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Cyclic AMP (cAMP) is a second messenger that translates extracellular signals into tightly regulated biological responses. The cAMP binding domain (CBD) is a conserved regulatory switch that binds to cAMP and allosterically controls multiple cellular functions. All CBDs share a common architecture comprised of α- and β-subdomains. cAMP binds to the phosphate binding cassette (PBC) nested within the β-subdomain. In mammals the main cAMP receptors are protein kinase A (PKA), guanine exchange factors (EPAC) and ion channel proteins, including both the hyperpolarization-activated cyclic nucleotide-dependent channels (HCN channels) and the cyclic nucleotide-gated channels (CNG channels). Impaired activities of these proteins are associated with diabetes, cardiovascular diseases, cancer and Alzheimer's disease. Therefore, these proteins represent promising therapeutical targets. However, the mechanism of their cAMP-dependent allosteric control is not completely understood. In the present thesis we have studied the allosteric mechanism of activation in PKA and EPAC using an NMR-based approach and we have proposed a model explaining how cAMP allosterically controls the activity of PKA and EPAC. Binding of cAMP to the Regulatory (R) subunit of PKA facilitates the release of the Catalytic (C) subunit. According to our model, binding of cAMP triggers long range perturbations that propagate from the PBC to the R:C interface through both direct and indirect pathways. The indirect pathway involves two key relay sites located at the C-terminus of β2 (1163) and at the N-terminus of β3 (D170). D170 functions as an electrostatic switch that mediates the communication between the PBC and the helical subdomain, whereas 1163 controls the global unfolding. Hence, removal of cAMP uncouples the α- and β-subdomains by breaking the circuitry of cooperative interactions radiating from the PBC. The proposed model was further validated by the cAMP agonist Sp-cAMPS and the cAMP antagonist Rp-cAMPS. It was observed that Rp-cAMPS, in which the equatorial exocylic oxygen is replaced by sulphur, does not activate a necessary indirect allosteric pathway, while its diastereoisomer (Sp-cAMPS) with opposite phosphorus chirality behaves similarly to cAMP activating all allosteric pathways. Our data also showed that the cAMP-antagonist stabilizes a ternary inhibitory complex between the effector ligand and both the regulatory and the catalytic subunits of PKA. At this point it is still not understood how the proposed model of cAMP allostery is conserved in other cAMP binding proteins such as EPAC. EPAC is a multidomain guanine nucleotide exchange factor specific for small GTP-binding proteins and is directly activated by cAMP. We have probed how cAMP docks into the EPAC1 CBD and how its signal allosterically propagates from the cAMP binding site to the helical subdomain, which mediates the inhibitory interactions between the regulatory and catalytic regions of EPAC. Our comparative NMR investigation of cAMP signalling in PKA and EPAC revealed key functionally significant differences between these two systems that will facilitate the design of EPAC-selective therapeutics. / Thesis / Doctor of Philosophy (PhD)

The sirtuins are a family of NAD⁺-dependent deacetylase enzymes which are implicated in various illnesses including cancer and neurodegenerative diseases. Part I of this thesis describes the synthesis and biological evaluation of inhibitors of the SIRT1 and SIRT2 isoforms of this important family of enzymes. Chapter 1 gives an overview of sirtuin biology and the physiological roles of these enzymes. In particular the link between SIRT1 and cancer and SIRT2 and its role in the onset of neurodegenerative diseases is discussed. A review of the most potent and selective inhibitors of SIRT1 and SIRT2 is given including an introduction to the tenovin and cambinol classes of inhibitor. Chapter 2 describes various issues relating to the structure of the important chemical tool tenovin-6. The synthesis of analogues to improve the solubility, determine the preferred conformation and verify the products of metabolism of tenovin-6 is presented including their evaluation by in vitro and in cell methods. Part II of this chapter reports the design and use of a ¹H NMR method used to monitor the sirtuin-mediated deacetylation reaction. This was particularly relevant due to concerns raised about the possibility of false positive results obtained with the commercially available assay kit commonly used by the sirtuin community. This new ¹H NMR method was used to