Emergence of small molecule non-RGD-mimetic inhibitors for RGD integrins

The RGD integrins are recognized therapeutic targets for thrombosis, fibrosis, and cancer, amongst others. Current inhibitors are designed to mimic the tripeptide sequence (arginineglycine-aspartic acid) of the natural ligands; however, the RGD-mimetic antagonists for αIIbβ3 have been shown to cause partial agonism, leading to the opposite pharmacological effect. The challenge of obtaining oral activity and synthetic tractability with RGD-mimetic molecules, along with the issues relating to pharmacology, has left integrin-therapeutics in need of a new strategy. Recently, a new generation of inhibitor has emerged that lacks the RGD-mimetic. This 2 perspective will discuss the discovery of these non-RGD-mimetic inhibitors, and the progress that has been made in this promising new chemotype

The development of small molecule inhibitors for fibrosis drug discovery

PDF of supporting information as well as thesis. Previously restricted to Strathclyde users from 1 May 2017 until 1 May 2022Fibrotic diseases can be attributed to approximately 45% of deaths within western developed countries. This category of disease can affect nearly every tissue in the body, predominantly liver, kidney, and lung. The severity of fibrotic diseases is widely recognised but currently there is no accepted effective disease modifying treatment. There have been a number of potential drug targets identified in recent years, including the enzyme autotaxin (ATX) and the RGD integrins, which are known to play a key role in the pathogenesis. In collaboration with GlaxoSmithKline, the projects detailed in this report were aimed to develop small molecule inhibitors with drug like physicochemical properties for fibrosis drugdiscovery.;Chapter 1 focusses on the secreted enzyme ATX, which is responsible for the hydrolysis of lysophosphatidylcholine (LPC) to the bioactive lysophosphatidic acid (LPA) and choline. The ATX-LPA signalling pathway is implicated in cell survival, migration, and proliferation; thus, the inhibition of ATX is a recognised therapeutic target for a number of diseases including fibrotic diseases, cancer, and inflammation, amongst others. Many of the developed synthetic inhibitors for ATX have resembled the lipid chemotype of the native ligand; however, a small number of inhibitors have been described that deviate from this common scaffold. Herein, Chapter 1 details the structure-activity relationship (SAR) exploration of a previously reported small molecule ATX inhibitor through the design, synthesis, and biological evaluation of aseries of analogues.;Furthermore, using enzyme kinetics studies it is shown that analogues of this chemotype are noncompetitive inhibitors, and using a crystal structure with ATX the discrete binding mode was confirmed. This work has provided valuable insight into the binding of this chemotype, which could aid the design of novel ATX inhibitors with non-lipid-like scaffolds.;Chapter 2 describes a lead-optimisation project targeting the RGD subfamily of the integrin receptors. The RGD integrins are recognised therapeutic targets for thrombosis, fibrosis, and cancer, amongst others. Current inhibitors are designed to mimic the tripeptide sequence of the natural ligands (arginine-glycine-aspartic acid); however, the RGD-mimetic antagonists for one particular RGD integrin (αIIbβ3) have been shown to cause partial agonism, leading to the opposite pharmacological effect.The challenge of obtaining oral activity and synthetic tractability with RGD-mimetic molecules, along with the issues relating to pharmacology, has left integrintherapeutics in need of a new strategy. Recently, a new generation of inhibitor has emerged that lacks the RGD-mimetic.;The work detailed herein aimed to build on this emerging area, with the design, synthesis, and biological evaluation of novel small molecule inhibitors targeting the αvβ3 integrin. These compounds are shown to be accessed via synthetically divergent routes, allowing for the quick exploration of adiverse set of potential lead compounds. Initial efforts led to the identification offour promising lead-like inhibitors with pIC50 values ranging from 4.1-5.5 for the target integrin αvβ3. Unfortunately, the initial hit compound, that the subsequent compound design stemmed from, was later determined to be a false positive, and as a result work on the project ceased. Thus, Chapter 2 details a project that was misled due to false positive assay results.Fibrotic diseases can be attributed to approximately 45% of deaths within western developed countries. This category of disease can affect nearly every tissue in the body, predominantly liver, kidney, and lung. The severity of fibrotic diseases is widely recognised but currently there is no accepted effective disease modifying treatment. There have been a number of potential drug targets identified in recent years, including the enzyme autotaxin (ATX) and the RGD integrins, which are known to play a key role in the pathogenesis. In collaboration with GlaxoSmithKline, the projects detailed in this report were aimed to develop small molecule inhibitors with drug like physicochemical properties for fibrosis drugdiscovery.;Chapter 1 focusses on the secreted enzyme ATX, which is responsible for the hydrolysis of lysophosphatidylcholine (LPC) to the bioactive lysophosphatidic acid (LPA) and choline. The ATX-LPA signalling pathway is implicated in cell survival, migration, and proliferation; thus, the inhibition of ATX is a recognised therapeutic target for a number of diseases including fibrotic diseases, cancer, and inflammation, amongst others. Many of the developed synthetic inhibitors for ATX have resembled the lipid chemotype of the native ligand; however, a small number of inhibitors have been described that deviate from this common scaffold. Herein, Chapter 1 details the structure-activity relationship (SAR) exploration of a previously reported small molecule ATX inhibitor through the design, synthesis, and biological evaluation of aseries of analogues.;Furthermore, using enzyme kinetics studies it is shown that analogues of this chemotype are noncompetitive inhibitors, and using a crystal structure with ATX the discrete binding mode was confirmed. This work has provided valuable insight into the binding of this chemotype, which could aid the design of novel ATX inhibitors with non-lipid-like scaffolds.;Chapter 2 describes a lead-optimisation project targeting the RGD subfamily of the integrin receptors. The RGD integrins are recognised therapeutic targets for thrombosis, fibrosis, and cancer, amongst others. Current inhibitors are designed to mimic the tripeptide sequence of the natural ligands (arginine-glycine-aspartic acid); however, the RGD-mimetic antagonists for one particular RGD integrin (αIIbβ3) have been shown to cause partial agonism, leading to the opposite pharmacological effect.The challenge of obtaining oral activity and synthetic tractability with RGD-mimetic molecules, along with the issues relating to pharmacology, has left integrintherapeutics in need of a new strategy. Recently, a new generation of inhibitor has emerged that lacks the RGD-mimetic.;The work detailed herein aimed to build on this emerging area, with the design, synthesis, and biological evaluation of novel small molecule inhibitors targeting the αvβ3 integrin. These compounds are shown to be accessed via synthetically divergent routes, allowing for the quick exploration of adiverse set of potential lead compounds. Initial efforts led to the identification offour promising lead-like inhibitors with pIC50 values ranging from 4.1-5.5 for the target integrin αvβ3. Unfortunately, the initial hit compound, that the subsequent compound design stemmed from, was later determined to be a false positive, and as a result work on the project ceased. Thus, Chapter 2 details a project that was misled due to false positive assay results

