Martin J Scanlon

• PhD (University of Nottingham)
• Professor (Associate) at Monash University (Australia)

Fatty acid-binding protein 4 (FABP4) is a key lipid binding protein expressed in microglia, which has been demonstrated to play a critical role in microglial-mediated neuroinflammation, a component of many neurodegenerative diseases. Compounds able to inhibit the function of FABP4 have shown promise in reducing microglial-mediated neuroinflammation...

Disulfide bond protein A (DsbA) plays a pivotal role in catalysing the formation of disulfide bonds within the periplasm of most Gram-negative bacteria. As this process is required for the folding of multiple virulence-associated proteins, inhibitors of DsbA have the potential to be developed as novel anti-virulence agents. Despite extensive effort...

53BP1 is a DNA damage response protein recruited to sites of double strand breaks through recognition of dimethylated lysine on histone 4 by its tandem Tudor domains. Like 53BP1, BRCA-1 plays a role in the regulation of DNA repair pathways, and BRCA-1 mutations have been strongly linked to breast and ovarian cancer. Interestingly, mice null for 53B...

Covalent modification of protein targets has application in both protein activity profiling and in drug discovery. Covalent warheads typically contain an electrophile that selectively reacts with nucleophilic residues in a protein target, such as cysteine, serine and threonine. Expanding this to other amino acids is an emerging strategy in covalent...

Non-coding RNAs account for up to 98 % of the human transcriptome. It has become increasingly clear that non-coding RNAs play diverse and critical roles in many important cellular functions. Although modulation of non-coding RNAs using small molecules is a promising therapeutic strategy, there are relatively few well-characterised RNA-ligand struct...

The development of low-affinity fragment hits into higher-affinity leads is a major hurdle in fragment-based drug design. Here, we demonstrate the Rapid Elaboration of Fragments into Leads (REFiL) by applying an integrated workflow that provides a systematic approach to generate higher-affinity binders without the need for structural information. T...

Fragment-based drug design relies heavily on structural information for the elaboration and optimisation of hits. The ability to identify neighbouring binding hot spots, energetically favourable interactions and conserved binding motifs in protein structures through X-ray crystallography can inform the evolution of fragments into lead-like compound...

Structures of protein–ligand complexes provide critical information for drug design. Most protein–ligand complex structures are determined using X-ray crystallography, but where crystallography is not able to generate a structure for a complex, NMR is often the best alternative. However, the available tools to enable rapid and robust structure dete...

Objectives Neisseria gonorrhoeae is an exclusively human pathogen that commonly infects the urogenital tract resulting in gonorrhoea. Empirical treatment of gonorrhoea with antibiotics has led to multidrug resistance and the need for new therapeutics. Inactivation of lipooligosaccharide phosphoethanolamine transferase A (EptA), which attaches phosp...

Disulfide bond protein A (DsbA) is an oxidoreductase enzyme that catalyzes the formation of disulfide bonds in Gram-negative bacteria. In Escherichia coli, DsbA (EcDsbA) is essential for bacterial virulence, thus inhibitors have the potential to act as antivirulence agents. A fragment-based screen was conducted against EcDsbA and herein we describe...

Disulfide-bond-forming proteins (Dsbs) play a crucial role in the pathogenicity of many Gram-negative bacteria. Disulfide-bond-forming protein A (DsbA) catalyzes the formation of the disulfide bonds necessary for the activity and stability of multiple substrate proteins, including many virulence factors. Hence, DsbA is an attractive target for the...

DsbA enzymes catalyze oxidative folding of proteins that are secreted into the periplasm of Gram-negative bacteria, and they are indispensable for the virulence of human pathogens such as Vibrio cholerae and Escherichia coli. Therefore, targeting DsbA represents an attractive approach to control bacterial virulence. X-ray crystal structures reveal...

DsbA enzymes catalyze oxidative folding of proteins that are secreted into the periplasm of Gram-negative bacteria, and they are indispensable for the virulence of human pathogens such as Vibrio cholerae and Escherichia coli. Therefore, targeting DsbA represents an attractive approach to control bacterial virulence. X-ray crystal structures reveal...