validate the inhibition of SIRT2 by tenovin-6. Chapter 3 describes the parallel synthesis and evaluation of tenovin analogues as inhibitors of SIRT1 and SIRT2. This study identified that replacement of the t-butyl substituent of tenovin-6 with the 3,5-dihalogen-4-alkoxy substitution pattern led to a variety of analogues having SIRT2 selectivity. As well as the collection of valuable SAR data, in cell data is also presented for the analogues. Chapter 4 provides attempts to rationalise the SAR data collected in Chapters 2 and 3 through a computational study. The molecular docking software GOLD was used to predict the binding site of the tenovin scaffold and hence rationalise the observed potencies of various analogues. Chapter 5 reports the synthesis and biological evaluation of triazole and cambinol analogues as SIRT1 and SIRT2 inhibitors. Part I details the synthesis and in vitro testing of a series of ring constrained tenovin analogues based on the 1,4-disubstituted triazole using click chemistry. A series of 1,5-disubstituted analogues were also synthesised. Part II describes the synthesis of S-alkylated cambinol analogues and the effect of N3-methylation upon activity and selectivity towards SIRT1. Part II of this thesis details the synthesis and biological testing of novel potent allosteric inhibitors of RmlA. RmlA is the first enzyme in the L-rhamnose biosynthetic pathway in bacteria. L-rhamnose is an important component of the bacterial cell wall and as such RmlA is therefore an important target in the discovery of novel anti-bacterial compounds. Chapter 7 provides an overview of the RmlA enzyme including its role in L-rhamnose biosynthesis and why it is an attractive target for anti-bacterial drug discovery. No small molecule inhibitors of RmlA have been reported previously. Chapter 8 describes the design and synthesis of pyrimidine-2,4-dione analogues as novel allosteric inhibitors of RmlA. SAR data is generated and rationalised by X-ray crystallographic techniques to study the structures of complexes of RmlA with various analogues. Analogues were also tested for their ability to inhibit the growth of the important human pathogen Mycobacterium tuberculosis.

VIH-1, l’agent étiologique du Syndrome de l’Immunodéficience Acquise, est un rétrovirus qui infecte les cellules immunitaires et détourne leur machinerie cellulaire pour se répliquer rapidement. Lors de l’infection, le génome ARN est rétrotranscrit en ADN par la transcriptase inverse virale (RT), puis l’insertion du génome proviral dans l’ADN de la cellule hôte est une étape obligatoire du cycle viral catalysée par l’enzyme virale Intégrase (IN). L’interaction de l’IN avec son cofacteur essentiel, la protéine nucléaire LEDGF/p75, dirige l’intégration à l’intérieur de gènes dans des régions fortement exprimées de la chromatine, ce qui permet la production efficace de nouveaux virions. Les Inhibiteurs Allostériques Intégrase-LEDGF (INLAIs) sont une nouvelle classe de molécules antirétrovirales se liant à l’IN au site de liaison de LEDGF/p75. Conçus pour inhiber compétitivement l’interaction protéine-protéine IN-LEDGF/p75, ils inhibent également les activités enzymatiques de l’Intégrase et augmentent son niveau de multimérisation.Nous avons étudié plusieurs nouvelles séries d’INLAIs de la société Mutabilis, et avons pu démontrer que ces molécules inhibent l’intégration, mais ont aussi un effet antirétroviral plus puissant et indépendant de LEDGF/p75 post-intégration au cours de la maturation des virions, qui conduit à la production de virus non infectieux, ayant une morphologie excentrique caractérisée par un défaut d’encapsidation du génome viral. Lors de l’infection de cellules par ces virus, le cycle viral s’arrête à l’étape de rétrotranscription du génome viral. Nous avons montré que ces virions contiennent pourtant un génome viral stable et fonctionnel, une RT active et l’ARNtLys3 qui sert d’amorce à la rétrotranscription, et ont également conservé leur immunoréactivité pour les lymphocytes B et T. En évaluant l’impact du polymorphisme de l’IN au voisinage du site de liaison, nous avons identifié le variant polymorphe Ala125, pour lequel l’INLAI MUT-A perd concomitamment son effet sur la maturation des virions et sur la multimérisation de l’IN, tandis qu’il inhibe aussi bien l’intégration et l’interaction IN-LEDGF, prouvant que l’effet tardif des INLAIs est associé à l’induction de la multimérisation de l’IN. Nous avons pu associer la multimérisation de l’IN à une déstabilisation du dimère par les INLAIs en analysant les co-structures de MUT-A avec les intégrases polymorphes. Les INLAIs, outre