About 70% of currently used drugs are natural products or derivatives of natural products. In contrast to synthetic compounds, natural product compounds are not generated by chemical synthesis, but instead isolated from living microorganisms, such as bacteria and fungi. The cyclic peptides constitute one of the major groups of natural product compounds. Drugs such as Vancomycin, Daptomycin, Caspofungin, Cyclosporin, and Bleomycin all belong to this group and their successes in the clinic demonstrate that cyclic peptides can be antibiotics and antifungal compounds, as well as immunosuppressive and even cancer drugs.

The structure(s) of a cyclic peptide is usually narrowly defined by the producing organism. Hence, without added modifications, only a limited potential exists for the exploration of structural variations that might lead to efficacy improvements or optimization of other pharmacological properties. What is commonly referred to as structure activity relationship (SAR) studies in the development of synthetic compounds can normally not be done on a cyclic peptide. Although changes can, and have been made to cyclic peptides, the specific structure(s) and complexity of these molecules makes them unsuitable for modification by conventional synthetic chemistry approaches.

Chemical modification of cyclic peptide lead compounds: AureoGen’s research is focused on identifying and using innovative chemistry methodologies to generate novel antibiotics from existing cyclic peptide templates. Until recently, the structural modifications required to convert a native cyclic peptide into a functional drug required expensive and complicated, multi-step synthesis strategies. This hampered the development of many promising drug candidates, and also prevented optimization, such as reduction of side effects, of existing drugs. AureoGen has identified several novel chemistry methodologies that allow efficient and cost-effective modification of cyclic peptide compounds without the complex, multiple-step synthesis approaches used in the past. These chemistries allow AureoGen to generate new chemical entities (NCEs) with improved pharmacological properties from existing cyclic peptide lead compounds. AureoGen’s methodologies allow for efficient in-depth exploration of the pharmacological properties (SAR studies) of virtually any cyclic peptide.

AureoGen’s chemistry efforts are currently directed towards reducing the side effects of an antibiotic with potent efficacy against Gram-negative infections and towards improving the pharmacokinetic properties of a candidate drug for the treatment of drug-resistant Gram-positive infections, such as methicillin resistant Staphylococcus aureus (MRSA) (see Drug Development Programs).

NRPS engineering: As an alternative to chemistry, recent advances in the understanding of the biology and genetics of cyclic peptide producing organisms have produced tools and knowledge that allow introduction of a wide range of modifications to a cyclic peptide by altering the sequence of the genes encoding the non-ribosomal peptide synthase (NRPS) complexes responsible for its synthesis in the producer organism. Engineering of NRPS genes allows for the design of organisms capable of producing cyclic peptides with all the properties of a finished drug molecule, or molecules requiring only minor chemical modifications. In addition, the technology allows for efficient and cost-effective in-depth exploration (SAR) of the pharmacological properties of virtually any cyclic peptide template, as well as simple and cost effective generation of entirely novel cyclic peptides.

Chemo-enzymatic synthesis (CES): The CES technology provides another alternative approach to generating structurally modified cyclic peptides. The technology combines the use of solid-phase peptide synthesis (for the synthesis of linear precursor peptides) with a cyclization reaction catalyzed by a recombinant, NRPS gene-derived thioesterase (TE) domain. Recent research has demonstrated that TE domains, i.e. the domains responsible for the head-to-tail cyclization of the linear cyclic peptide precursors, in (bacterial) NRPS complexes can be efficiently expressed, as separate proteins with fully retained activity. Remarkably, it has also been shown that such recombinant TE domains often are promiscuous and capable of (in vitro) generation of cyclic peptides from a wide range of linear precursors, provided the amino acids at, or close to, the ends (of the precursors) are identical, or similar to those in the native substrate. Consequently, this approach allows for of a wide variety of amino acids, as well as other structural elements, to be incorporated into a cyclic peptide. It also allows for a considerable variation in the number of residues, in the linear precursor. Thus, the permissive properties of recombinant TE domain enzymes can be exploited for the generation of a wide variety of cyclic peptides and the CES approach has the potential to be used for rapid and efficient exploration of the structure-activity relationships (SARs) of a cyclic peptide without the need for more time consuming synthetic chemistry or genetic engineering efforts.