Bacterial thiol-disulfide oxidoreductase DsbA is essential for bacterial virulence factor assembly and has been identified as a viable antivirulence target. Herein, we report a structure-based elaboration of a benzofuran hit that bound to the active site groove of Escherichia coli DsbA. Substituted phenyl groups were installed at the 5- and 6-posit...

Glucocorticoids are steroid hormones that are essential for life in mammals. Therapeutically, they are some of the most cost-effective drugs for the treatment of inflammatory diseases ranging from skin rashes to COVID-19, but their use is limited by adverse effects. Glucocorticoids exert their effects via the glucocorticoid receptor, a type I nucle...

The coronaviral non-structural protein 9 (Nsp9) is essential for viral replication; it is the primary substrate of Nsp12’s pseudokinase domain within the viral replication transcription complex, an association that also recruits other components during different stages of RNA reproduction. In the unmodified state, Nsp9 forms an obligate homodimer v...

D i S ulfide B ond forming proteins (DSB) play a crucial role in the pathogenicity of many Gram-negative bacteria. Disulfide bond protein A (DsbA) catalyzes the formation of disulfide bonds necessary for the activity and stability of multiple substrate proteins, including many virulence factors. Hence, DsbA is an attractive target for the developme...

The presence of suitable cavities or pockets on protein structures is a general criterion for a therapeutic target protein to be classified as ‘druggable’. Many disease-related proteins that function solely through protein–protein interactions lack such pockets, making development of inhibitors by traditional small-molecule structure-based design m...

A bottleneck in fragment-based lead development is the lack of systematic approaches to elaborate the initial fragment hits, which usually bind with low affinity to their target. Herein we describe analysis using X-ray crystallography of a diverse library of compounds prepared using microscale parallel synthesis. This approach yielded an 8-fold inc...

The development of low-affinity fragment hits into higher affinity leads is a major hurdle in fragment-based drug design. Here we demonstrate an approach for the Rapid Elaboration of Fragments into Leads (REFiL) applying an integrated workflow that provides a systematic approach to generate higher-affinity binders without the need for structural in...

A fragment-based drug discovery approach was taken to target the thiol-disulfide oxidoreductase enzyme DsbA from Escherichia coli (EcDsbA). This enzyme is critical for the correct folding of virulence factors in many pathogenic Gram-negative bacteria, and small molecule inhibitors can potentially be developed as anti-virulence compounds. Biophysica...

Apical membrane antigen 1 (AMA1) is essential for the invasion of host cells by malaria parasites. Several small‐molecule ligands have been shown to bind to a conserved hydrophobic cleft in Plasmodium falciparum AMA1. However, a lack of detailed structural information on the binding pose of these molecules has hindered their further optimisation as...

Peroxisome proliferator-activated receptor α (PPARα) is a transcriptional regulator of lipid metabolism. GW7647 is a potent PPARα agonist that must reach the nucleus to activate this receptor. In cells expressing human fatty acid–binding protein 1 (FABP1), GW7647 treatment increases FABP1’s nuclear localization, and potentiates GW7647-mediated PPAR...

Cyclic d / l peptides (CPs) assemble spontaneously via backbone H‐bonding to form extended nanostructures. These modular materials have great potential as versatile bionanomaterials. However, the useful development of CP nanomaterials requires practical methods to direct and control their assembly. In this work, we present novel, heterogeneous, cov...

Cyclic D/L peptides (CPs) assemble spontaneously through backbone hydrogen bonding to form extended nanostructures. These modular materials have great potential as versatile bionanomaterials. However, the useful development of CP nanomaterials requires practical methods to direct and control their assembly. In this work, we present novel, heterogen...

The worldwide incidence of neisserial infections, particularly gonococcal infections, is increasingly associated with antibiotic resistant strains. In particular, extensively drug-resistant Neisseria gonorrhoeae strains that are resistant to third-generation cephalosporins are a major public health concern. There is a pressing clinical need to iden...

Significance Most antibiotics do not interfere with viral infections. Rifampicin is a notable exception, as it inhibits several poxviruses, including the causative agent of smallpox. However, the inhibition of viral assembly is unrelated to the antibacterial activity of rifampicin against microbial RNA polymerases. Here, we reveal how the antibioti...

Fragment-based drug design (FBDD) has become firmly established as a viable approach to the identification of starting points for the development of potent and selective compounds that modulate protein activity. As of 2017, the United States Food and Drug Administration have approved two molecules derived from FBDD for therapeutic use, many more ar...

