding12. The problem binding to proteins. These criteria were, arises when modifications made to a on the one hand, used to select one of compound to reduce its serum shift also the analogs that surfaced as potent dual reduce its potency. 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 <0.01 <0.01 ParE E. coli 0.240 0.073 MIC (µg/ml) S. pneumoniae 0.016 0.016 M. catarrhalis 0.031 <0.008 H. in uenzae 0.18 0.13 E. coli 64 24 Figure 13. A substituent on the thiazole ring S. aureus 0.32 0.036 reduces pKa value of the carboxylic acid and Clearance (ml/min/kg) overall clearance rates. The effect may be Rat 66 17 due to impaired recognition by transporters. Dog 26 0.64 Note that the fluorine on the piperidine was substituted with a methoxyl group in the Mouse 99 10 course of optimization work (extracted from pK 3.6 4.2 a Basarab et al. 2014).

14 Unexpectedly, the lead compound of dual inhibition of GyrB and ParE, as well the series also covalently bound liver as excellent antibacterial activity against proteins in vitro, which could reduce several Gram-positive and two Gram- the amount of drug available to interact negative bacteria6. By approaching the with the target and potentially lead to problem from a different perspective, the safety issues. Studies suggested that the benzimidazole ureas drew closer to the compound was being metabolized via right balance. cytochrome P oxidation and the resulting modification of the urea that binds in Along similar lines, the pyrrolamide team the adenine pocket produced a reactive improved bioavailability of an analog metabolite that bound to protein. In a that went into Phase I clinical trials by classic example of the tradeoff between disguising the carboxylic acid bound inhibitory potency and other properties, to the thiazole ring interacting in the attempts to replace the urea diminished salt bridge pocket. They reasoned that the antibacterial activity of the resulting anion transporters, membrane proteins analogs, in some instances several fold6. in kidney and liver cells that remove negatively charged compounds from body To solve the problem, instead of removing fluids, recognized the carboxylic acid and the site of metabolism, the team decided thus cleared early pyrrolamide analogs at to reduce the amount of compound being high rates. The team added a functional converted and bound to liver proteins. group adjacent to the carboxylic acid to To do this, they incorporated a functional form an intramolecular hydrogen bond, group on the compound that would essentially reducing the likelihood that redirect metabolic activity away from the carboxylic acid would dissociate and the urea — a veritable molecular decoy. thus, become an anion (Figure 13). As a Careful to ensure that the newly designed result, clearance rates in multiple study compound remained planar and had as models were significantly lower for the low a hydrophobicity as possible, they new analog and its antibacterial activity achieved zero binding to liver proteins remained strong15. and the compound maintained potent

15 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 activity of a compound due to poor series were needed to match hydrophobic membrane penetration, even if enzyme interaction sites in GyrB and ParE to inhibitory potencies were equally high. achieve good enzyme potency, and also LogD measurements, which reflect to enable cell membrane permeation, the extent of polarity (negative values but excessive initial hydrophobicity led indicate high polarity), were used to guide to poor physical properties such as low the optimal polarity needed to achieve solubility and/or high protein binding. both membrane penetration and good physical properties. High logD values (>3) Case in point, analysis of the azaindole indicate excessive hydrophobicity which series revealed improvements in could cause physical properties (solubility antibacterial activity that tracked with and/or protein binding) to suffer, while changes in hydrophobicity. The team values too low (much below zero) could realized that excessively polar compounds, cause membrane permeability to suffer. though more soluble and less protein Compounds having LogD values between bound, could not penetrate bacterial cell 0 and 2 seemed like the best compromise. membranes11. Based on this tradeoff, the team chose a This observation was corroborated compound for efficacy experiments that by work with the pyridylureas. In the exhibited moderate solubility but strong process of finding functional groups antibacterial activity. that would strengthen interactions at the Selecting a compound having a low salt bridge pocket, the team noticed that clearance rate from the body was another charged, polar carboxylic acids at this factor in the selection. Low clearance site generated more soluble compounds rates are often correlated with higher but analogs with a neutral, less polar extents of protein binding, although carboxamide achieved higher antibacterial if protein binding is too high, then activity7 (Figure 14). antibacterial activity in the body suffers, In fact a correlation emerged between so balancing clearance and extent of the acidity of a compound and its antibacterial activity in this regard is also a antibacterial activity -- the higher the compromise. acidity, the weaker the antibacterial Figure 14. Carboxylic acids interacting in the R salt bridge pocket made pyridylurea analogs more soluble and more effective at inhibiting

GyrB and ParE, but their antibacterial activity IC50 (µM) was limited, especially compared to analogs GyrB S. aureus <0.01 <0.01 with a carboxamide group. Higher acidity appeared to limit the ability of the compound GryB E. coli 0.51 0.017 to permeate bacterial membranes ParE E. coli <0.01 0.96 (extracted from Basarab et al. 2013). ParE S. pneumoniae <0.01 0.012 MIC (µg/ml) S. pneumoniae 2.0 0.09 M. catarrhalis 64 0.7 H. inuenzae >64 5.5 E. coli >64 >64 Log D -0.62 2.0 16 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 the functional group is removed after on one or two important properties administration of the prodrug. In the in order to maximize the therapeutic case of the benzimidazole urea, the efficacy of a compound is necessary developed prodrug had good solubility to accommodate development goals. from the added phosphate group. Once For example, a top candidate of the administered, the phosphate group pyrrolopyrimidine series featuring a single was hydrolyzed via normal metabolic ring at the salt bridge site had lower processes, leaving the active antibacterial inhibitory potential and antibacterial to reach its target and achieve high activity than analogs with two rings. exposure in animal models6. However, testing the compounds against What is clear from examining the work mutated E. coli with compromised cell of these five antibacterial development membrane and efflux pumps showed programs is that a wealth of knowledge that the activity of all but the single- has accumulated about how to achieve ringed compound was impacted by balanced dual inhibition of GyrB and efflux and limited ability to permeate ParE, while maintaining adequate the bacterial membrane14. The smaller solubility and small serum shift. It is candidate would be the right choice if a also clear that, although this knowledge goal of the development program was to improves the likelihood of developing demonstrate effectiveness against Gram- a successful novel DNA gyrase and negative bacteria. topoisomerase IV inhibitor, nothing can Creativity is also essential for balancing substitute the need to test and re-test all factors that make a successful promising compounds. Achieving an antibacterial. The introduction of a antibacterial that successfully hits these metabolic decoy in the benzimidazole two targets and mitigates resistance urea series is an example of an development will require time — time unconventional approach to a problem to characterize the target, time to that could have severely compromised examine different mechanisms by which the potency and safety of the series. a compound can inhibit that target, and The same team was also creative about time to understand toxicity by thoroughly solving solubility issues. Their optimized investigating development paths compound with strong antibacterial that failed. attributes had very low solubility at We cannot afford to lose the arms race physiological pH. Therefore, the team against pathogenic bacteria. Our best created a prodrug by adding a phosphate bet will be to recruit the best minds, group to the compound. A prodrug is implement the best technology, and a version of a compound that includes above all, take the time to find the sweet a functional group used to transiently spot of, hopefully, several effective killers. alter an undesirable property. Typically,

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