leur intérêt thérapeutique sont de remarquables réactifs qui ont permis de démontrer le rôle essentiel de l’intégrase à trois étapes clés du cycle viral du VIH-1 : la rétrotranscription, l’intégration et la maturation des virions. / HIV-1, the causative agent of AIDS, is a retrovirus that infects immune cells and hijacks their cell machinery to achieve rapid replication. In the course of infection, the RNA genome is reverse transcribed into DNA by the viral Reverse Transcriptase (RT) before the obligatory insertion of the proviral genome into the host cell DNA catalyzed by the viral enzyme Integrase (IN). The interaction of IN with its essential cofactor, the nuclear protein LEDGF/p75, targets integration within gene introns in highly transcribed chromatine regions, which allows efficient production of new virions. IN-LEDGF Allosteric Inhibitors (INLAIs) are a novel class of antiretroviral molecules binding IN at the LEDGF/p75-binding site. Designed to competitively inhibit IN-LEDGF/p75 protein-protein interaction, they are also capable of inhibiting IN enzymatic activities and raising the IN multimerization level.We studied several new INLAI series from the company Mutabilis. We could demonstrate that these molecules inhibit integration, but also have a more potent, LEDGF-independent, antiretroviral effect during virion maturation, resulting in the production of non-infectious virions. Virions produced upon INLAI treatment have an eccentric morphology characterized by an encapsidation defect of the viral genome, and lead to an infection block at reverse transcription. Yet, we showed that these virions package a stable and functional viral genome, an active RT and the tRNALys3 primer for reverse transcription, and also keep their immunoreactivity towards B- and T-cell lymphocytes. When evaluating the influence of polymorphism at the edge of the binding site, we identified the IN Ala125 polymorphic variant which causes the concomitant loss of MUT-A effect on virion maturation and IN multimerization, whereas inhibition of integration and IN-LEDGF interaction are maintained. This proves that INLAIs exert their late stage effect through induction of IN multimerization. We could associate IN multimerization to INLAI-induced dimer destabilization by analyzing MUT-A co-structures with polymorphic integrases. Beside their therapeutic interest INLAIs are highly valuable reagents that allowed to demonstrate the essential role of integrase at three key steps of the HIV-1 replication cycle, reverse transcription, integration and virus maturation.

Dysfunction of the N-methyl-D-aspartate (NMDA) receptor is thought to contribute to the cognitive deficits of many neurodegenerative diseases and psychiatric disorders. Cognitive symptoms of Alzheimer's disease can be treated with NMDA receptor antagonists or drugs targeting the cholinergic system; however, there are no effective treatments for cognitive deficits of schizophrenia or Huntington's disease. With the discovery of a potent and selective allosteric modulator of the NMDA receptor, there is the possibility of new treatments based on NMDA receptor functional-enhancement through neuroactive steroids, closely related in structure to the endogenous neurosteroid, cerebrosterol. The aim of this thesis was to examine steroidal modulation of the NMDA receptor both in vitro and in vivo. In chapter 2, NMDA receptor enhancement of both the synthetic and endogenous neuroactive steroids was assessed in neurons maintained in cell culture using calcium imaging techniques. Sulphation of the steroids greatly increased the efficacy of NMDA receptor enhancement compared to the unsulphated steroids. Chapters 3 and 4 investigate the potential for neuroactive steroids to treat cognitive impairments of Huntington's disease. Using a mouse model, tests were selected that were analogous to those in which patients are impaired; however, no impairments were found in the mouse model. Chapter 5, therefore, used a different model of cognitive impairment – namely, rats with a set-shifting impairment, as is seen in many psychiatric and neurological disorders, including Huntington's disease – to assess the effect of the synthetic steroid administration. Unfortunately, the rats did not show the expected impairment. The lack of reliable animal models compromised testing the efficacy of these promising NMDA receptor positive allosteric modulators. Nevertheless, the promising in vitro results suggest that there could still be therapeutic potential. In addition, the compound is a useful research tool for exploring NMDA receptor function in health and disease.

