Eric Marsault

Macrocycles Are Great Cycles: Applications, Opportunities, and Challenges of Synthetic Macrocycles in Drug Discovery

Macrocycles occupy a unique segment of chemical space. In the past decade, their chemical diversity expanded significantly, supported by advances in bioinformatics and synthetic methodology. As a consequence, this structural type has now been successfully tested on most biological target classes. The goal of this article is to put into perspective the current applications, opportunities, and challenges associated with synthetic macrocycles in drug discovery. Historically, macrocyclic drug candidates have originated primarily from two sources. The first, natural products, provided unique drugs such as erythromycin, rapamycin, vancomycin, cyclosporin, and epothilone. Excellent reviews are dedicated to this class and how it inspired further synthetic and medicinal chemistry efforts; thus, it will not be covered here. From a molecular evolution standpoint, the medicinal chemistry of macrocyclic natural products usually involved direct use as a therapeutic agent or functionalization of the natural product scaffold by hemisynthesis. It parallels significant advances in the total synthesis of macrocyclic natural products during the past 2 decades. The second traditional source of macrocycles stems from peptides, some of which are natural products and, hence, also belong to the first category. Macrocyclization was recognized early in peptide chemistry as an efficient way to restrict peptide conformation, reduce polarity, increase proteolytic stability, and consequently improve druggability. Chemists accessed macrocyclic peptides with different geometries (head to tail, side chain to side chain, head to side chain), including the incorporation of nonpeptidic groups. Compelling examples of macrocyclic scaffolding of peptides include the works on somatostatins, melanocortins, and integrins, among others. Macrocyclic peptides generated several drugs from synthetic or natural sources, including octreotide, cyclosporine, eptifibatide, and caspofungin. Purely peptidic, depsipeptidic, and peptoid macrocycles will also not be covered in this article; the reader is instead referred to previous reviews. It is well understood that the boundary between synthetic macrocycles and the above categories is not always clear-cut; as a result, examples presented in the following sections could occasionally belong to one of these categories. In these cases, they were selected owing to their relevance to the perspective. Macrocycles are defined herein as molecules containing at least one large ring composed of 12 or more atoms. On the basis of standard molecular descriptors, macrocycles as a class are at the outskirts of the window generally considered optimal for good PK-ADME properties using these criteria. Indeed, their molecular weights tend to be on the higher end (often in the 500-900 g 3mol -1 range), their numbers of H-bond donors and acceptors, as well as their polar surface area (PSA), tend to be on the far side of the accepted druglike spectrum. For an equal number of heavy atoms, macrocycles inherently possess a lower number of rotatable bonds than their acyclic analogues, a beneficial feature for oral bioavailability (in the following, “acyclic” will be used in the sense of “nonmacrocyclic”). As a result, macrocycles are more conformationally restricted than their acyclic analogues, which potentially can impart higher target binding and selectivity and improved oral bioavailability (in this assessment, endocyclic bonds are considered to be nonrotatable, which is only an approximation; see ref 18). For a systematic chemoinformatic analysis of biologically active macrocycles, the reader is referred to the recent review of Brandt et al. Topologically, macrocycles have the unique ability to span large surface areas while remaining conformationally restricted compared to acyclic molecules of equivalent molecular weight. This characteristic makes them especially suited for targets displaying shallow surfaces, which can prove to be quite challenging for acyclic small molecules. Medicinal chemistry relies strategically on robust synthetic methods capable of producing an acceptable chemical diversity to adequately interrogate the chemical space of a biological target. Macrocycles are often (and rightly so) perceived as difficult to synthesize and hence deterred many medicinal chemists because of the lack of versatile synthetic platforms. The macrocyclization step is regularly plagued by low yields and often requires high dilution conditions to counterbalance entropic loss. In other words, the reduction in entropy responsible for beneficial conformational restrictions to the final molecule comes at a price during synthesis: what goes around comes around. Accordingly, the first part of this article is dedicated to the drug discovery aspects ofmacrocycles and highlights salient features of their medicinal chemistry. This section is organized by target class, a choice aimed at providing the reader an appreciation of the structural diversity generated for each class. To give the reader an appreciation of the tools available to construct macrocyclic scaffolds, the site and method of the pivotal macrocyclization step are indicated in the figures. Readers are referred to the source articles for further details. In the second part, the technologies and synthetic approaches that already have demonstrated utility or possess a high potential for macrocycle-based

Elucidation of the Structure–Activity Relationships of Apelin: Influence of Unnatural Amino Acids on Binding, Signaling, and Plasma Stability

Apelin is the endogenous ligand of the APJ receptor, a member of the G‐protein‐coupled receptor family. The apelin–APJ complex has been detected in many tissues and is emerging as a promising target for several pathophysiological conditions. There is currently little information on the structure–activity relationship (SAR) of the apelin hormone. In an effort to better delineate SAR, we synthesized analogues of apelin‐13 modified at selected positions with unnatural amino acids, with a particular emphasis on the C‐terminal portion. Analogues were then tested in binding and functional assays by evaluating Gi/o‐mediated decreases in cAMP levels and by assessing β‐arrestin2 recruitment to the APJ receptor. The plasma stability of new compounds was also assessed. Several analogues were found to possess increased binding and higher stability than the parent peptide.

