Lesley A. Howell

Queen Mary University of London

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Mark Searcey

University of East Anglia

Peter J. McCormick

Queen Mary University of London

Maria A. O’Connell

University of East Anglia

Andrew M. Beekman

University of East Anglia

Enric I. Canela

Universitat de Barcelona

Estefanía Moreno

Universitat de Barcelona

Sergi Ferré

National Institute on Drug Abuse

Vicent Casadó

Universitat de Barcelona

Joaquín Botta

William Harvey Research Institute

Carmen Lluís

Universitat de Barcelona

Cooperative Institutions

University of East Anglia

Universitat de Barcelona

Norwich Research Park

Biomedical Research Networking Center on Neurodegenerative Diseases

Centro de Investigación Biomédica en Red

Instituto de Salud Carlos III

Institut de Biomedicina de la Universitat de Barcelona

National Institute on Drug Abuse

Norwich University

National Institutes of Health

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Identification of a Cannabinoid Receptor 2 Allosteric Site Using Computational Modeling and Pharmacological Analysis

ACS Pharmacology & Translational Science (2025)

Zara Farooq Pietro Delre Stylianos Iliadis Giuseppe Felice Mangiatordi Marialessandra Contino

Emerging evidence has demonstrated that cannabinoid receptor 2 (CB2) is involved in a number of diseases, such as neurodegenerative disorders and various types of cancer, making it an attractive pharmacological target. Classically, a protein active site or an orthosteric binding site, where the endogenous ligand binds to, is used as a target for the design of most small-molecule drugs. This can present challenges when it comes to phylogenetically related proteins that have similar orthosteric binding sites, such as the cannabinoid receptors. An alternative approach is to target sites that are unique to these receptors yet still impact receptor function, known as allosteric binding sites. Using an inactive-state human cannabinoid receptor 2 crystal structure (PDB ID:5ZTY), we identified a putative CB2 allosteric site using computational approaches. In vitro signaling assays using known allosteric modulators and CB2 agonists have been used to verify the in silico results. This identification opens promising avenues for the development of selective and specific CB2 ligands for therapeutic purposes.

Identification

10.1021/acsptsci.4c00547

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Citations (0)

Solid-Phase Synthesis of Duocarmycin Analogues and the Effect of C-Terminal Substitution on Biological Activity

The Journal of Organic Chemistry (2015)

Michael J. Stephenson Lesley A. Howell Maria A. O’Connell Keith R. Fox Claire Adcock

The duocarmycins are potent antitumor agents with potential for use in the development of antibody-drug conjugates (ADCs) as well as being clinical candidates in their own right. In this article, we describe the synthesis of a duocarmycin monomer (DSA) that is suitably protected for utilization in solid-phase synthesis. The synthesis was performed on a large scale, and the resulting racemic protected Fmoc-DSA subunit was separated by supercritical fluid chromatography (SFC) into the single enantiomers; its application to solid-phase synthesis methodology gave a series of monomeric and extended duocarmycin analogues with amino acid substituents. The DNA sequence selectivity was similar to that in previous reports for both the monomeric and extended compounds. Substitution at the C-terminus of duocarmycin caused a decrease in antiproliferative activity for all of the compounds studied. An extended compound containing an alanine at the C-terminus was converted to the primary amide or to an extended structure containing a terminal tertiary amine, but this had no beneficial effects on biological activity.