Drug Development Programs

Antifungal Program

AureoGen’s primary antifungal lead compound is the nine amino acid cyclic peptide Aureobasidin A (AbA). AbA, which is produced by the Aureobasidium pullulans strain BP-1938, is potent fungicidal compound that is very well tolerated in animals and man. AbA also has a unique mode of action (MoA)

in that it targets the enzyme inositol phoporylceramide (IPC) synthase in the fungal sphingolipid biosynthesis pathway. Although there are notable similarities between the fungal and mammalian sphingolipid biosynthesis pathways, up to, and through the phytoceramide synthesis step, the IPC synthase-catalyzed addition of inositolphosphate is a decidedly fungi-specific reaction. Mammalian cells instead conjugate phosphocholine to the 1-hydroxyl group on ceramide, to form sphingomyelin. The uniqueness of the IPC synthase-catalyzed reaction, coupled with the fact that mutational studies have demonstrated that the enzyme is essential in fungi, make IPC synthase a very attractive target for antifungal drugs.

Structure of Aureobasidin A

Although AbA is a very efficinent inhibitor of IPC synthase, the activity spectrum of the native compound is not perfect. Native AbA very active (and efficacious) against virtually all Candida species, including C. albicans. It is also active against most Cryptococcus species, as well as several other fungi. By contrast, the native compound shows little activity against most aspergilli, and most notably it is essentially inactive against A. fumigatus. Since Candida spp. and Aspergillus spp. are the two most common human pathogens, and since broad-spectrum antibiotics are preferred in the clinic, native AbA's lack of efficacy against aspergilli has (to date) hampered its development into a marketed drug. The reason for A. fumigatus' resistance to AbA is not that the IPC synthase in A. fumigatus is resistant to the compound, but rather that this organism has a pump(s) capable of efficiently clearing the drug – poisoning of the pump(s) make the organism susceptible to the compound. An AbA derivative(s) capable of avoiding or blocking the A. fumigatus pump(s) would have greatly enhanced development potential.

AureoGen has invented a novel synthetic chemistry process that allows efficient cost-effective modification of AbA. In contrast to previous approaches, AureoGen’s process does not require disassembly of the peptide backbone of the molecule and it allows generation of derivatives with as few as 2-3 synthesis steps. Using this approach, AureoGen has to date synthesized 58 novel derivatives – all NCEs and all owned by AureoGen Biosciences - several of which have more than three orders of magnitude improved activities against A. fumigatus. AureoGen has also evaluated selected derivatives in animal models of candidiasis and aspergillosis which identified several compounds with clear efficacy against both pathogens.

Although AureoGen’s synthesis efforts on AbA has produced derivatives with clear development potential, the number of compounds synthesized to date represents only a small fraction of what can be produced with AureoGen’s chemistry – there is an excellent potential to produce additional compounds with further improved properties.

AureoGen licensed the AbA project (chemistry and compounds) to Merck for continued development, on December 21, 2015.

Antibacterial Program

I. Polymyxin B (PMB1)

Drug-resistant infections by Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae and other Gram-negative pathogens have increased drastically in recent years. At the same time an increasing lack of antibiotics with true efficacy against, in particular drug-resistant strains of P. aeruginosa and A. baumannii, has become desperate enough to prompt a re-evaluation and re-introduction of drugs which for many years have been considered to toxic for

Structure of polymyxin B

clinical use. The most notable of these (re-introduced) drugs are two compounds in the polymyxin family: colistin (polymyxin E) and polymyxin B. Currently, colistin and polymyxin B are the only drugs with real efficacy against certain strains of P. aeruginosa and A. baumannii. Still, although the essentially desperate situation discussed above has forced the re-introduction of these compounds in the clinic, their toxicities are a considerable concern in the clinic and better tolerated derivatives would greatly increase both their applicability and use. A better tolerated polymyxin B (or colistin) derivative would also allow higher dose levels which would help overcome the occasionally observed low levels of resistance to these drugs. The primary toxicities associated with the polymyxins (and the primary reason for the limitations in their clinical use) are nephrotoxitiy and neurotoxicity. The nephrotoxicity is generally considered more significant and clinically relevant.

