Bioorganic & Medicinal Chemistry Letters 24 (2014) 413–418
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BMCL Digest
New natural products as new leads for antibacterial drug discovery q Dean G. Brown ⇑, Troy Lister, Tricia L. May-Dracka AstraZeneca Pharmaceuticals, 35 Gatehouse Dr, Waltham, MA 02451, United States
a r t i c l e
i n f o
Article history: Received 22 November 2013 Revised 13 December 2013 Accepted 14 December 2013 Available online 22 December 2013
a b s t r a c t Natural products have been a rich source of antibacterial drugs for many decades, but investments in this area have declined over the past two decades. The purpose of this review article is to provide a recent survey of new natural product classes and the mechanisms by which they work. Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
Keywords: Natural products Antibacterial Antimicrobial
In the past decade, only seven new chemical entities (NCE) have been approved for therapy for treatment of bacterial infections.1 The number of approvals are down substantially from the peak years in the mid-1980s, where on average, four new drugs were introduced to the market each year.2 The plausible reasons for this decline are most likely a combination of factors from a changing regulatory environment, increased drug safety standards, as well as failure of modern drug discovery techniques, such as high throughput screening (HTS) against the essential bacterial genome, to deliver on the promise of large numbers of new and novel targets in antibacterial space.3,4 O’Shea and Moser have published on the physical properties of approved antibacterial drugs which stands in contrast to typical ‘Lipinski-like’ molecules found in most company screening collections (e.g., antibacterial drugs with Gram-negative activity have log D = 2.8 vs 1.6 for non-antibacterial drugs).5 Aside from being highly polar and zwitterionic in many cases, many of these are also covalent inhibitors (e.g., b-lactams) or contain other chemical features postulated to aid in outer membrane penetration of bacteria. In comparison to mammalian cells, the cell wall of bacteria (especially Gram-negative) present a formidable barrier for traditional small molecule drug-like space which is typically represented in corporate HTS collections. This barrier is due to both highly polar outer membranes and prolific efflux pumps that remove foreign compounds. Of all the drugs currently approved as antibacterial NCE’s, a significant percentage of those are either natural products themselves q This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ⇑ Corresponding author. Tel.: +1 781 839 4101; fax: +1 781 839 4390. E-mail address: [email protected] (D.G. Brown).
or were derived from a natural product scaffold. Inspection of the 148 compounds utilized by O’Shea and Moser in their analysis indicates that roughly 66% of these compounds fall into one of the two natural product categories listed above. In line with the decline in natural product NCE’s seeking registration, there has also been a steady decline in major infrastructure investments for natural product isolation amongst large pharmaceutical companies. Up until the late 1990s and early 2000s many major pharmaceuticals companies possessed or collaborated with fully integrated natural product groups capable of large-scale fermentation, isolation, characterization and semi-synthesis (e.g., Eli Lilly, Merck, Bayer). Several reasons may explain why this paradigm has shifted. One reason could be that many of the sources of soil microorganisms capable of being cultured have already been examined and the risk of spending significant energy ‘re-discovering’ old natural products is too high to justify further investments. Another reason may be the facilitation of HTS and large corporate collections which were speculated to expedite the discovery process (e.g., HTS hits would be more amenable to synthetic analogs in the Hit-to-lead stage). And yet another possible reason may be a mind-set that commercial markets for novel antibacterial agents are not large and that the pre-clinical discovery investments should be made in accordance with the predicted market and return on investment. As such, natural products infrastructure may have been too expensive to continue to invest in. However, the need for new antibacterial agents remains high, with ever increasing rates of resistance of the current drugs on the market today. If new agents are not discovered, many of the current therapies will no longer work in the future, even for common infections. The purpose of this review is to highlight some of the new sources of natural products and discuss their proposed mechanisms as well as future trends. It is hoped that molecules, such
0960-894X/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.12.059
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as those mentioned in this review, will spark a renewed interest in natural products as sources for future directions in antibacterial drug discovery. Furthermore, with significant advances in synthetic organic chemistry (e.g., click chemistry and selective transition metal organocatalysis such as cross-metathesis, C–H bond activation), the opportunities for semi-synthesis are much greater than those in the 1950–1990s and thus new advances in this area are ripe for opportunity. Furthermore, advances in chemical biology tools and proteomics also lend themselves to the type of phenotypic screening typically done with antibacterial extracts and may open up new areas previously undiscovered. Secretory pathways: Signal peptidase I and II inhibitors. The secretory pathway in bacteria is responsible for translocation of pre-proteins from the cytoplasm for assembly into cell-wall structures.6 These pre-proteins contain signal peptides which are cleaved off by bacterial enzymes called signal peptidases.7 An attractive feature of this pathway is that it is located in the periplasmic space of the Gram-negative cell wall envelope, and as such, drug molecules need only to penetrate the outer membrane of Gram-negative bacteria to engage the target. Inhibition of this pathway is proposed to lead to cell death by accumulation of pre-proteins. Two targets for which natural product inhibitors have recently been reported are type 1 signal peptidase (SPase I: membrane-bound serine protease with a Lys-Ser catalytic dyad) and type II signal peptidase (SPase II: membrane-bound aspartyl protease)7 The arylomycins (1, Fig. 1) were identified from a strain of Streptomyces and are lipohexapeptides which were found to inhibit bacterial SPase I.8,9 These compounds have activity against wild-type Staphylococcus epidermis, (MIC = 0.25 lg/mL) but do not have
significant activity in wild-type Staphylococcus aureus, Escherichia coli or Pseudomonas aeruginosa. It was demonstrated that these latter three organisms have a proline residue in the active site rather than a serine in the case of S. epidermis.10 This change no longer accommodates an important H-bond to the peptidic side chain of arylomycin. Activity was regained in the proline to serine mutants of these bacteria (S. aureus MIC = 2 lg/mL, E. coli MIC = 4 lg/mL and P. aeruginosa MIC = 8 lg/mL) and conversely lost in S. epidermis by mutating from serine to proline (S. epidemeris S29P MIC = 8 lg/ mL, a 32-fold loss). Binding data in E. coli is consistent with this hypothesis, where 1 has a KD for E. coli Spase I of 979 ± 69 nM for the proline wild-type species as compared to 39 ± 15 nM for the serine mutant (P84S).10 The arylomycins also exist in glycosylated forms, which does not appear to interfere with antibacterial activity and may instead offer solubilizing properties.11 The arylomycins have also been shown to synergize when co-administered with aminoglycosides, but not with other classes of antibiotics.12 Two other molecules have been identified recently as inhibitors of SPase 1 from Staphylococcus (SPase termed SpsB).13 Actinocarbasin (2) is related to arylomycin, but is polysulfated, glycosylated and has different side-chains extending from the macrocycle than that of arylomycin. This compound has an MIC range from 0.5 to 128 lg/mL against 10 isolates of methicillin-resistant Staphylococcus aureus (MRSA). Krisynomycin (3) is a cyclic depsipeptide with antimicrobial activity against clinical isolates of MRSA (MIC range from 16 to 128 lg/mL against 24 clinical isolates). Of note is the fact that they both potentiate the activity of the b-lactam antibiotic imipenem, with restoration of imipenem potency against MRSA at concentrations 16-fold below their MIC. These compounds do not appear to synergize with other classes of antibiotics an interesting
Me HO HO
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6, Myxovirescin A1, C-14 C=C, C-25 = (R) 7, Myxovirescin A2, C-14 C=C, C-25 = (S) 8, Dihydromyxovirescin = C-14 C−C, C-25 = (R)
Figure 1. Type 1 signal peptidase inhibitors arylomycin 1, actinocarbasin 2, & krisynomycin 3. type II signal peptidase inhibitors globomycin 4 and 5 and myxovirescin scaffolds 6, 7, 8.