The p75 splice variant of lens epithelium‐derived growth factor (LEDGF) is a 75 kDa protein that is recruited by the human immunodeficiency virus (HIV) to tether the pre‐integration complex to the host chromatin and promote integration of proviral DNA into the host genome. We have designed a series of small cyclic peptides that are structural mimic...

The p75 splice variant of lens epithelium‐derived growth factor (LEDGF) is a 75 kDa protein that is recruited by the human immunodeficiency virus (HIV) to tether the pre‐integration complex to the host chromatin and promote integration of proviral DNA into the host genome. We have designed a series of small cyclic peptides that are structural mimic...

The cytoplasmic trafficking of docosahexaenoic acid (DHA), a cognitively‐beneficial fatty acid, across the blood‐brain barrier (BBB) is governed by fatty acid‐binding protein 5 (FABP5). Lower levels of brain DHA have been observed in Alzheimer's disease (AD), which is associated with diminished BBB expression of FABP5. Therefore, upregulating FABP5...

In bacteria, the DnaG primase is responsible for synthesis of short RNA primers used to initiate chain extension by replicative DNA polymerase(s) during chromosomal replication. Among the proteins with which Escherichia coli DnaG interacts is the single-stranded DNA-binding protein, SSB. The C-terminal hexapeptide motif of SSB (DDDIPF; SSB-Ct) is h...

Herein we describe a method for the design, purchase, and assembly of a fragment-screening library from a list of commercially available compounds. The computational tools used in assessment of compound properties as well as the workflow for compound selection are provided for reference as implemented in commercially available software that is free...

At a time when the antibiotic drug discovery pipeline has stalled, antibiotic resistance is accelerating with catastrophic implications for our ability to treat bacterial infections. Globally we face the prospect of a future when common infections can once again kill. Anti-virulence approaches that target the capacity of the bacterium to cause dise...

SPRY domain-containing SOCS box proteins SPSB1, 2, and 4 mediate the proteasomal degradation of inducible nitric oxide synthase (iNOS) and thereby modulate the amount of NO available for combating infectious organisms. A highly conserved Asp-Ile-Asn-Asn-Asn (DINNN) motif found at the N-terminus of iNOS binds to SPSB2 with nanomolar affinity. The de...

Significance statement: Genetic deletion of fatty acid-binding protein 5 (FABP5) in mice reduces uptake of exogenous docosahexaenoic acid (DHA) into brain endothelial cells and brain capillaries and reduces brain parenchymal levels of endogenous DHA. Therefore, FABP5 in the brain endothelial cell is a crucial contributor to the brain levels of DHA...

We describe a general approach to determine the binding pose of small molecules in weakly bound protein-ligand complexes by deriving distance constraints between the ligand and methyl groups from all methyl-containing residues of the protein. We demonstrate that using a single sample, which can be prepared without the use of expensive precursors, i...

SPSB2 knock-out mice have been found to exhibit prolonged expression of iNOS in macrophage cells and enhanced killing of persistent pathogens, suggesting that inhibitors of SPSB2-iNOS interaction have potential as novel anti-infectives. In this study, we describe the design, synthesis and characterization of cyclic peptidomimetic inhibitors of the...

CFA/I pili are representatives of a large family of related pili that mediate adherence of enterotoxigenic Escherichia coli to intestinal epithelial cells. They are assembled via the alternate chaperone-usher pathway and consist of two subunits, CfaB, which comprises the pilus shaft and a single pilus-tip associated subunit, CfaE. The current model...

SPSB2 mediates the proteasomal degradation of iNOS. Inhibitors of SPSB2-iNOS interaction are expected to prolong iNO1S lifetime and thereby enhance killing of persistent pathogens. Here, we describe the synthesis and characterization of two redox-stable cyclized peptides containing the DINNN motif required for SPSB2 binding. Both analogues bind wit...

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) plays an important role in the invasion by merozoites of human red blood cells during a malaria infection. A key region of PfAMA1 is a conserved hydrophobic cleft formed by 12 hydrophobic residues. As anti-apical membrane antigen 1 antibodies and other inhibitory molecules that target this hy...