COMPUTATIONAL TECHNIQUES TO EVALUATE AT ATOMIC LEVEL THE MECHANISM OF MOLECULAR BINDING

Integrins are an important class of transmembrane receptors that relay signals bidirectionally across the plasma membrane, regulating several cell functions and playing a key role in diverse pathological processes. Specifically, integrin subtype \u3b1IIb\u3b23 is involved in thrombosis and stroke, while subtypes \u3b1v\u3b23 and \u3b15\u3b21 play an important role in angiogenesis and tumor progression. They therefore emerged as attractive pharmacological targets. In the past decades several peptides and peptidomimetics targeting these proteins and based on the integrin recognition motif RGD (Arg-Gly-Asp) have been developed, whereby their affinity and selectivity for a specific integrin subtype have been fine-tuned by modulation of RGD flanking residues, by cyclization or by introduction of chemical modifications. Thus far, the design and development of RGD-based cyclopeptides have been mainly based on empirical approaches, requiring expensive and time-consuming synthesis campaigns. In this field, the employment of computational tools, that could be valuable to accelerate the drug design and optimization process, has been limited by the inherent difficulties to predict in silico the three-dimensional structure and the inhibitory activity of cyclopeptides. However, recent improvements in both computational resources and in docking and modeling techniques are expected to open new perspectives in the development of cyclopeptides as modulators of protein-protein interactions and, particularly, as integrin inhibitors. Within this PhD project, I have investigated the applicability of computational techniques in predicting and rationalizing how the environment of the recognition-motif in cyclopeptides (i.e. flanking residues and introduction of chemical modification) could influence their integrin affinity and selectivity. These features can regulate integrin affinity both by favoring direct interactions with the receptor and/or by modulating the three-dimensional conformation properties of the recognition motif. To take into account both these aspects, I have proposed and optimized a multi-stage computational protocol in which an exhaustive conformational sampling of the investigated cyclopeptides is followed by docking calculations and re-scoring techniques. Specifically: i) the exhaustive sampling could be achieved by using Metadynamics in its Bias Exchange variant (BE-META), an enhanced sampling technique which represents a valuable methodology for the acceleration of rare events, allowing to cross the high free energy barriers characteristic of cyclopeptides and providing reliable estimations of the populations of the accessible conformers. ii) The docking calculations, complemented with the re-scoring technique MM-GB/SA (Molecular Mechanics Generalized Born Surface Area) and the cluster analysis of the decoy poses, allow to evaluate the ability of each peptide to engage interactions with the receptors and to rank the docking poses according to their binding ability; iii) a joint analysis of the previous outcomes results in a reliable ranking of cyclopeptides based on their binding affinity and in the rationalization of their structure-activity relationship. This computational protocol has been exploited in two different applications, illustrated within the thesis. In the first application the protocol has been applied to rationalize how the introduction of chemical modifications, specifically backbone N-methylation, impacts on the equilibrium conformation and consequently on the integrin affinity of five RGD containing cyclic hexapeptides, which were previously generated by the group of professor Kessler to modulate their selectivity for \u3b1IIb\u3b23 integrin. The study revealed that backbone N-methylation affects the preferences of the \u3c6 dihedral angle of the methylated residue, specifically favoring the adoption of additional conformations, characterized by a 180\ub0 twist of the peptide bond plane preceding the methylated residue. These twists of dihedral angles were found to have relevant consequences on the cyclopeptides conformation, influencing the formation of intra-molecular hydrogen bonds as well as