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contrast to arylomycin mentioned above. It is proposed that the potentiating effect is due to prevention of signal-peptidase mediated secretion of necessary proteins utilized in b-lactam resistance pathways. Two natural product lead structures have been identified which engage SPase II: (a) globomycins 4 & 514–16 and (b) myxovirescins 6, 7, 8 (or antibiotic TA, produced by myxobacteria).17 Of interest is that these two distinct classes of macrocyles (cyclic peptide and polyketide) inhibit the same target. Although this target is an aspartyl protease, neither structure is reminiscent of any traditional aspartyl protease inhibitor from other therapy areas (e.g., HIV protease, renin, b-secretase). Both chemotypes possess activity against E. coli (4, E. coli SANK70569 MIC = 12.5 lg/mL; 5, E. coli SANK70569 MIC = 1.56 lg/mL; 6, NCTC10418 E. coli MIC = 6.25 lg/mL; 8 E. coli NCTC10418 MIC = 3.12 lg/mL).18,19 It is interesting that the increase in the size of the lipophilic tail for globomycin increases activity by roughly 10-fold (4 to 5). This raises an interesting question on how these molecules penetrate the polar lipopolysacchride outer membrane of E. coli given their size, and if an increase in lipohpilic size enables penetration for these macrocyles. It is possible that an unknown active transport mechanism enables the bacterial penetration, but this has not been reported to date in the literature. A solid phase synthesis of globomycin has been reported which has provided analogs as well as some SAR development.20 Syntheses and stereochemical assignment of myxovirescin A1 is also known.19,21 Protein synthesis inhibitors Inhibitors of protein synthesis encompass several important classes of therapeutically useful antibiotics. For example, tetracyclins, macrolides, and aminoglycosides are all classified as protein synthesis inhibitors which disrupt the function of the 70S ribosome at varying stages thereby halting protein synthesis. The ribosome is a multimolecular complex, composed of a small (30S) and large (50S) subunit; it is functionally diverse which allows for many opportunities for the disruption of mRNA translation into proteins (initiation, aminoacyl tRNA entry, proofreading, peptidyl transfer, ribosomal translocation, and termination).22 Although this is a rich target class of antibacterial agents, resistance and undesirable side effects have led to a continual search for new inhibitors with novel mechanisms of action. Of significance in the field is the determination of high resolution Xray crystal structures of the ribosome. In 2000 the crystal structure of the 50S from Haloarcula marismortui (2.4 Å)23a and 30S from Thermus thermophilus (3 Å)23b were solved, and in 2005 the 30S of the more relevant pathogen E. coli (3.5 Å)23c was solved. This has been a technically very challenging area compared to enzyme structural biology, but these continual advances in ribosome structural biology will most certainly enable this field for more routine structure based drug design. A recent high throughput screen of 6700 microbial extracts led to the identification of orthoformimycin (9).24 Isolated from Streptomyces griseus, orthoformimycin contains a novel orthoformate ester, a functional group that had previously only been observed in fungal and bacterial secondary metabolites. Orthoformimycin exhibits moderate-to-poor antibacterial activity against several organisms, including Bacillus subtilis (MIC = 70 lg/mL). Although further investigations are needed to fully elucidate its mechanism of action, initial data indicates a novel 50S binding mode that inhibits translational elongation. Another promising protein synthesis inhibitor is bottromycin A (10) and analogs thereof. Although this natural product was first isolated in 1957, it’s absolute configuration was only recently confirmed through an enantioselective total synthesis.25 Bottromycin shows potent activity against MRSA and vancomycin-resistant Enterococci (VRE) (MICs = 1.0 and 0.5 lg/mL, respectively).26 Along with recent synthetic progress on bottromycin, the gene cluster responsible for biosynthesis has also been indentified.27 This
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antibacterial activity is the consequence of a unique mechanism of action that involves blocking aminoacyl tRNA from connecting to the A site on the 50S ribosome. Despite compelling in vitro activity, bottromycin faces significant challenges for its use as a therapeutic as in vivo studies have shown rapid methyl ester hydrolysis in plasma (R-group of 10 in Fig. 2), with the resulting carboxylic acid metabolite exhibiting attenuated activity (MRSA MIC = 64 lg/mL). A recent publication, however, has demonstrated that replacing the methyl ester with a propyl ketone (11) retains activity against drug resistant organisms and shows improved plasma stability.26 Antimicrobial peptides Antimicrobial peptides (AMP’s) have long been recognized as Nature’s defence against invasive bacterial infection.28 Perhaps better categorized as host-defense peptides, they elicit the ability to modulate immune response through various mechanisms to complement intrinsic, although often weak (in vivo), antimicrobial activity. AMP’s are synthesized at the ribosome as a gene-encoded precursor peptide followed by post-translational proteolytic activation. Highly diverse, AMP’s are broadly categorized based on their secondary structure, which is amphipathic in design due to spatially organized clusters of hydrophobicity and cationic character. More specifically, AMP’s are between 1050 amino acids, positively charged (between+2 and +9) and comprised of P30% hydrophobic residues. These structural features underpin the typically accepted mode of action of these molecules, which is binding to and disruption of the microbe’s (and host) cell membrane, although additional mechanisms may be at play. AMP’s have not fared well as therapeutics due to a combination of poor in vivo activity, extensive proteolysis, high manufacturing costs, and unmanageable safety margins. Modification of these natural products is proving increasingly successful at overcoming many of the short-falls hindering use as therapeutics. A recent example from Polyphor AG describes the chemical evolution of protegrin 1 (PG-1) to POL7080 (structure unknown), a candidate for serious Pseudomonas aeruginosa infections that has recently successfully completed phase 1 trials.29 PG-1 was identified from porcine leukocytes as an antimicrobial with moderate activity against Gram-positive and Gram-negative bacteria, fungi and some enveloped viruses. With a mechanism consistent with membrane disruption via pore formation, PG-1 also exhibited significant hemolysis and, as such, had limited clinical use. PG-1 contains 18 amino acids and is ordered into an anti-parallel b-strand by two disulfide bridges. Polyphor AG developed a D-proline–L-proline template grafted into a peptidomimetic scaffold to simulate and stabilize the b-hairpin conformation exhibited by PG-1. From this concept they generated a diverse library of peptidomimetic macrocycles. One sequence variant in this library (L8-1) exhibited broad spectrum antibacterial activity with reduced hemolysis. Iterative rounds of synthesis generated analogues with an increasingly potent and selective profile (L19-45, L26-19, L27-11 (12), POL7001), ultimately producing POL7080, a PG-1 peptidomemetic with P. aeruginosa (ATCC 27853) MIC of 0.008 lg/mL and ED50 of 0.250.55 mg/kg in a mouse septicaemia model. POL7080 is inactive against other Gram-negative and Gram-positive bacteria, is non-hemolyic at 100 lg/mL and has high plasma stability across species. Utilizing various chemo-proteomic and genetic techniques, Polyphor AG established that progenors of POL7080 (and thus by extrapolation, POL7080 itself) exhibit antibacterial activity by disrupting the LPS trafficking pathway in P. aeruginosa through binding the essential outer membrane protein, LptD. Seemingly justifying the considerable excitement generated by the discovery of POL7080, Roche recently announced a licensing agreement with Polyphor AG for upto $547 M to co-develop POL7080. A distinct, yet equally diverse group of AMP’s are alternatively produced in microbes by non-ribosomal-peptide synthetases. Exhibiting intrinsic, potent antibacterial activity, polymyxin B,