Pseudomonas aeruginosa is an opportunistic human pathogen for which new antimicrobial drug options are urgently sought. P. aeruginosa disulfide-bond protein A1 (PaDsbA1) plays a pivotal role in catalyzing the oxidative folding of multiple virulence proteins and as such holds great promise as a drug target. As part of a fragment-based lead discovery...

The brain has a limited ability to synthesize the essential polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) from its omega-3 fatty acid precursors. Therefore, in order to maintain brain concentrations of this PUFA at physiological levels, plasma-derived DHA must be transported across the blood-brain barrier (BBB). While DHA is able to...

Proteolysis has a critical role in transmitting information within a biological system and therefore an important element of biology is to determine the subset of proteins amenable to proteolysis. Until recently, it has been thought that proteases cleave native protein substrates only within solvent exposed loops, but recent evidence indicates that...

Inhibitors of the mitochondrial branched chain aminotransferase may have therapeutic potential in the treatment of diet-induced obesity and dyslipidemia. To explore the pharmacology of this metabolic pathway requires a potent and selective molecule that is well tolerated and has appropriate pharmacokinetic properties. The combination of fragment-ba...

Shigella flexneri secretes an enterotoxic, SPATE family autotransporter (AT), SigA, which has cytopathic activity towards cultured epithelial cells. Its cytopathic activity is due to its ability to degrade the cytoskeletal protein, α-fodrin. The mechanisms by which AT toxins target cells and tissues differ and the details of how SigA acts are not k...

To examine the expression of fatty acid binding proteins (FABPs) at the human blood-brain barrier (BBB) and to assess their ability to bind lipophilic drugs. mRNA and protein expression of FABP subtypes in immortalized human brain endothelial (hCMEC/D3) cells were examined by RT-qPCR and Western blot, respectively. FABPs that were found in hCMEC/D3...

The biogenesis of membranes from constituent proteins and lipids is a fundamental aspect of cell biology. In the case of proteins assembled into bacterial outer membranes, an overarching question concerns how the energy required for protein insertion and folding is accessed at this remote location of the cell. The translocation and assembly module...

The use of the click reaction for the introduction of conjugate groups, such as affinity or fluorescent labels, to a peptide for the study of peptide biochemistry and pharmacology is widespread. However, the nature and location of substituted 1,2,3-triazoles in peptide sequences may markedly affect conformation or binding as compared with native se...

Nuclear hormone receptors (NHRs) regulate the expression of proteins that control aspects of reproduction, development and metabolism, and are major therapeutic targets. However, NHRs are ubiquitous and participate in multiple physiological processes. Drugs that act at NHRs are therefore commonly restricted by toxicity, often at non-target organs....

The transfer of antibiotic resistance between bacteria is mediated by mobile genetic elements such as plasmids and transposons. TnpX is a member of the large serine recombinase subgroup of site-specific recombinases and is responsible for the excision and insertion of mobile genetic elements that encode chloramphenicol resistance in the pathogens C...

The DsbA:DsbB redox machinery catalyzes disulfide bond formation in secreted proteins and is required for bacterial virulence factor assembly. Both enzymes have been identified as targets for antivirulence drugs. Here we report synthetic analogues of ubiquinone (dimedone derivatives) that inhibit disulfide bond formation (IC50 ~1 μM) catalysed by E...

We have identified a class of molecules, known as 2-aminothiazoles (2-ATs), as frequent-hitting fragments in biophysical binding assays. This was exemplified by 4-phenylthiazol-2-amine being identified as a hit in 14/14 screens against a diverse range of protein targets, suggesting that this scaffold is a poor starting point for fragment-based drug...

http://onlinelibrary.wiley.com/doi/10.1002/anie.201410341/full Adams, L. A.‡, Sharma, P‡., Mohanty, B., Ilyichova, O. V., Mulcair, M. D., Williams, M. L., Gleeson, E. C., Totsika, M., Doak, B. C., Caria, S., Rimmer, K., Horne, J., Shouldice, S. R., Vazirani, M., Headey, S. J., Plumb, B. R., Martin, J. L., Heras, B., Simpson, J. S. and Scanlon, M....

Apical membrane antigen 1 (AMA1) interacts with RON2 to form a protein complex that plays a key role in the invasion of host cells by malaria parasites. Blocking this protein-protein interaction represents a potential route to control malaria and related parasitic diseases, but the polymorphic nature of AMA1 has proven to be a major challenge for v...