some structural features which are known to be fundamental in integrin binding. Both structural analysis and docking calculations allowed to identify the \u201cbioactive\u201d conformation (i.e. an extended RGD conformation able to recapitulate the canonical electrostatic and the additional stabilizing hydrophobic interactions). Of note, the cyclopeptides that are pre-organized, already in their free state, in this bioactive conformation are the ones displaying the best \u3b1IIb\u3b23 binding affinity in terms of IC50 values, confirming that pre-organization of cyclopeptides in solution can strongly affect their binding strength to the receptor and demonstrating that the knowledge of their conformational equilibrium is fundamental to provide reliable affinity predictions. In the second application, I have focused my attention on cyclopeptides harboring a recently discovered integrin recognition motif: isoDGR (isoAsp-Gly-Arg), deriving from the spontaneous deamidation of NGR (Asp-Gly-Arg) sequence present in integrin natural ligands. As a preliminary step, I have systematically tested the accuracy of eight Molecular Mechanics force fields in reproducing the equilibrium properties of isoDGR-based cyclopeptides, for which NMR experiments have been acquired. The comparison between simulated and NMR-derived data (i.e. chemical shifts and J scalar couplings) revealed that, while most of the investigated force fields can properly reproduce the equilibrium conformational properties of cyclic peptides, only two of them (i.e. the AMBER force fields ff99sb-ildn and ff99sb*-ildn) are able to recover the NMR observables characteristics of the non-standard residue isoAspartate with an accuracy close to the systematic uncertainty. Overall, these results suggest that the transferability of force field parameters to non standard amino acids is not straightforward. However, two force fields allowed to obtain a satisfactory accuracy and have been therefore employed for the subsequent investigation. I thus applied the computational protocol to rationalize the diverse selectivity and affinity profiles for integrins \u3b1v\u3b23 and \u3b15\u3b21, both related to cancer, displayed by three isoDGR-based cyclic hexapeptides. These molecules differ in the residues flanking the isoDGR motif and show appealing tumor-homing properties; specifically it has been shown that one of these, c(CGisoDGRG), can be coupled with human serum albumin through a chemical linker to be used as a drug delivery agent for functionalized gold nanoparticles. Herein, I investigated the role of the chemical linker in improving affinity and selectivity of c(CGisoDGRG) for \u3b1v\u3b23. The application of the multi-stage protocol allowed to propose an explanation for the different selectivity profiles displayed by these molecules, where the direct interactions engaged by the flanking residues and/or their steric hindrance seem to be largely responsible for the observed different affinities. As a last result, through the combination of MD and NMR techniques, I demonstrated that the chemical linker improved the \u3b1v\u3b23 affinity of c(CGisoDGRG) by engaging direct interactions with the receptor and I proposed two possible complex models, which well-reproduce data from Saturation Transfer Difference experiments. Overall, in this PhD work I have shown that the combination of different computational techniques, BE-META, docking and MM-GB/SA re-scoring, could be a reliable approach to perform structure-activity relationship studies in cyclopeptides. Specifically, the proposed protocol is able to predict the influence of the recognition motif environment (i.e. chemical modification and flanking residues) on integrin affinities. These two features regulate integrin affinity differently: the first one by conformational modulation of the recognition motif, the second by engaging direct interactions with the receptor. Of note, the approach can deal with both these mechanisms of affinity modulation. We expect that the protocol herein described could be used in future to screen novel peptides library or to complement biochemical experiments during the drug optimization stages, assisting organic chemists in the design of more effective integrin-targeting peptides