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Figure 2. Protein synthesis inhibitors orthoformimycin and bottromycin.
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Figure 3. Peptidomemetic macrocycles.
vancomycin, and daptomycin are scarce examples of unadulterated natural product AMP’s that have successfully passed clinical trials and have been commercialized. Although safety remains an issue with these agents, their potent, broad spectrum activity and marginal pharmaceutical properties endears them with last line of defence status for highly drug resistant bacteria (Fig. 3). Topoisomerase inhibitors Type II topoisomerase is a clinically validated antibacterial drug target and inhibition blocks DNA synthesis. At least two pathways involving this protein are known; namely, disrupting the topoisomerase-DNA complex (so-called topoisomerase poisons) and/or interrupting catalytic turnover
(topoisomerase inhibitors).30 Fluoroquinolones, for example, are considered topoisomerase poisons. In 2011, Merck disclosed a novel natural product, kibdelomycin (13), that was isolated from S. aureus collected from a soil sample from the Central Africa Republic.31 Kibdelomycin is a relatively complex natural product comprised of five distinct structural subunits: a tetramic acid bridging a novel sugar residue through a N-glycosidic linkage and to a decalin subunit through an enol linkage. The decalin is in turn connected to a hexopyranose by an O-glyosidic bond and the hexapyranose is capped via a 3,4-dichloro-pyrrolamide. The natural product exhibits potent whole cell activity against wild-type
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Gram-positive bacteria (MIC = 2.0 and 0.5 lg/mL for S. aureus and MRSA, respectively). Moderate levels of inhibition were also observed in a TolC efflux pump mutant of E. coli (MIC = 32 lg/ mL), but no activity against wild type Gram-negative pathogens was evident (E. coli MIC >64 lg/mL), likely due to poor permeability and/or efflux. The activity associated with kibdelomycin is similar to that observed in the structurally unrelated natural products, novobiocin (14) and clorobiocin (15).32 The mechanism of action is hypothesized to involve inhibition of the ATPase activity of type II DNA topoisomerases, which leads to obstruction of DNA synthesis and cell death. Independently, researchers at AstraZeneca discovered pyrrolamide 16 through a fragment-based lead generation approach (FBLG).33 X-ray co-crystallography of early pyrrole containing hits from the FBLG in the S. aureus GyrB N-terminal ATP binding domain showed a hydrogen bond between the pyrrole-NH and aspartic acid-81. In an effort to improve potency of these early hits, a 3,4-dichloro substituent was installed on the pyrrole to increase hydrophobic interactions in the adenine pocket and to reduce the pKa of the NH group to make it a better hydrogen bond donor. This modification lead to >150 fold increase in potency over the initial hit. Pyrrolamide 16 exhibits single digit micromolar MIC activity across a range of Gram-positive bacteria. This represents an interesting example whereby the properties of a fully synthetic DNA gyrase inhibitor drug candidate were altered by the incorporation of a functional group (3,4-dichloropyrrole amide) that was subsequently identified as a component of a natural product that exhibits the same mode of action; thus medicinal chemistry mimicking nature in parallel discovery pathways (Fig. 4). Emerging classes of new natural product space Despite the decline in research dedicated to natural products discovery and development, natural products with compelling biological profiles and new structural diversity continue to be discovered as exemplified by the following two recent examples (Fig. 5). In 2011, Rene De Mot and co-workers at the Katholieke Universiteit Leuven disclosed a new amphipathic salicylate containing antibiotic, promysalin (17).34 Promysalin is uniquely comprised of a 2-pyrroline carboxylate that bridges a single salicylic acid moiety and a 2,8-dihydroxymyristamide side chain. These three distinct structural features are derived from three different central metabolites that are elaborated and combined by a previously unknown
gene cluster combination. Produced by Pseudomonas putida, promysalin facilitates swarming characteristics and biofilm formation in the producing strain, but is antagonistic to other Pseudomonads including MDR clinical isolates of P. aeruginosa (IC50 = 0.16 lg/mL and minimum bactericidal activity (MBC) = 100 lg/mL against PA14). Indeed, of the 16 Pseudomonas species tested, 12 had sensitive strains, and in a broader panel of P. aeruginosa environmental and clinical isolates, all 83 were sensitive. This intriguing microbiological profile warrants further investigation, and additional work is required to elucidate the absolute stereochemistry, mechanism of action and structure activity relationships of promysalin. At the 53rd ICAAC meeting (Denver, Colorado 2013) a group of researchers from Fundacion Medina, Cubist Pharmaceuticals and the University of California, San Diego presented a poster disclosing a family of novel natural product antibiotics (MDN– 0057–60).35 Isolated from the crude fermentation extract of a filamentous fungus, these natural products possess two isonitrile functional groups, which although rarely found in natural products, are not unprecedented. Indeed, MK4588, an antibiotic natural product isolated from Leptospaeria fungus by Gomi and co-workers in 1990, would appear to be related.36 MDN-0057 exhibits potent broad spectrum Gram-negative activity with MIC ranging 0.2564 lg/mL against E. coli, Acinetobacter baumannii, P. aeruginosa and K. pneumonia clinical isolates. MIC values are significantly lower in efflux pump mutant or permeabalized strains of these organisms indicating a propensity for efflux and poor permeability for this chemotype. MDN-0057 did not inhibit S. aureus (>128 lg/ mL) or eukaryotic cell growth (HepG2 and Fa2N4 >512 lg/mL) and was inactive in the hERG K channel at 50 lM. A study of the impact of MDN-0057 on macromolecular synthesis indicated a concentration dependant inhibition of cell wall synthesis, but not RNA, DNA, protein or lipid synthesis. Additionally, cytology experiments showed that incubation of E. coli cells with MDN-0057 rendered the cells more permeable to DAPI and sytox green (dyes) thus reinforcing a cell wall target related mode of action. In summary, natural product research continues to be an important cornerstone for antibacterial drug discovery. With modern advances in selective organic synthesis, ribosome crystallography, chemical biology tools for target elucidation, and novel methods for uncovering new natural products, this area can continue to provide new medicines towards unmet patient needs.