<p>Degradation of mRNA is a highly regulated step important for proper gene expression. Degradation of eukaryotic mRNA is initiated by shortening of the 3’ end located poly(A) tail. Poly(A)-specific ribonuclease (PARN) is an oligomeric enzyme that degrades the poly(A) tail with high processivity. A unique property of PARN is its ability to interact not only with the poly(A) tail but also with the 5’ end located mRNA cap structure. A regulatory role in protein synthesis has been proposed for PARN based on its ability to bind the cap that is required for efficient initiation of eukaryotic mRNA translation. Here we have investigated how the cap structure influences PARN activity and how PARN binds the cap. We show that the cap activates PARN and enhances the processivity of PARN. Further we show that the cap binding complex (CBC) inhibits PARN activity through a protein-protein interaction. To investigate the cap binding property of PARN, we identified the cap binding site at the molecular level using site-directed mutagenesis and fluorescence spectroscopy. We identified tryptophan 475, located within the RNA recognition motif (RRM) of PARN, as crucial for cap binding. A crystal structure of PARN bound to cap revealed that cap binding is mediated by the nuclease domain and the RRM of PARN. Tryptophan 475 binds the inverted 7-Me-guanosine residue through a stacking interaction. Involvement of the nuclease domain in cap binding suggests that the cap site and the active site overlap. Mutational analysis showed that indeed amino acids involved in cap binding are crucial for hydrolytic activity of PARN. Taken together, we show that PARN is an allosteric enzyme that is activated by the cap structure and that the allosteric cap binding site in one PARN subunit corresponds to the active site in the other PARN subunit.</p>

Antibiotic resistance is a worsening threat to human health. Increasing our understanding of the mechanisms causing this resistance will be of great benefit in designing methods to evade resistance and in developing new classes of antibiotics. In this thesis, I have used the TetR Family Transcriptional Regulators (TFRs), which constitute one of the largest antibiotic resistance regulator families, as a model system to study the structure, function and evolution of antibiotic resistance determinants. I performed a thorough examination of the variation and conservation seen in TFR sequences and structures using computational approaches. Through structure comparison, I have identified the most conserved features shared by the TFR family that are crucial for their stability and function. Based on my findings on conserved TFR structural features, a quantitative assay of binding affinity determination was developed. Through sequence comparison and a residue contact map method, I discovered the existence of a conserved residue network that correlates well with the known allostery pathway of TetR. This predicted allosteric communication network was experimentally tested in TtgR. I have also developed methods to identify TFR operator sequences through genomic comparisons and validated my prediction through experiments. In addition, I have developed an in vivo system that can be used to identify and characterize proteins that mediate resistance to almost any antibiotic. This system is simple, fast, and scalable for high-throughput applications, and could be used to discover a wide range of novel antibiotic resistance mechanisms. The principles that I applied to the TFR family could also be applied to other protein families.

The α7 nicotinic acetylcholine receptor (nAChR) subtype is abundantly expressed in the central nervous system (CNS) and in the periphery. Positive allosteric modulators (PAMs) of the α7 increase the response to an agonist and are divided into two types depending on whether they also decrease desensitization of the receptor (type II) or not (type I). Therefore, this study aims to investigate whether the enhancement of endogenous α7 nAChR function will result in a beneficial effect in nociceptive, inflammatory and chronic neuropathic pain models. While NS1738 and PNU-120596 were not active to reduce acute thermal pain, measured by hot-plate and tail-flick tests, only PNU-120596 dose-dependently attenuated paw-licking behavior in the formalin test. Our results with selective (MEK) inhibitor U0126 argues for an important role of extracellular signal-regulated kinase (ERK1/2) pathways activation in PNU-120596’s antinociceptive effects in formalin-induced pain. The α7 antagonist MLA, via intrathecal and intraplantar administration, reversed PNU-120596’s effects, confirming PNU-120596’s action through central and peripheral α7 nAChRs. Tolerance to PNU-120596 was not developed after chronic treatment of the drug. Furthermore, mixtures of PNU-120596 and choline, an endogenous α7 nAChR agonist, synergistically reduced formalin-induced pain, while interactions of non-antinociceptive doses of PNU-120596 and PHA-543613, a selective α7 nAChR agonist, or nicotine resulted in antinociception. In contrast, PNU-120596 failed to enhance nicotine-induced convulsions, -hypomotility and –antinociception in acute pain models. Surprisingly, it enhanced nicotine-induced hypothermia via α7 nAChRs. In the carrageenan inflammatory test both NS1738 and PNU-120596 significantly reduced thermal hyperalgesia, while only PNU-120596 significantly reduced edema. Importantly, PNU-120596 reversed established thermal hyperalgesia and edema induced by carrageenan. In the chronic neuropathic pain (CCI) model, PNU-120596 had long-lasting (up to 6 hrs), dose-dependent anti-hyperalgesic and anti-allodynic effects after a single injection, while NS1738 was inactive. Subcutaneous and intrathecal administration of MLA reversed PNU-120596’s effects, suggesting the involvement of α7 nAChRs. Finally, PNU-120596 enhanced an ineffective dose of selective agonist PHA-543613 to produce anti-allodynic effects in the CCI model. Our results show a fundamental in vivo difference between type I and II α7 nAChR PAMs, and demonstrate type II’s potential for the treatment of chronic inflammatory pain.