Platelet glycoprotein VI in the regulation of thrombus growth

Platelet glycoprotein VI (GPVI) is the principal signalling receptor for collagen, which is exposed upon damage to the extracellular matrix and at site of rupture of an atherosclerotic plaque. GPVI has recently been shown to be also a receptor for fibrinogen and fibrin, which mediate platelet aggregation and clot formation. In mice, GPVI deficiency protects form arterial thrombosis without causing excessive bleeding. Individuals with an inherited deficiency in GPVI have a mild bleeding diathesis. Together, these observations suggest a more important role of GPVI in thrombosis over haemostasis. The research described in this thesis investigates the relative contribution of GPVI in adhesion and platelet aggregation under flow on collagen and fibrin(ogen). As overarching hypothesis, I propose that GVI is a relevant signalling receptor for fibrin and fibrinogen in supporting thrombus propagation and stability. The first part is a critical assessment of the evidence that fibrin and fibrinogen bind to monomeric or dimeric GPVI in view of the earlier existing controversies. This is followed by the exploration of the role of GPVI in platelets adhesion to collagen using blood from Chilean patients with a homozygous insertion mutation in the GP6 gene which causes lack of GPVI expression on the platelet surface. The results show a critical role for GPVI in supporting platelet aggregation and phosphtidylserine exposure on collagen and non-collagen surfaces, but not adhesion which, on collagen, is mediated by the integrin α2β1. Further, it is estimated that in Chile there are over 4,000 individuals GPVI-deficient, of whom only a handful are known to have a bleeding diathesis. The next study investigates the contribution of GPVI to platelet adhesion and thrombus growth on fibrin, fibrinogen, using the Syk inhibitor, PRT-060318, and an anti-GPVI Fab, 9O12. The results show that GPVI contributes to platelet activation in response to fibrin and fibrinogen, that induces platelet secretion but low level of Ca2+ rises and that it is not required for platelet adhesion. Further, they show that in response to fibrin, GPVI acts in concert with the integrin αIIbβ3 in a non-redundant way. Successively, the action of two small molecule inhibitors of GPVI, losartan and honokiol, on platelet activation is investigated. The results show that both compounds are not selective GPVI antagonists. The following study explores the contribution of GPVI and integrin αIIbβ3 to the stability of a preformed thrombus under flow conditions. A comparison is made between inhibitors of Syk, Src, Btk and those of secondary mediators, ADP and TxA2. The results demonstrate a critical role for Syk in supporting aggregate stability, likely through fibrinogen-induced activation of both GPVI and integrin αIIbβ3. In addition, Syk appears to act in synergy with ADP and TxA2 and to be independent of collagen-induced GVPI activation, as a blocking nanobody has minor effect. Overall, the research in this thesis provides evidence that the role of GPVI extends beyond the onset of thrombus formation. GPVI activation in response to fibrin(ogen) contributes to platelet aggregation and it is key to thrombus stability. This conclusion further supports the argument that blocking GPVI may effectively prevent arterial thrombosis. Furthermore, this research highlights the importance of the integrin αIIbβ3 in supporting GPVI activation, suggesting that blocking signalling downstream of both receptors, can provide an alternative therapeutic strategy for the treatment of arterial thrombosis