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Figure 4. Topoisomerase inhibitors from natural sources (13, 14, and 15) and synthetic preparation (16).
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20, MDN-0058, R = OH 22, MK4588 21, MDN-0060, R = H
Figure 5. New natural product space.
References and notes 1. Spellberg, B.; Blaser, M.; Guidos, R. J.; Boucher, H. W.; Bradley, J. S.; Eisenstein, B. I.; Gerding, D.; Lynfield, R.; Reller, L. B.; Rex, J.; Schwartz, J. S.; Septimus, E.; Tenover, F. C.; Gilbert, D. N. IDSA Public Policy Paper Oxford University Press 2011, 52, S397. 2. Spellberg, B.; Powers, J. H.; Brass, E. P.; Miller, L. G.; Edwards, J. E., Jr. Clin. Inf. Dis. 2004, 38, 1279. 3. Payne, D. J.; Gwynn, M. N.; Holmes, D. J.; Pompliano, D. L. Nat. Rev. Drug Disc. 2007, 6, 29. 4. Gwynn, M. N.; Portnoy, A.; Rittenhouse, S. F.; Payne, D. J. Ann. N.Y. Acad. Sci. 2010, 1213, 5. 5. O’Shea, R.; Moser, H. E. J. Med. Chem. 2008, 51, 2871. 6. Driessen, A. J. M.; Nouwen, N. Ann. Rev. Biochem. 2008, 77, 643. 7. Paetzel, M.; Karla, A.; Strynadka, N. C. J.; Dalbey, R. E. Chem. Rev. 2002, 102, 4549. 8. Schimana, J.; Gebhardt, K.; Höltzel, A.; Schmid, D. G.; Süssmuth, R.; Müller, J.; Pukall, R.; Fielder, H.-P. J. Antibiot. (Tokyo) 2002, 55, 565. 9. Paetzel, M.; Goodall, J. J.; Kania, M.; Dalbey, R. E.; Page, M. G. P. J. Biol. Chem. 2004, 279, 30781. 10. Smith, P. A.; Roberts, T. C.; Romesburg, F. E. Chem. Biol. 2010, 17, 1223. 11. Liu, J.; Luo, C.; Smith, P. A.; Chin, J. K.; Page, M. G. P.; Paetzel, M.; Romesberg, F. E. J. Am. Chem. Soc. 2011, 133, 17869. 12. Smith, P. A.; Romesberg, F. E. Antimicrob. Agents Chemother. 2012, 56, 5054. 13. Therien, A. G.; Huber, J. L.; Wilson, K. E.; Beaulieu, P.; Caron, A.; Claveau, D.; Deschamps, K.; Donald, R. G. K.; Galgoci, A. M.; Gallant, M.; Gu, X.; Kevin, N. J.; Lafleur, J.; Leavitt, P. S.; Lebeau-Jacob, C.; Lee, S. S.; Lin, M. M.; Michels, A. A.; Ogawa, A. M.; Painter, R. E.; Parish, C. A.; Park, Y.-W.; Benton-Perdomo, L.; Petcu, M.; Phillips, J. W.; Powles, M. A.; Skorey, K. I.; Tam, J.; Tan, C. M.; Young, K.; Wong, S.; Waddell, S. T.; Miesel, L. Antimicrob. Agents Chemother. 2012, 56, 4662. 14. Inukai, M.; Enokita, R.; Torikata, A.; Ankara, M.; Iwado, S.; Arai, M. J. Antibiot. 1978, 31, 410. 15. Inukai, M.; Nakajima, M.; Osawa, M.; Haneishi, T.; Arai, M. J. Antibiot. (Tokyo) 1978, 31, 421. 16. Dev, I. K.; Harvey, R. J.; Ray, P. H. J. Biol. Chem. 1985, 260, 5891. 17. Xiao, Y.; Gerth, K.; Müller, R.; Wall, D. Antimicrob. Agents Chemother. 2014, 2012, 56. 18. Kiho, T.; Nakayama, M.; Yasuda, K.; Miyakoshi, S.; Inukai, M.; Kogen, H. Bioorg. Med. Chem. 2004, 12, 337. 19. Content, S.; Dutton, C. J.; Roberts, L. Bioorg. Med. Chem. Lett. 2003, 13, 321. 20. Sarabia, F.; Chammaa, S.; García-Ruiz, C. J. Org. Chem. 2011, 76, 2132. 21. (a) Fürstner, A.; Bonnekessel, M.; Blank, J. T.; Radkowski, K.; Seidel, G.; Lacombe, F.; Gabor, B.; Mynott, R. Chem.-Eur. J. 2007, 13, 8762; (b) Williams, D. R.; McGill, J. M. J. Org. Chem. 1990, 55, 3457.