Apical membrane antigen 1 (AMA1) of the human malaria parasite Plasmodium falciparum has been implicated in invasion of the host erythrocyte. It interacts with malarial rhoptry neck (RON) proteins in the moving junction that forms between the host cell and the invading parasite. Agents that block this interaction inhibit invasion and may serve as p...

Significance The four-helix bundle (4HB) domain of Mixed Lineage Kinase Domain-Like (MLKL) bears two clusters of residues that are required for cell death by necroptosis. Mutations within a cluster centered on the α4 helix of the 4HB domain of MLKL prevented its membrane translocation, oligomerization, and ability to induce necroptosis. This cluste...

The decoration of the lipid A headgroups of the lipooligosaccharide (LOS) by the LOS phosphoethanolamine (PEA) transferase (LptA) in Neisseria spp. is central for resistance to polymyxin. The structure of the globular domain of LptA shows that the protein has five disulphide bonds, indicating that it is a potential substrate of the protein oxidatio...

The aqueous cytoplasm of cells poses a potentially significant barrier for many lipophilic drugs to reach their sites of action. Fatty acid binding proteins (FABPs) bind to poorly water-soluble fatty acids (FAs) and lipophilic compounds and facilitate their intracellular transport. Several structures of FA in complex with FABPs have been described,...

We established an efficient means of probing ligand-induced conformational change in the malaria drug target AMA1 using 19F NMR. AMA1 was labeled with 5-fluorotryptophan (5F-Trp) and the resulting 5F-Trp resonances were assigned by mutagenesis of the native Trp residues. By introducing additional Trp residues at strategic sites within a ligand-resp...

We have determined that a previously reported class of pyrrolo[2,3-d]pyrimidine-4-amines exhibit low-binding to apical membrane antigen 1 (AMA1) and suffer from unattractive qualities, such as aggregation. We attempted to remove these traits by generating molecules with improved solubility, however this did not translate into enhanced binding affin...

SPRY domain-containing SOCS box protein 2 (SPSB2) regulates inducible nitric oxide synthase (iNOS) by targeting it for proteasomal degradation. Inhibiting this interaction prolongs the intracellular lifetime of iNOS, leading in turn to enhanced killing of infectious pathogens such as bacteria and parasites. SPSB2 recognizes a linear motif (DINNN) i...

New antibacterials need new approaches to overcome the problem of rapid antibiotic resistance. Here we review the development of potential new antibacterial drugs that do not kill bacteria or inhibit their growth, but combat disease instead by targeting bacterial virulence.

In mammals, the αβT cell receptor (TCR) signaling complex is composed of a TCRαβ heterodimer that is noncovalently coupled to three dimeric signaling molecules, CD3ϵδ, CD3ϵγ, and CD3ζζ. The nature of the TCR signaling complex and subunit arrangement in different species remains unclear however. Here we present a structural and biochemical analysis...

Bacterial DsbA enzymes catalyze oxidative folding of virulence factors, and have been identified as targets for antivirulence drugs. However, DsbA enzymes characterized to date exhibit a wide spectrum of redox properties and divergent structural features compared to the prototypical DsbA enzyme of Escherichia coli DsbA (EcDsbA). Nonetheless, sequen...

We have determined the structure of the human integrin α1I domain bound to a triple-helical collagen peptide. The structure of the α1I-peptide complex was investigated using data from NMR, small angle x-ray scattering, and size exclusion chromatography that were used to generate and validate a model of the complex using the data-driven docking prog...

The design of a suitable library is an essential prerequisite to establish a fragment-based screening capability. Several pharmaceutical companies have described their approaches to establishing fragment libraries; however there are few detailed reports of both design and analysis of performance for a fragment library maintained in an academic sett...

The three pillars of rational drug design from a fragment library are an efficient screen, a robust assay, and atomic-resolution structures of the protein–ligand complexes. However, not all targets are amenable to structure determination by X-ray crystallography or NMR spectroscopy. In particular, targets involved in diseases of protein misfolding...

Protein thermal shift is a relatively rapid and inexpensive technique for the identification of low molecular weight compound interactions with protein targets. An increase in the melting temperature of the target protein in the presence of a test ligand is indicative of a promising ligand–protein interaction. Due to its simplicity, protein thermal...