Matriptase Proteolytically Activates Influenza Virus and Promotes Multicycle Replication in the Human Airway Epithelium

ABSTRACT Influenza viruses do not encode any proteases and must rely on host proteases for the proteolytic activation of their surface hemagglutinin proteins in order to fuse with the infected host cells. Recent progress in the understanding of human proteases responsible for influenza virus hemagglutinin activation has led to the identification of members of the type II transmembrane serine proteases TMPRSS2 and TMPRSS4 and human airway trypsin-like protease; however, none has proved to be the sole enzyme responsible for hemagglutinin cleavage. In this study, we identify and characterize matriptase as an influenza virus-activating protease capable of supporting multicycle viral replication in the human respiratory epithelium. Using confocal microscopy, we found matriptase to colocalize with hemagglutinin at the apical surface of human epithelial cells and within endosomes, and we showed that the soluble form of the protease was able to specifically cleave hemagglutinins from H1 virus, but not from H2 and H3 viruses, in a broad pH range. We showed that small interfering RNA (siRNA) knockdown of matriptase in human bronchial epithelial cells significantly blocked influenza virus replication in these cells. Lastly, we provide a selective, slow, tight-binding inhibitor of matriptase that significantly reduces viral replication (by 1.5 log) of H1N1 influenza virus, including the 2009 pandemic virus. Our study establishes a three-pronged model for the action of matriptase: activation of incoming viruses in the extracellular space in its shed form, upon viral attachment or exit in its membrane-bound and/or shed forms at the apical surface of epithelial cells, and within endosomes by its membrane-bound form where viral fusion takes place.

Design and Synthesis of Potent, Selective Inhibitors of Matriptase

Matriptase is a member of the type II transmembrane serine protease family. Several studies have reported deregulated matriptase expression in several types of epithelial cancers, suggesting that matriptase constitutes a potential target for cancer therapy. We report herein a new series of slow, tight-binding inhibitors of matriptase, which mimic the P1-P4 substrate recognition sequence of the enzyme. Preliminary structure-activity relationships indicate that this benzothiazole-containing RQAR-peptidomimetic is a very potent inhibitor and possesses a good selectivity for matriptase versus other serine proteases. A molecular model was generated to elucidate the key contacts between inhibitor 1 and matriptase.

Conjugation of a brain-penetrant peptide with neurotensin provides antinociceptive properties

Neurotensin (NT) has emerged as an important modulator of nociceptive transmission and exerts its biological effects through interactions with 2 distinct GPCRs, NTS1 and NTS2. NT provides strong analgesia when administered directly into the brain; however, the blood-brain barrier (BBB) is a major obstacle for effective delivery of potential analgesics to the brain. To overcome this challenge, we synthesized chemical conjugates that are transported across the BBB via receptor-mediated transcytosis using the brain-penetrant peptide Angiopep-2 (An2), which targets LDL receptor-related protein-1 (LRP1). Using in situ brain perfusion in mice, we found that the compound ANG2002, a conjugate of An2 and NT, was transported at least 10 times more efficiently across the BBB than native NT. In vitro, ANG2002 bound NTS1 and NTS2 receptors and maintained NT-associated biological activity. In rats, i.v. ANG2002 induced a dose-dependent analgesia in the formalin model of persistent pain. At a dose of 0.05 mg/kg, ANG2002 effectively reversed pain behaviors induced by the development of neuropathic and bone cancer pain in animal models. The analgesic properties of ANG2002 demonstrated in this study suggest that this compound is effective for clinical management of persistent and chronic pain and establish the benefits of this technology for the development of neurotherapeutics.

Discovery and Structure–Activity Relationship of a Bioactive Fragment of ELABELA that Modulates Vascular and Cardiac Functions

ELABELA (ELA) was recently discovered as a novel endogenous ligand of the apelin receptor (APJ), a G protein-coupled receptor. ELA signaling was demonstrated to be crucial for normal heart and vasculature development during embryogenesis. We delineate here ELAs structure-activity relationships and report the identification of analogue 3 (ELA(19-32)), a fragment of ELA that binds to APJ, activates the Gαi1 and β-arrestin-2 signaling pathways, and induces receptor internalization similarly to its parent endogenous peptide. An alanine scan performed on 3 revealed that the C-terminal residues are critical for binding to APJ and signaling. Finally, using isolated-perfused hearts and in vivo hemodynamic and echocardiographic measurements, we demonstrate that ELA and 3 both reduce arterial pressure and exert positive inotropic effects on the heart. Altogether, these results present ELA and 3 as potential therapeutic options in managing cardiovascular diseases.