22. For a review, see: Poehlsgaard, J.; Douthwaite, S. Nat. Rev. Microbiol. 2005, 3, 870. 23. (a) Ban, N.; Nissen, P.; Hansen, J.; Moore, P. B.; Steitz, T. A. Science 2000, 289, 905; (b) Wimberly, B.; Brodersen, D. E.; Clemons, W. M., Jr.; Morgan-Warren, R. J.; Carter, A. P.; Vonrhein, C.; Hartsch, T.; Ramakrishnan, V. Nature 2000, 407, 327; (c) Schuwirth, B. S.; Borovinskaya, M. A.; Hau, C. W.; Zhang, W.; VilaSanjurjo, A.; Holton, J. M.; Doudna Cate, J. H. Science 2005, 310, 827. 24. Maffioli, S. I.; Fabbretti, A.; Brandi, L.; Savelsbergh, A.; Monciardini, P.; Abbondi, M.; Rossi, R.; Donadio, S.; Gualerzi, C. O. ACS Chem. Biol. 1939, 2013, 8. 25. Shimamura, H.; Gouda, H.; Nagai, T.; Hirose, M.; Ichioka, M.; Furuya, Y.; Kobayashi, Y.; Hirono, S.; Sunazuka, T.; Omura, S. Angew. Chem., Int. Ed. 2009, 48, 914. 26. Kobayashi, Y.; Ichioka, M.; Hirose, T.; Nagai, K.; Matsumoto, A.; Matsui, H.; Hanaki, H.; Masuma, R.; Takahashi, Y.; Omura, S.; Sunazuka, T. Bioorg. Med. Chem. Lett. 2010, 20, 6116. 27. Crone, W. J. K.; Leeper, F. J.; Truman, A. W. Chem. Sci. 2012, 3, 3516. 28. Nguyen, L. T.; Haney, E. F.; Vogel, H. J. Trends Biotechol. 2011, 29, 464. 29. Srinivas, N.; Jetter, P.; Ueberbacher, B. J.; Werneburg, M.; Zerbe, K.; Steinmann, J.; Van der Meijden, B.; Bernardini, F.; Lederer, A.; Dias, R. L. A.; Misson, P. E.; Henze, H.; Zumbrunn, J.; Gombert, F. O.; Obrecht, D.; Hunziker, P.; Schauer, S.; Ziegler, U.; Käch, A.; Eberl, L.; Riedel, K.; DeMarco, S. J.; Robinson, J. A. Science 2010, 327, 1010. 30. Bisacchi, G. S.; Dumas, J. Ann. Rep. Med. Chem. 2009, 44, 379. 31. Phillips, J. W.; Goetz, M. A.; Smith, S. K.; Zink, D. L.; Polishook, J.; Onishi, R.; Salowe, S.; Wiltsie, J.; Allocco, J.; Sigmund, J.; Dorso, K.; Lee, S.; Skwish, S.; de la Cruz, M.; Martin, J.; Vicente, F.; Genilloud, O.; Lu, J.; Painter, R. E.; Young, K.; Overbye, K.; Donald, R. G. K.; Singh, S. B. Chem Biol. 2011, 18, 955. 32. (a) Li, S.-M.; Heide, L. Curr. Med. Chem. Anti-Infect. Agents 2004, 3, 279; (b) Heide, L.; Gust, B.; Anderle, C.; Li, S.-M. Curr. Top. Med. Chem. 2008, 8, 667. 33. 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.; UriaNickelsen, M.; Demeritt, J.; Loch, J. T., III; Hull, K.; Blodgett, A.; Illingworth, R. N.; Prince, B.; Boriack-Sjodin, P. A.; Hauck, S.; MacPherson, L. J.; Ni, H.; Sherer, B. Antimicrob. Agents Chemother. 2012, 56, 1240. 34. Li, W.; Estrada-de los Santos, P.; Matthijs, S.; Xie, G.-L.; Busson, R.; Cornelis, P.; Rozenski, J.; De Mot, R. Chem. Biol. 2011, 18, 1320. 35. Genilloud, O.; Vicente, F.; Reyes, F.; Bills, G.; De la Cruz, M.; Tormo, J.; Martin, J.; Gonzalez, V.; El Aouad, N.; de Pedro, N.; Perez del Palacio, J.; Diaz, C.; Monteiro, M.; Conrad, M.; Cassidy, K.; Cotroneo, N.; Cappellucci, L.; Knight-Connoni, V. Conf. Antimicrob. Agents Chemother 2013, 89. Presentation F1215. 36. Itoh, J.; Takeuchi, Y.; Gomi, S.; Inouye, S.; Mikawa, T.; Yoshikawa, N.; Ohishi, H. J. Antibiotics 1990, 43, 456.
Contents lists available at ScienceDirect
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BMCL Digest
New natural products as new leads for antibacterial drug discovery q Dean G. Brown ⇑, Troy Lister, Tricia L. May-Dracka AstraZeneca Pharmaceuticals, 35 Gatehouse Dr, Waltham, MA 02451, United States
a r t i c l e
i n f o
Article history: Received 22 November 2013 Revised 13 December 2013 Accepted 14 December 2013 Available online 22 December 2013
a b s t r a c t Natural products have been a rich source of antibacterial drugs for many decades, but investments in this area have declined over the past two decades. The purpose of this review article is to provide a recent survey of new natural product classes and the mechanisms by which they work. Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
Keywords: Natural products Antibacterial Antimicrobial
In the past decade, only seven new chemical entities (NCE) have been approved for therapy for treatment of bacterial infections.1 The number of approvals are down substantially from the peak years in the mid-1980s, where on average, four new drugs were introduced to the market each year.2 The plausible reasons for this decline are most likely a combination of factors from a changing regulatory environment, increased drug safety standards, as well as failure of modern drug discovery techniques, such as high throughput screening (HTS) against the essential bacterial genome, to deliver on the promise of large numbers of new and novel targets in antibacterial space.3,4 O’Shea and Moser have published on the physical properties of approved antibacterial drugs which stands in contrast to typical ‘Lipinski-like’ molecules found in most company screening collections (e.g., antibacterial drugs with Gram-negative activity have log D = 2.8 vs 1.6 for non-antibacterial drugs).5 Aside from being highly polar and zwitterionic in many cases, many of these are also covalent inhibitors (e.g., b-lactams) or contain other chemical features postulated to aid in outer membrane penetration of bacteria. In comparison to mammalian cells, the cell wall of bacteria (especially Gram-negative) present a formidable barrier for traditional small molecule drug-like space which is typically represented in corporate HTS collections. This barrier is due to both highly polar outer membranes and prolific efflux pumps that remove foreign compounds. Of all the drugs currently approved as antibacterial NCE’s, a significant percentage of those are either natural products themselves q This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ⇑ Corresponding author. Tel.: +1 781 839 4101; fax: +1 781 839 4390. E-mail address: [email protected] (D.G. Brown).