Solid-Phase Synthesis

Amide

Peptide Synthesis

Alanine

10.1021/acs.joc.5b01373

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Citations (13)

Synthesis and evaluation of 9-aminoacridines derived from benzyne click chemistry

Bioorganic & Medicinal Chemistry Letters (2009)

Lesley A. Howell Aaron Howman Maria A. O’Connell Anja Müeller Mark Searcey

Aryne

Duplex (building)

10.1016/j.bmcl.2009.08.070

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Citations (23)

Design and development of stapled transmembrane peptides that disrupt the activity of G-protein–coupled receptor oligomers

Journal of Biological Chemistry (2019)

Joaquín Botta Lučka Bibič Patrick M. Killoran Peter J. McCormick Lesley A. Howell

Membrane proteins can associate into larger complexes. Examples include receptor tyrosine complexes, ion channels, transporters, and G protein–coupled receptors (GPCRs). For the latter, there is abundant evidence indicating that GPCRs assemble into complexes, through both homo- and heterodimerization. However, the tools for studying and disrupting these complexes, GPCR or otherwise, are limited. Here, we have developed stabilized interference peptides for this purpose. We have previously reported that tetrahydrocannabinol-mediated cognitive impairment arises from homo- or heterooligomerization between the GPCRs cannabinoid receptor type 1 (CB1R) and 5-hydroxytryptamine 2A (5-HT2AR) receptors. Here, to disrupt this interaction through targeting CB1–5-HT2A receptor heteromers in HEK293 cells and using an array of biochemical techniques, including calcium and cAMP measurements, bimolecular fluorescence complementation assays, and CD-based helicity assessments, we developed a NanoLuc binary technology (NanoBiT)-based reporter assay to screen a small library of aryl-carbon–stapled transmembrane-mimicking peptides produced by solid-phase peptide synthesis. We found that these stapling peptides have increased α-helicity and improved proteolytic resistance without any loss of disrupting activity in vitro, suggesting that this approach may also have utility in vivo. In summary, our results provide proof of concept for using NanoBiT to study membrane protein complexes and for stabilizing disrupting peptides to target such membrane complexes through hydrocarbon-mediated stapling. We propose that these peptides could be developed to target previously undruggable GPCR heteromers. Membrane proteins can associate into larger complexes. Examples include receptor tyrosine complexes, ion channels, transporters, and G protein–coupled receptors (GPCRs). For the latter, there is abundant evidence indicating that GPCRs assemble into complexes, through both homo- and heterodimerization. However, the tools for studying and disrupting these complexes, GPCR or otherwise, are limited. Here, we have developed stabilized interference peptides for this purpose. We have previously reported that tetrahydrocannabinol-mediated cognitive impairment arises from homo- or heterooligomerization between the GPCRs cannabinoid receptor type 1 (CB1R) and 5-hydroxytryptamine 2A (5-HT2AR) receptors. Here, to disrupt this interaction through targeting CB1–5-HT2A receptor heteromers in HEK293 cells and using an array of biochemical techniques, including calcium and cAMP measurements, bimolecular fluorescence complementation assays, and CD-based helicity assessments, we developed a NanoLuc binary technology (NanoBiT)-based reporter assay to screen a small library of aryl-carbon–stapled transmembrane-mimicking peptides produced by solid-phase peptide synthesis. We found that these stapling peptides have increased α-helicity and improved proteolytic resistance without any loss of disrupting activity in vitro, suggesting that this approach may also have utility in vivo. In summary, our results provide proof of concept for using NanoBiT to study membrane protein complexes and for stabilizing disrupting peptides to target such membrane complexes through hydrocarbon-mediated stapling. We propose that these peptides could be developed to target previously undruggable GPCR heteromers.

10.1074/jbc.ra119.009160

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Citations (27)

Targeting Higher‐Order DNA: Beyond the G‐Quadruplex

ChemBioChem (2009)

Lesley A. Howell Mark Searcey

There must be more to DNA than this: The G-quadruplex is a higher-order DNA structure that is of interest as a potential therapeutic target. However it is not the only structure to bind small-molecule ligands, and in this minireview we describe recent studies of molecules that target three-way and four-way junctions.

G-quadruplex

10.1002/cbic.200900243

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Citations (18)

A small molecule that induces assembly of a four way DNA junction at low temperature

Chemical Communications (2011)

Lesley A. Howell Zoë A. E. Waller Richard P. Bowater Maria A. O’Connell Mark Searcey

Small molecules that induce the formation of higher order DNA structures have potential therapeutic and nanotechnology applications. Screening of a click library has identified the first compound to induce the formation of a Holliday junction structure at room temperature without the need for a high temperature annealing step.