des-Formylflustrabromine (dFBr), isolated from the marine organism Flustra foliacea, is the first selective, positive allosteric modulator (PAM) of α4β2 nicotinic acetylcholine receptors that potentiates the action of the neurotransmitter acetylcholine (ACh). Most agonists for this receptor population are not selective and can activate other nACh receptors. A selective PAM, which activates α4β2 nACh receptors only in the presence of ACh, might find application in the treatment of of various neurological diseases such as Alzheimer’s disease or autism. dFBr was examined and found to produce a biphasic dose-response curve over a wide concentration range (i.e., potentiation at low concentration, but inhibition of the ACh-induced response at high concentrations). Our goal was to examine various structural features of dFBr required for potentiation; a secondary goal was to examine the same for inhibition. To understand the structural requirements of dFBr, a systematic ‘deconstruction reconstruction and elaboration’ approach (see p. 48) was employed to determine the contribution of various structural components of dFBr to its activity at α4β2 nACh receptors. Novel compounds were synthesized and characterized. Human α4β2 nACh receptors were expressed in Xenopus oocytes and the actions of dFBr and its analogs were measured using a two-electrode voltage clamp technique. Dose-response curves were obtained for the compounds in the absence and presence of 100 μM ACh. Structural features of dFBr optimal and/or required for PAM action at 42 nACh receptors were identified. A novel reconstructed analog with all the essential features for PAM action was synthesized and submitted for biological testing. Elaborated analogs of dFBr further helped in identification of various structural features important for PAM action and the inhibition of action of ACh. The ‘deconstruction reconstruction and elaboration’ approach (see p.48) identified important structural features of dFBr that modify its actions as a PAM or an antagonist (NAM? or channel blocker?) at α4β2 nACh receptors. This information should be useful for the subsequent design of novel analogs to evaluate their potential for the treatment of neurological disorders associated with ACh.

Thrombin is a key enzyme of the coagulation cascade exhibiting important roles in both pro-coagulation and anti-coagulation processes. Most clinically used anticoagulant drugs, including polymeric heparin, warfarin, hirudin, argatroban and the recently approved dabigatran, aim to reduce thrombin activity. There are several binding domains on thrombin including the active site, anion-binding exosites I and II, and the sodium binding site. We hypothesized that thrombin may be better regulated through an allosteric process mediated by small molecules binding to either exosite I or II. An appropriately designed allosteric regulator that reduces the procoagulant signal in a finely tuned manner may maintain a delicate balance between procoagulant and anticoagulant signals in blood resulting reduced bleeding complications. In this work, we synthesized and studied a library of potent, small, aromatic molecules as allosteric inhibitors of thrombin. Of the 28 potential inhibitors, 11 molecules inhibited thrombin with reasonable potency. Structure activity relationship studies showed that sulfation at the 5-position of the benzofuran scaffold was essential for targeting thrombin. Michaelis-Menten kinetic studies indicated a non-competitive, allosteric mechanism of inhibition. Site-directed mutagenesis, competitive binding and molecular modeling studies led to the identification of the most plausible binding pose for a potent sulfated dimer. To further improve the potency, a small library of sulfated benzofuran trimers was synthesized and studied for thrombin inhibition. Further, to find new scaffold to inhibit thrombin allosterically, docking-based virtual screening approach was used. All these molecules were found to be moderately potent thrombin inhibitors and can serve as lead to develop allosteric inhibitor. Overall, this work presents the first small, synthetic, sulfated aromatic molecules as potent allosteric modulators of thrombin. Finally, this work also highlights the opportunity of exploring allosteric modulators of other coagulation enzymes, e.g., factors Xa, IXa and XIa, based on the sulfated benzofuran scaffold.