Characterization of a protein: protein interaction between RNF181 and the platelet integrin, αIIbβ3

The integrin αIIbβ3 is a heterodimeric protein that exists in an inactive, resting state within the membrane of quiescent platelets. In response to platelet activation within a pro-thrombotic environment, the integrin αIIbβ3 becomes active, serves as a receptor for a number of ligands including fibrinogen, and orientates platelet – platelet cohesion. The αIIb and β3 subunits maintain the integrin in a resting conformational state by forming a tight clasp at their cytoplasmic tails (CT’s). Disruption of this clasp by platelet activation initiates the transition of the integrin from an inactive conformation to an active ligand binding state. The highly conserved αIIb membrane proximal motif (989KVGFFKR995) resides at the binding interface of the αIIb and β3 CT contacts and acts as a docking site for integrin binding proteins. These binding partners regulate the association of the integrin αIIb and β3 CT’s and hence, the activation state of the integrin. Therefore, since αIIb and β3 CT seperation is a fundamental feature of integrin activation, much research has emphasized on the dynamic relationship between the conserved motif of the αIIb CT and its binding partners. RNF181 was previously demonstrated to be a potential integrin binding protein as it had an affinity for the 989KVGFFKR995 motif. Given the preliminary nature of this earlier work, this thesis focused on exploring a possible, functional relationship between RNF181 and αIIbβ3. Using a variety of experimental approaches, strong evidence for a functional co-association of integrin αIIbβ3 and RNF181 was revealed. Using confocal microscopy, co-localization between the two proteins in platelets and integrin αIIbβ3 expressing CHO cells was demonstrated. Using CO-IP’s, Thermophoresis and ITC, a direct affinity between RNF181 and αIIbβ3 was demonstrated. The interaction was observed to have a moderate and transient affinity. Using bioinformatics and peptide arrays, the precise binding sites that allowed RNF181 to interact with the integrin αIIb cytoplasmic tail was explored. In particular, short linear peptide sequences that mediate their binding capacity were identified. Using platelet function assays, it was demonstrated that palmitoylated RNF181 derived peptides potently and specifically inhibited platelet / integrin αIIbβ3 activation. Finally, through a preliminary study using siRNA, a role for RNF181 as being a positive regulator of integrin αIIbβ3 activation was suggested

Role of bacterial lipopolysaccharides in the modulation of platelet and megakaryocyte function

Cardiovascular disease is a group of inflammatory diseases that is responsible for the plurality of deaths in the world. In ischaemic diseases, aberrant platelet activation leads to thrombus formation and occlusion of key arteries. Furthermore, platelets are critically involved in sepsis, where thrombocytopaenia is correlated with worse patient outcomes. Therefore, immune receptors, such as Toll-like receptor 4 (TLR4), have been identified as potential targets for reducing platelet activation by decoupling immune functions from haemostasis. Multiple experimental techniques were used to determine the impact of ultrapure lipopolysaccharide (LPS) chemotypes on platelet and megakaryocyte function as LPS is a specific ligand for TLR4. To determine whether LPS modulates platelet activity, LPS and platelets were co-incubated under different conditions. We determined that ultrapure LPS derived from various species of Gram-negative bacteria cannot significantly modulate platelet activity, as measured by aggregation, fibrinogen binding, and P-selectin exposure, except under specific conditions. This is potentially due to activation dependent cell�surface expression of TLR4. Subsequently, to investigate whether LPS induces inflammatory signalling in megakaryocytes, a novel megakaryocyte reporter cell line, Meg-01R, was developed and used to determine that ultrapure LPS is not sufficient on its own to modulate megakaryocyte function. Characterisation of this cell line suggests that MyD88-dependent signalling pathways are active in Meg-01R cells but TLR4 is not present in sufficient quantities at the cell surface. As LL37 directly binds to LPS and is also a strong platelet agonist, we investigated the impact of LPS on LL37- induced platelet activation. Here, we discovered a TLR4-independent cell-independent formation of LL37-LPS micelles that prevents LL37-induced platelet activation. Based on these results, LPS and TLR4 are not sufficient, in vitro, to decouple the immune function of platelets from haemostasis or induce changes in megakaryocyte function however it may still play an important role in conjunction with other immune receptors