or were derived from a natural product scaffold. Inspection of the 148 compounds utilized by O’Shea and Moser in their analysis indicates that roughly 66% of these compounds fall into one of the two natural product categories listed above. In line with the decline in natural product NCE’s seeking registration, there has also been a steady decline in major infrastructure investments for natural product isolation amongst large pharmaceutical companies. Up until the late 1990s and early 2000s many major pharmaceuticals companies possessed or collaborated with fully integrated natural product groups capable of large-scale fermentation, isolation, characterization and semi-synthesis (e.g., Eli Lilly, Merck, Bayer). Several reasons may explain why this paradigm has shifted. One reason could be that many of the sources of soil microorganisms capable of being cultured have already been examined and the risk of spending significant energy ‘re-discovering’ old natural products is too high to justify further investments. Another reason may be the facilitation of HTS and large corporate collections which were speculated to expedite the discovery process (e.g., HTS hits would be more amenable to synthetic analogs in the Hit-to-lead stage). And yet another possible reason may be a mind-set that commercial markets for novel antibacterial agents are not large and that the pre-clinical discovery investments should be made in accordance with the predicted market and return on investment. As such, natural products infrastructure may have been too expensive to continue to invest in. However, the need for new antibacterial agents remains high, with ever increasing rates of resistance of the current drugs on the market today. If new agents are not discovered, many of the current therapies will no longer work in the future, even for common infections. The purpose of this review is to highlight some of the new sources of natural products and discuss their proposed mechanisms as well as future trends. It is hoped that molecules, such
0960-894X/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.12.059
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as those mentioned in this review, will spark a renewed interest in natural products as sources for future directions in antibacterial drug discovery. Furthermore, with significant advances in synthetic organic chemistry (e.g., click chemistry and selective transition metal organocatalysis such as cross-metathesis, C–H bond activation), the opportunities for semi-synthesis are much greater than those in the 1950–1990s and thus new advances in this area are ripe for opportunity. Furthermore, advances in chemical biology tools and proteomics also lend themselves to the type of phenotypic screening typically done with antibacterial extracts and may open up new areas previously undiscovered. Secretory pathways: Signal peptidase I and II inhibitors. The secretory pathway in bacteria is responsible for translocation of pre-proteins from the cytoplasm for assembly into cell-wall structures.6 These pre-proteins contain signal peptides which are cleaved off by bacterial enzymes called signal peptidases.7 An attractive feature of this pathway is that it is located in the periplasmic space of the Gram-negative cell wall envelope, and as such, drug molecules need only to penetrate the outer membrane of Gram-negative bacteria to engage the target. Inhibition of this pathway is proposed to lead to cell death by accumulation of pre-proteins. Two targets for which natural product inhibitors have recently been reported are type 1 signal peptidase (SPase I: membrane-bound serine protease with a Lys-Ser catalytic dyad) and type II signal peptidase (SPase II: membrane-bound aspartyl protease)7 The arylomycins (1, Fig. 1) were identified from a strain of Streptomyces and are lipohexapeptides which were found to inhibit bacterial SPase I.8,9 These compounds have activity against wild-type Staphylococcus epidermis, (MIC = 0.25 lg/mL) but do not have
significant activity in wild-type Staphylococcus aureus, Escherichia coli or Pseudomonas aeruginosa. It was demonstrated that these latter three organisms have a proline residue in the active site rather than a serine in the case of S. epidermis.10 This change no longer accommodates an important H-bond to the peptidic side chain of arylomycin. Activity was regained in the proline to serine mutants of these bacteria (S. aureus MIC = 2 lg/mL, E. coli MIC = 4 lg/mL and P. aeruginosa MIC = 8 lg/mL) and conversely lost in S. epidermis by mutating from serine to proline (S. epidemeris S29P MIC = 8 lg/ mL, a 32-fold loss). Binding data in E. coli is consistent with this hypothesis, where 1 has a KD for E. coli Spase I of 979 ± 69 nM for the proline wild-type species as compared to 39 ± 15 nM for the serine mutant (P84S).10 The arylomycins also exist in glycosylated forms, which does not appear to interfere with antibacterial activity and may instead offer solubilizing properties.11 The arylomycins have also been shown to synergize when co-administered with aminoglycosides, but not with other classes of antibiotics.12 Two other molecules have been identified recently as inhibitors of SPase 1 from Staphylococcus (SPase termed SpsB).13 Actinocarbasin (2) is related to arylomycin, but is polysulfated, glycosylated and has different side-chains extending from the macrocycle than that of arylomycin. This compound has an MIC range from 0.5 to 128 lg/mL against 10 isolates of methicillin-resistant Staphylococcus aureus (MRSA). Krisynomycin (3) is a cyclic depsipeptide with antimicrobial activity against clinical isolates of MRSA (MIC range from 16 to 128 lg/mL against 24 clinical isolates). Of note is the fact that they both potentiate the activity of the b-lactam antibiotic imipenem, with restoration of imipenem potency against MRSA at concentrations 16-fold below their MIC. These compounds do not appear to synergize with other classes of antibiotics an interesting
Me HO HO
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Figure 1. Type 1 signal peptidase inhibitors arylomycin 1, actinocarbasin 2, & krisynomycin 3. type II signal peptidase inhibitors globomycin 4 and 5 and myxovirescin scaffolds 6, 7, 8.