Macrocyclic Cell Penetrating Peptides: A Study of Structure- Penetration Properties

Arginine-rich cell penetrating peptides are short cationic peptides able to cross biological membranes despite their peptidic character. In order to optimize their penetration properties and further elucidate their mechanisms of cellular entry, these peptides have been intensively studied for the last two decades. Although several parameters are simultaneously involved in the internalization mechanism, recent studies suggest that structural modifications influence cellular internalization. Particularly, backbone rigidification, including macrocyclization, was found to enhance proteolytic stability and cellular uptake. In the present work, we describe the synthesis of macrocyclic arginine-rich cell penetrating peptides and study their cellular uptake properties using a combination of flow cytometry and confocal microscopy. By varying ring size, site of cyclization, and stereochemistry of the arginine residues, we studied their structure-uptake relationship and showed that the mode and site of cyclization as well as the stereochemistry influence cellular uptake. This study led to the identification of a hepta-arginine macrocycle as efficient as its linear nona-arginine congener to enter cells.

Experimental treatment of Staphylococcus aureus bovine intramammary infection using a guanine riboswitch ligand analog

Staphylococcus aureus is a leading cause of intramammary infections (IMI). We recently demonstrated that Staph. aureus strains express the gene guaA during bovine IMI. This gene codes for a guanosine monophosphate synthetase and its expression is regulated by a guanine riboswitch. The guanine analog 2,5,6-triaminopyrimidine-4-one (PC1) is a ligand of the guanine riboswitch. Interactions between PC1 and its target result in inhibition of guanosine monophosphate synthesis and subsequent death of the bacterium. The present study describes the investigational use of PC1 for therapy of Staph. aureus IMI in lactating cows. The in vitro minimal inhibitory concentration of PC1 ranged from 0.5 to 4 μg/mL for a variety of Staph. aureus and Staphylococcus epidermidis strains and required a reducing agent for stability and full potency. A safety assessment study was performed, whereby the healthy quarters of 4 cows were infused with increasing doses of PC1 (0, 150, 250, and 500 mg). Over the 44 h following infusions, no obvious adverse effect was observed. Ten Holstein multiparous cows in mid lactation were then experimentally infused into 3 of the quarters with approximately 50 cfu of Staph. aureus strain SHY97-3906 and infection was allowed to progress for 2 wk before starting PC1 treatment. Bacterial counts reached then about 10(3) to 10(4) cfu/mL of milk. Infected quarters were treated with 1 of 3 doses of PC1 (0, 250, or 500 mg) after each morning and evening milking for 7d (i.e., 14 intramammary infusions of PC1). During the treatment period, milk from PC1-treated quarters showed a significant reduction in bacterial concentrations. However, this reduction of Staph. aureus count in milk was not maintained during the 4 wk following the end of the treatment and only 15% of the PC1-treated quarters underwent bacteriological cure. The somatic cell count and the quarter milk production were not affected by treatments. Although bacterial clearance was not achieved following treatment with PC1, these results demonstrate that the Staph. aureus guanine riboswitch represents a relevant and promising drug target for a novel class of antibiotics for the animal food industry.

Efficient parallel synthesis of macrocyclic peptidomimetics.

A new method for solid phase parallel synthesis of chemically and conformationally diverse macrocyclic peptidomimetics is reported. A key feature of the method is access to broad chemical and conformational diversity. Synthesis and mechanistic studies on the macrocyclization step are reported.

C-Terminal modifications of apelin-13 significantly change ligand binding, receptor signaling, and hypotensive action.

Apelin is the endogenous ligand of the APJ receptor, a member of the G protein-coupled receptor family. This system plays an important role in the regulation of blood pressure and cardiovascular functions. To better understand the role of its C-terminal Phe(13) residue on ligand binding, receptor signaling, and hypotension, we report a series of modified analogues in which Phe(13) was substituted by unnatural amino acids. These modifications delivered new compounds exhibiting higher affinity and potency to inhibit cAMP accumulation compared to apelin-13. In particular, analogues Bpa(13) or (α-Me)Phe(13) were 30-fold more potent to inhibit cAMP accumulation than apelin-13. Tyr(OBn)(13) substitution led to a 60-fold improvement in binding affinity and induced stronger and more sustained drop in blood pressure compared to apelin-13. Our study identified new potent analogues of apelin-13, which represent valuable probes to better understand its structure-function relationship.