The role of formyl peptide receptors in the regulation of platelet function

Formyl peptide receptors (FPRs) belong to the family of G protein-coupled receptors (GPCRs) and play crucial roles in the regulation of innate immunity and host defence. FPRs include three family members; FPR1, FPR2/ALX and FPR3. They bind a wide variety of structurally and chemically diverse ligands that can exert various functions. Despite a plethora of research focusing on the role of FPRs in the regulation of immunity, there is a paucity of studies on their roles on the regulation of platelet haemostatic function. Here, we demonstrate the impact of both FPR1 and FPR2/ALX on the modulation of platelet reactivity, haemostasis and thrombosis. By using selective pharmacological inhibitors for FPR1 and FPR2/ALX, and Fpr1- and Fpr2/3-deficient mice, we were able to establish instrumental roles for these receptors in the regulation of the normal platelet haemostatic function. Additionally, we report a crucial role for fMLF in the regulation of platelet function through FPR1 signalling. fMLF exerted a priming effect on platelet activation through inducing distinct functions and enhances thrombus formation under arterial flow conditions. These effects were diminished in the presence of FPR1-selective pharmacological inhibitors and in platelets obtained from Fpr1- deficient mice. In addition, we investigated the role of LL37 in the regulation of platelet function and its modulation on platelet reactivity under pathological conditions, such as psoriasis, via acting through FPR2/ALX. We demonstrate that LL37 activates a range of platelet functions, enhances thrombus formation, and shortens the tail-bleeding time in mice. Moreover, we report the overexpression of mCRAMP (an LL37 murine orthologue) in affected skin and plasma of a murine [imiquimod (IMQ)-induced] model of human psoriasis and its ability to enhance platelet responses via Fpr2/3. We also report a role for Annexin A1 and its N-terminal peptide, Ac2-26, in the regulation of platelet function through FPR2/ALX. Ac2-26 induced the activation of various platelet functions. Moreover, AnxA1-deficient mice demonstrate enhanced functional responses towards Ac2-26, which may be attributable to the overexpression of Fpr2/3 in these mice. Since both FPR1 and FPR2/ALX and their ligands play critical roles in various pathological conditions, their influence on the modulation of platelet activation and thrombus formation will provide novel insights into the mechanisms that control platelet-mediated complications under various disease settings

Hematology

Hematology encompasses the physiology and pathology of blood and of the blood-forming organs. In common with other areas of medicine, the pace of change in hematology has been breathtaking over recent years. There are now many treatment options available to the modern hematologist and, happily, a greatly improved outlook for the vast majority of patients with blood disorders and malignancies. Improvements in the clinic reflect, and in many respects are driven by, advances in our scientific understanding of hematological processes under both normal and disease conditions. Hematology - Science and Practice consists of a selection of essays which aim to inform both specialist and non-specialist readers about some of the latest advances in hematology, in both laboratory and clinic

Women in Bioorganic Chemistry

Issues relating to the gender schism and its effect on the career advancement of women in the Academy, especially in the field of STEM disciplines, deserve our attention and the efforts of all the scientific community to mitigate the gender gap. In order to embrace gender equality, recognize the career progression of women, and to celebrate the achievements of women in the field of bioorganic chemistry, we present contributions both from highly renowned female scientists and young female researchers who are in the early stages of their careers. This Special Issue includes fifteen manuscripts, including eleven high-quality research articles and four comprehensive review articles in the area of bioorganic chemistry, published from mid-2020 to early 2022. The scope of this Special Issue covers a wide range of topics at the organic chemistry–biology interface, including the synthesis and derivatization of natural compounds and their analogues, and the investigation of their biological activities in the human health field (for instance as antitumorals, antioxidants and antimicrobial agents), as well as their possible application in the crop protection field as agrochemicals. An example of nanoparticle-based biomaterial is also included. The techniques employed, besides organic synthesis, are in silico studies (docking procedures and molecular modeling), FT-IR spectroscopy, laser diffraction, PET, fluorescence, STD-NMR studies, enzymatic evaluation, experiments on cell lines and in vivo studies on mice

Mechanisms of Vascular Disease: A Reference Book for Vascular Specialists

New updated edition first published with Cambridge University Press. This new edition includes 29 chapters on topics as diverse as pathophysiology of atherosclerosis, vascular haemodynamics, haemostasis, thrombophilia and post-amputation pain syndromes