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contrast to arylomycin mentioned above. It is proposed that the potentiating effect is due to prevention of signal-peptidase mediated secretion of necessary proteins utilized in b-lactam resistance pathways. Two natural product lead structures have been identified which engage SPase II: (a) globomycins 4 & 514–16 and (b) myxovirescins 6, 7, 8 (or antibiotic TA, produced by myxobacteria).17 Of interest is that these two distinct classes of macrocyles (cyclic peptide and polyketide) inhibit the same target. Although this target is an aspartyl protease, neither structure is reminiscent of any traditional aspartyl protease inhibitor from other therapy areas (e.g., HIV protease, renin, b-secretase). Both chemotypes possess activity against E. coli (4, E. coli SANK70569 MIC = 12.5 lg/mL; 5, E. coli SANK70569 MIC = 1.56 lg/mL; 6, NCTC10418 E. coli MIC = 6.25 lg/mL; 8 E. coli NCTC10418 MIC = 3.12 lg/mL).18,19 It is interesting that the increase in the size of the lipophilic tail for globomycin increases activity by roughly 10-fold (4 to 5). This raises an interesting question on how these molecules penetrate the polar lipopolysacchride outer membrane of E. coli given their size, and if an increase in lipohpilic size enables penetration for these macrocyles. It is possible that an unknown active transport mechanism enables the bacterial penetration, but this has not been reported to date in the literature. A solid phase synthesis of globomycin has been reported which has provided analogs as well as some SAR development.20 Syntheses and stereochemical assignment of myxovirescin A1 is also known.19,21 Protein synthesis inhibitors Inhibitors of protein synthesis encompass several important classes of therapeutically useful antibiotics. For example, tetracyclins, macrolides, and aminoglycosides are all classified as protein synthesis inhibitors which disrupt the function of the 70S ribosome at varying stages thereby halting protein synthesis. The ribosome is a multimolecular complex, composed of a small (30S) and large (50S) subunit; it is functionally diverse which allows for many opportunities for the disruption of mRNA translation into proteins (initiation, aminoacyl tRNA entry, proofreading, peptidyl transfer, ribosomal translocation, and termination).22 Although this is a rich target class of antibacterial agents, resistance and undesirable side effects have led to a continual search for new inhibitors with novel mechanisms of action. Of significance in the field is the determination of high resolution Xray crystal structures of the ribosome. In 2000 the crystal structure of the 50S from Haloarcula marismortui (2.4 Å)23a and 30S from Thermus thermophilus (3 Å)23b were solved, and in 2005 the 30S of the more relevant pathogen E. coli (3.5 Å)23c was solved. This has been a technically very challenging area compared to enzyme structural biology, but these continual advances in ribosome structural biology will most certainly enable this field for more routine structure based drug design. A recent high throughput screen of 6700 microbial extracts led to the identification of orthoformimycin (9).24 Isolated from Streptomyces griseus, orthoformimycin contains a novel orthoformate ester, a functional group that had previously only been observed in fungal and bacterial secondary metabolites. Orthoformimycin exhibits moderate-to-poor antibacterial activity against several organisms, including Bacillus subtilis (MIC = 70 lg/mL). Although further investigations are needed to fully elucidate its mechanism of action, initial data indicates a novel 50S binding mode that inhibits translational elongation. Another promising protein synthesis inhibitor is bottromycin A (10) and analogs thereof. Although this natural product was first isolated in 1957, it’s absolute configuration was only recently confirmed through an enantioselective total synthesis.25 Bottromycin shows potent activity against MRSA and vancomycin-resistant Enterococci (VRE) (MICs = 1.0 and 0.5 lg/mL, respectively).26 Along with recent synthetic progress on bottromycin, the gene cluster responsible for biosynthesis has also been indentified.27 This
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antibacterial activity is the consequence of a unique mechanism of action that involves blocking aminoacyl tRNA from connecting to the A site on the 50S ribosome. Despite compelling in vitro activity, bottromycin faces significant challenges for its use as a therapeutic as in vivo studies have shown rapid methyl ester hydrolysis in plasma (R-group of 10 in Fig. 2), with the resulting carboxylic acid metabolite exhibiting attenuated activity (MRSA MIC = 64 lg/mL). A recent publication, however, has demonstrated that replacing the methyl ester with a propyl ketone (11) retains activity against drug resistant organisms and shows improved plasma stability.26 Antimicrobial peptides Antimicrobial peptides (AMP’s) have long been recognized as Nature’s defence against invasive bacterial infection.28 Perhaps better categorized as host-defense peptides, they elicit the ability to modulate immune response through various mechanisms to complement intrinsic, although often weak (in vivo), antimicrobial activity. AMP’s are synthesized at the ribosome as a gene-encoded precursor peptide followed by post-translational proteolytic activation. Highly diverse, AMP’s are broadly categorized based on their secondary structure, which is amphipathic in design due to spatially organized clusters of hydrophobicity and cationic character. More specifically, AMP’s are between 1050 amino acids, positively charged (between+2 and +9) and comprised of P30% hydrophobic residues. These structural features underpin the typically accepted mode of action of these molecules, which is binding to and disruption of the microbe’s (and host) cell membrane, although additional mechanisms may be at play. AMP’s have not fared well as therapeutics due to a combination of poor in vivo activity, extensive proteolysis, high manufacturing costs, and unmanageable safety margins. Modification of these natural products is proving increasingly successful at overcoming many of the short-falls hindering use as therapeutics. A recent example from Polyphor AG describes the chemical evolution of protegrin 1 (PG-1) to POL7080 (structure unknown), a candidate for serious Pseudomonas aeruginosa infections that has recently successfully completed phase 1 trials.29 PG-1 was identified from porcine leukocytes as an antimicrobial with moderate activity against Gram-positive and Gram-negative bacteria, fungi and some enveloped viruses. With a mechanism consistent with membrane disruption via pore formation, PG-1 also exhibited significant hemolysis and, as such, had limited clinical use. PG-1 contains 18 amino acids and is ordered into an anti-parallel b-strand by two disulfide bridges. Polyphor AG developed a D-proline–L-proline template grafted into a peptidomimetic scaffold to simulate and stabilize the b-hairpin conformation exhibited by PG-1. From this concept they generated a diverse library of peptidomimetic macrocycles. One sequence variant in this library (L8-1) exhibited broad spectrum antibacterial activity with reduced hemolysis. Iterative rounds of synthesis generated analogues with an increasingly potent and selective profile (L19-45, L26-19, L27-11 (12), POL7001), ultimately producing POL7080, a PG-1 peptidomemetic with P. aeruginosa (ATCC 27853) MIC of 0.008 lg/mL and ED50 of 0.250.55 mg/kg in a mouse septicaemia model. POL7080 is inactive against other Gram-negative and Gram-positive bacteria, is non-hemolyic at 100 lg/mL and has high plasma stability across species. Utilizing various chemo-proteomic and genetic techniques, Polyphor AG established that progenors of POL7080 (and thus by extrapolation, POL7080 itself) exhibit antibacterial activity by disrupting the LPS trafficking pathway in P. aeruginosa through binding the essential outer membrane protein, LptD. Seemingly justifying the considerable excitement generated by the discovery of POL7080, Roche recently announced a licensing agreement with Polyphor AG for upto $547 M to co-develop POL7080. A distinct, yet equally diverse group of AMP’s are alternatively produced in microbes by non-ribosomal-peptide synthetases. Exhibiting intrinsic, potent antibacterial activity, polymyxin B,
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Figure 2. Protein synthesis inhibitors orthoformimycin and bottromycin.