Aggregation of small organic compounds is a problem encountered in a variety of assay screening formats where it often results in detection of false positives. A saturation transfer difference-NMR-detected screen of a commercially available fragment library, followed by biochemical assay, identified several inhibitors of the enzyme ketopantoate red...

Apical membrane antigen 1 (AMA1) is an essential component of the moving junction complex used by Plasmodium falciparum to invade human red blood cells. AMA1 has a conserved hydrophobic cleft that is the site of key interactions with the rhoptry neck protein complex. Our goal is to develop small molecule inhibitors of AMA1 with broad strain specifi...

The RNA-binding protein TIAR is an mRNA-binding protein that acts as a translational repressor, particularly important under conditions of cellular stress. It binds to target mRNA and DNA via its RNA recognition motif (RRM) domains and is involved in both splicing regulation and translational repression via the formation of "stress granules." TIAR...

The α1β1 integrin receptor binds to its main extracellular ligand, collagen, through an inserted domain in its α-subunit called the αI domain (αI). αI contains a metal binding site that allows collagen to coordinate to the domain through a divalent metal ion. Here we report the backbone assignments of the apo and Mg2+ bound state of the isolated hu...

Apical membrane antigen 1 (AMA1) is an essential component of the moving junction complex used by Plasmodium falciparum to invade human red blood cells. AMA1 has a conserved hydrophobic cleft that is the site of key interactions with the rhoptry neck protein complex. Our goal is to develop small molecule inhibitors of AMA1 with broad strain specifi...

Fragment screening is becoming widely accepted as a technique to identify hit compounds for the development of novel lead compounds. In neighboring laboratories, we have recently, and independently, performed a fragment screening campaign on the HIV-1 integrase core domain (IN) using similar commercially purchased fragment libraries. The two campai...

We recently reported that dense gas processing of the protein ovalbumin (OVA) resulted in the formation of particles that were insoluble in water and which retained their immunogenicity in vivo. In the present study, the colloidal properties of these pure protein particles were investigated to in part inform rational formulation approaches. The col...

The enzyme TcpG is a periplasmic protein produced by the Gram-negative pathogen Vibrio cholerae. TcpG is essential for the production of ToxR-regulated proteins, including virulence-factor pilus proteins and cholera toxin, and is therefore a target for the development of a new class of anti-virulence drugs. Here, the 1.2 Å resolution crystal struct...

Several poorly water-soluble drugs have previously been shown to bind to intestinal (I-FABP) and liver fatty acid binding protein (L-FABP) in vitro. The purpose of this study was to examine the potential role of drug binding to FABPs on intestinal permeability and gut wall metabolism in vivo. The intestinal permeability of ibuprofen, progesterone a...

This study demonstrates a critical role for N-methylation in cyclosporin biosynthesis and maintenance of the biologically active cyclosporin conformation. The structural requirements for the AdoMet binding to CySyn were defined. N-methylation of specific amide positions in the cyclosporin backbone is critical for the complete assembly and cyclizati...

Fragment-based screening has been used to identify a novel ligand binding site on HIV-1 integrase. Crystal structures of fragments bound at this site (shown) have been used to design elaborated second-generation compounds that bind with higher affinity and good ligand efficiency.

Bis(1,2,3-triazole)s have attracted recent interest as coordinating ligands for transition metals. Here we report a rapid, modular method for the synthesis of 1,1'-disubstituted-4,4'-linked unsymmetrical bis(1,2,3-triazole)s. The method employs sequential copper catalyzed azide-alkyne cycloaddition and deprotection steps on a monosilylbutadiyne. TM...

Fatty-acid binding proteins (FABPs) are abundantly expressed proteins that bind a range of lipophilic molecules. They have been implicated in the import and intracellular distribution of their ligands and have been linked with metabolic and inflammatory responses in the cells in which they are expressed. Despite their high sequence identity, human...

Small heat-shock proteins (sHsps) are molecular chaperones that play an important protective role against cellular protein misfolding by interacting with partially unfolded proteins on their off-folding pathway, preventing their aggregation. Polyglutamine (polyQ) repeat expansion leads to the formation of fibrillar protein aggregates and neuronal c...