10.1039/c1cc12922h

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Citations (34)

Small‐Molecule and Peptide Inhibitors of the Pro‐Survival Protein Mcl‐1

ChemMedChem (2015)

Andrew M. Beekman Lesley A. Howell

Abstract The ability of protein–protein interactions to regulate cellular processes in both beneficial and detrimental ways has made them obvious drug targets. The Bcl‐2 family of proteins undergo a series of protein–protein interactions which regulate the intrinsic cell‐death pathway. The pro‐survival members of the Bcl‐2 family, including Bcl‐2, Bcl‐x L , and Mcl‐1, are commonly overexpressed in a number of human cancers. Effective modulators of members of the Bcl‐2 family have been developed and are undergoing clinical trials, but the efficient modulation of Mcl‐1 is still not represented in the clinic. In addition, Mcl‐1 is a major cause of resistance to radio‐ and chemotherapies, including inhibitors that target other Bcl‐2 family members. Subsequently, the inhibition of Mcl‐1 has become of significant interest to the scientific community. This review covers the progress made to date in modulating the activity of Mcl‐1, by both stapled peptides and small molecules. The development of peptides as drug candidates, and the advancement of experimental and computational techniques used to discover small molecules are also highlighted.

Protein family

Bcl-2 Family

Cell Survival

10.1002/cmdc.201500497

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Citations (41)

Author response: Modulation of dopamine D1 receptors via histamine H3 receptors is a novel therapeutic target for Huntington's disease

David Moreno‐Delgado Mar Puigdellívol Estefanía Moreno Mar Rodríguez-Ruiz Joaquín Botta

Modulation (music)

10.7554/elife.51093.sa2

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Citations (1)

In Silico Peptide-Directed Ligand Design Complements Experimental Peptide-Directed Binding for Protein-Protein Interaction Modulator Discovery

Lesley A. Howell Andrew M. Beekman

Using the protein-protein interaction of Mcl-1/Noxa, two methods for efficient modulator discovery are directly compared. In silico peptide-directed ligand design is evaluated against experimental peptide-directed, allowing for the discovery of two new inhibitors of Mcl-1/Noxa with cellular activity. In silico peptide-directed ligand design demonstrates an in vitro hit rate of 80%. The two rapid and efficient methods demonstrate complementary features for protein-protein interaction modulator discovery.

10.26434/chemrxiv.12525503.v1

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Orexin–Corticotropin-Releasing Factor Receptor Heteromers in the Ventral Tegmental Area as Targets for Cocaine

Journal of Neuroscience (2015)

Gemma Navarro César Quiroz David Moreno‐Delgado Adam Sierakowiak Kimberly A. McDowell

Release of the neuropeptides corticotropin-releasing factor (CRF) and orexin-A in the ventral tegmental area (VTA) play an important role in stress-induced cocaine-seeking behavior. We provide evidence for pharmacologically significant interactions between CRF and orexin-A that depend on oligomerization of CRF 1 receptor (CRF 1 R) and orexin OX 1 receptors (OX 1 R). CRF 1 R–OX 1 R heteromers are the conduits of a negative crosstalk between orexin-A and CRF as demonstrated in transfected cells and rat VTA, in which they significantly modulate dendritic dopamine release. The cocaine target σ 1 receptor (σ 1 R) also associates with the CRF 1 R–OX 1 R heteromer. Cocaine binding to the σ 1 R–CRF 1 R–OX 1 R complex promotes a long-term disruption of the orexin-A–CRF negative crosstalk. Through this mechanism, cocaine sensitizes VTA cells to the excitatory effects of both CRF and orexin-A, thus providing a mechanism by which stress induces cocaine seeking.

Orexin receptor

Orexin-A

Crosstalk

10.1523/jneurosci.4364-14.2015

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Citations (69)