The overall aim of AureoGen’s polymyxin B project is to prepare a polymyxin B derivative with significantly reduced nephrotoxicity. The approach involves using newly invented medicinal chemistry strategies that allow for targeted modifications of specific amino acid side chains (in the polymyxin B structure) that are known to be important for the nephrotoxicity. The aim is to explore structural modifications and that will reduce the toxicity without (significantly) impacting the compound’s excellent antibacterial activity, i.e. to identify (a) polymyxin B derivative(s) that retain(s) all, or most, of the parent compound’s antibacterial activity and efficacy, but that is significantly less toxic.

II. EPP.  EPP is a cyclic lipopeptide that is produced by a soil bacterium. The compound has potent, broad-spectrum activity against most Gram-positive organisms. AureoGen has carried out a comprehensive in vitro evaluation of EPP which revealed that in addition to the wild type organisms identified in previous studies, the compound also has robust activity against 22 common, contemporary, clinically relevant resistance phenotypes, from four different pathogen groups. A complete lack of cross-resistance (in this evaluation) strongly suggests that EPP has a novel mode of action (MoA). AureoGen has also generated comprehensive in vivo data suggesting that EPP is an efficacious antibiotic capable of eradicating established localized, as well as systemic, lethal infections in mice. This investigation also revealed that although EPP’s pharmacokinetic (PK) properties (consistent with the compound’s efficacy) in mice are reasonable, the compound is cleared much faster in rats and dogs, indicating that maintaining therapeutic plasma concentrations (of native EPP) in man may be problematic. EPP also appears to be very well tolerated – intravenous (IV) LD50 in mice is 560 mg/kg and a two week toxicity evaluation in rats failed to detect any drug-related, adverse effects. However, the value of this latter investigation may, to some extent, be compromised by the rapid clearance of the drug in rats. Thus, overall, available data suggest that EPP has many of the properties required for development into a drug for the treatment of the difficult-to-treat drug-resistant infections, most notably methicillin resistant Staphylococcus aureus (MRSA) infections, encountered in the clinic today. However, the data also suggest that development of EPP into a human therapeutic will require improvement of the compound’s PK properties. The overall objective of AureoGen’s EPP project is to use state-of-the-art medicinal chemistry to generate derivatives of EPP that have improved PK properties, and, if possible, also improved antibacterial activities. Such compounds will be NCEs with all the properties required for development into clinically useful antibiotics and commercial products.

III. Bacitracin. The cyclic peptide bacitracin is a potent, bactericidal antibiotic compound isolated from B. licheniformis. The compound is quite efficacious and has a broad target spectrum. Essentially all Gram-positive human pathogens, including most multi-drug resistant organisms, including MRSA and vancomycin resistant enterococci (VRE), as well as certain Gram-negative bacteria, are susceptible to bacitracin (Hickey, 1964; O’Donovan, 1994). The compound even has efficacy towards Mycobacterium tuberculosis (e.g. Rieber et al., 1969; Storm and Strominger, 1973; Crick et al., 2000). Also notable is the fact that despite several decades of widespread use, bacterial resistance to Bacitracin is quite rare. This makes Bacitracin an ideal lead template for development of a novel antibiotic.

The bacitracin USP used in the clinic is nephrotoxic when administered systemically, and although the drug has a history of systemic use, current use is mostly limited to topical applications. It is known that the active component in bacitracin USP, bacitracin A, is not in itself nephrotoxic, but is converted into a nephrotoxic metabolite, bacitracin F, in circulation. The structural changes associated with this conversion are well understood. Consequently, structural modifications designed to block the associated reactions can be envisioned quite readily. Nonetheless, bacitracin is a complex compound with properties and functionalities that are difficult to approach, in a cost-effective manner, by conventional synthetic chemistry. To resolve this problem AureoGen is using a novel Chemo-Enzymatic Synthesis (CES) approach to identify and implement the structural modifications required to block the conversion of bacitracin A to bacitracin F and thereby eliminate the nephrotoxicity associated with systemic use of the compound, at a cost compatible with commercial development. Successful completion of AureoGen’s bacitracin project will add a potent, broad-spectrum antibiotic, with a novel MoA that is an NCE with an excellent potential for commercial development, to the currently quite small inventory of drugs with efficacy against drug-resistant infections.