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Figure 3. Peptidomemetic macrocycles.
vancomycin, and daptomycin are scarce examples of unadulterated natural product AMP’s that have successfully passed clinical trials and have been commercialized. Although safety remains an issue with these agents, their potent, broad spectrum activity and marginal pharmaceutical properties endears them with last line of defence status for highly drug resistant bacteria (Fig. 3). Topoisomerase inhibitors Type II topoisomerase is a clinically validated antibacterial drug target and inhibition blocks DNA synthesis. At least two pathways involving this protein are known; namely, disrupting the topoisomerase-DNA complex (so-called topoisomerase poisons) and/or interrupting catalytic turnover
(topoisomerase inhibitors).30 Fluoroquinolones, for example, are considered topoisomerase poisons. In 2011, Merck disclosed a novel natural product, kibdelomycin (13), that was isolated from S. aureus collected from a soil sample from the Central Africa Republic.31 Kibdelomycin is a relatively complex natural product comprised of five distinct structural subunits: a tetramic acid bridging a novel sugar residue through a N-glycosidic linkage and to a decalin subunit through an enol linkage. The decalin is in turn connected to a hexopyranose by an O-glyosidic bond and the hexapyranose is capped via a 3,4-dichloro-pyrrolamide. The natural product exhibits potent whole cell activity against wild-type
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Gram-positive bacteria (MIC = 2.0 and 0.5 lg/mL for S. aureus and MRSA, respectively). Moderate levels of inhibition were also observed in a TolC efflux pump mutant of E. coli (MIC = 32 lg/ mL), but no activity against wild type Gram-negative pathogens was evident (E. coli MIC >64 lg/mL), likely due to poor permeability and/or efflux. The activity associated with kibdelomycin is similar to that observed in the structurally unrelated natural products, novobiocin (14) and clorobiocin (15).32 The mechanism of action is hypothesized to involve inhibition of the ATPase activity of type II DNA topoisomerases, which leads to obstruction of DNA synthesis and cell death. Independently, researchers at AstraZeneca discovered pyrrolamide 16 through a fragment-based lead generation approach (FBLG).33 X-ray co-crystallography of early pyrrole containing hits from the FBLG in the S. aureus GyrB N-terminal ATP binding domain showed a hydrogen bond between the pyrrole-NH and aspartic acid-81. In an effort to improve potency of these early hits, a 3,4-dichloro substituent was installed on the pyrrole to increase hydrophobic interactions in the adenine pocket and to reduce the pKa of the NH group to make it a better hydrogen bond donor. This modification lead to >150 fold increase in potency over the initial hit. Pyrrolamide 16 exhibits single digit micromolar MIC activity across a range of Gram-positive bacteria. This represents an interesting example whereby the properties of a fully synthetic DNA gyrase inhibitor drug candidate were altered by the incorporation of a functional group (3,4-dichloropyrrole amide) that was subsequently identified as a component of a natural product that exhibits the same mode of action; thus medicinal chemistry mimicking nature in parallel discovery pathways (Fig. 4). Emerging classes of new natural product space Despite the decline in research dedicated to natural products discovery and development, natural products with compelling biological profiles and new structural diversity continue to be discovered as exemplified by the following two recent examples (Fig. 5). In 2011, Rene De Mot and co-workers at the Katholieke Universiteit Leuven disclosed a new amphipathic salicylate containing antibiotic, promysalin (17).34 Promysalin is uniquely comprised of a 2-pyrroline carboxylate that bridges a single salicylic acid moiety and a 2,8-dihydroxymyristamide side chain. These three distinct structural features are derived from three different central metabolites that are elaborated and combined by a previously unknown
gene cluster combination. Produced by Pseudomonas putida, promysalin facilitates swarming characteristics and biofilm formation in the producing strain, but is antagonistic to other Pseudomonads including MDR clinical isolates of P. aeruginosa (IC50 = 0.16 lg/mL and minimum bactericidal activity (MBC) = 100 lg/mL against PA14). Indeed, of the 16 Pseudomonas species tested, 12 had sensitive strains, and in a broader panel of P. aeruginosa environmental and clinical isolates, all 83 were sensitive. This intriguing microbiological profile warrants further investigation, and additional work is required to elucidate the absolute stereochemistry, mechanism of action and structure activity relationships of promysalin. At the 53rd ICAAC meeting (Denver, Colorado 2013) a group of researchers from Fundacion Medina, Cubist Pharmaceuticals and the University of California, San Diego presented a poster disclosing a family of novel natural product antibiotics (MDN– 0057–60).35 Isolated from the crude fermentation extract of a filamentous fungus, these natural products possess two isonitrile functional groups, which although rarely found in natural products, are not unprecedented. Indeed, MK4588, an antibiotic natural product isolated from Leptospaeria fungus by Gomi and co-workers in 1990, would appear to be related.36 MDN-0057 exhibits potent broad spectrum Gram-negative activity with MIC ranging 0.2564 lg/mL against E. coli, Acinetobacter baumannii, P. aeruginosa and K. pneumonia clinical isolates. MIC values are significantly lower in efflux pump mutant or permeabalized strains of these organisms indicating a propensity for efflux and poor permeability for this chemotype. MDN-0057 did not inhibit S. aureus (>128 lg/ mL) or eukaryotic cell growth (HepG2 and Fa2N4 >512 lg/mL) and was inactive in the hERG K channel at 50 lM. A study of the impact of MDN-0057 on macromolecular synthesis indicated a concentration dependant inhibition of cell wall synthesis, but not RNA, DNA, protein or lipid synthesis. Additionally, cytology experiments showed that incubation of E. coli cells with MDN-0057 rendered the cells more permeable to DAPI and sytox green (dyes) thus reinforcing a cell wall target related mode of action. In summary, natural product research continues to be an important cornerstone for antibacterial drug discovery. With modern advances in selective organic synthesis, ribosome crystallography, chemical biology tools for target elucidation, and novel methods for uncovering new natural products, this area can continue to provide new medicines towards unmet patient needs.
O O
NH2
Me
O O
O
N
HO H
O
H
O
Me i-Pr
Me
Me
Me
HO
HO O
O Me
O
Me
O Me
HN
O O HN O Me
HO
Me
OH
OH
O
O
HO Me
NH
HO O
Cl
H 2N Cl
O HN
O
O Me
O
S N
O
O OH
N
O Cl
Me Me
Me
H N
HO O
O
O Me
O
Me Me
NH Cl
O HN
Cl Me
Me
13, Kibdelomycin
14, Novobiocin
15, Clorobiocin
16, AstraZeneca FBLG effort
Figure 4. Topoisomerase inhibitors from natural sources (13, 14, and 15) and synthetic preparation (16).
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R O
OH
O
N O
R O
O
O
O
Me
O HO
N
N C C
HO
N
N C
OH H
N C N
C
C
OH O
17, Promysalin
NH2
OMe
OMe
18, MDN-0057, R = OH 19, MDN-0059, R = H
OMe
20, MDN-0058, R = OH 22, MK4588 21, MDN-0060, R = H
Figure 5. New natural product space.
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