EP3154540A1 - Pharmaceutical compositions comprising an at2r agonist for treating pain - Google Patents

Abstract

The present invention relates to methods and pharmaceutical compositions for treating pain in subject in need thereof. In particular, the present invention relates to a method of treating pain in a subject thereof comprising administering the subject with at least one agonist of the type 2 angiotensin II receptors (AT2Rs)-TWIK-related arachidonic acid stimulated K+ channel (TRAAK) pathway.

Description

PHARMACEUTICAL COMPOSITIONS COMPRISING AN AT2R AGONIST FOR TREATING PAIN

FIELD OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for treating pain in subject in need thereof.

BACKGROUND OF THE INVENTION:

Pain is an unpleasant feeling often caused by intense or damaging stimuli. The International Association for the Study of Pain's widely used definition states: "Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage." For example, chronic pain is a common problem that presents a major challenge to healthcare providers because of its complex natural history, unclear etiology, and poor response to therapy. Chronic pain is a poorly defined condition. Most authors consider ongoing pain lasting longer than 6 months as diagnostic, and others have used 3 months as the minimum criterion. In chronic pain, the duration parameter is used arbitrarily. Some authors suggest that any pain that persists longer than the reasonable expected healing time for the involved tissues should be considered chronic pain. The pathophysiology of chronic pain is multifactorial and complex and still is poorly understood. Various neuromuscular, reproductive, gastrointestinal, and urologic disorders may cause or contribute to chronic pain. Sometimes multiple contributing factors may be present in a single patient. Chronic pain can result from musculoskeletal disorders such as osteoarthritis/degenerative joint disease/spondylosis, rheumatoid arthritis, lyme disease, reiter syndrome, disk herniation facet osteoarthropathy, fractures/compression fracture of lumbar vertebrae, faulty or poor posture, fibromyalgia, polymyalgia rheumatica, mechanical low back pain, chronic coccygeal pain, muscular strains and sprains, pelvic floor myalgia (levator ani spasm), piriformis syndrome, rectus tendon strain, hernias (e.g. obturator, sciatic, inguinal, femoral, spigelian, perineal, umbilical), abdominal wall myofascial pain (trigger points), chronic overuse syndromes (e.g., tendinitis, bursitis); neurological disorders such as, brachial plexus traction injury, cervical radiculopathy, thoracic outlet syndrome, spinal stenosis, arachnoiditis syndrome, metabolic deficiency myalgias, polymyositis, neoplasia of spinal cord or sacral nerve, cutaneous nerve entrapment in surgical scar, postherpetic neuralgia (shingles), neuralgia (e.g., iliohypogastric, ilioinguinal, or genitofemoral nerves), polyneuropathies, polyradiculoneuropathies, mononeuritis multiplex, chronic daily headaches, muscle tension headaches, migraine headaches, temporomandibular joint dysfunction, temporalis tendonitis, sinusitis, atypical facial pain, trigeminal neuralgia, glossopharyngeal neuralgia, nervus intermedius neuralgia, sphenopalatine neuralgia, referred dental or temporomandibular joint pain, abdominal epilepsy, abdominal migraine, urologic disorders, bladder neoplasm, chronic urinary tract infection, interstitial cystitis, radiation cystitis, recurrent cystitis, recurrent urethritis, urolithiasis, uninhibited bladder contractions (detrusor-sphincter dyssynergia), urethral diverticulum, chronic urethral syndrome, urethral carbuncle, prostatitis, urethral stricture, testicular torsion, peyronie disease; gastrointestinal disorders such as chronic visceral pain syndrome, gastroesophageal reflux, peptic ulcer disease, pancreatitis, chronic intermittent bowel obstruction, colitis, chronic constipation, diverticular disease, inflammatory bowel disease, irritable bowel syndrome; reproductive disorders (extrauterine) such as endometriosis, adhesions, adnexal cysts, chronic ectopic pregnancy, chlamydial endometritis or salpingitis, endosalpingiosis, ovarian retention syndrome (residual ovary syndrome), ovarian remnant syndrome, ovarian dystrophy or ovulatory pain, pelvic congestion syndrome, postoperative peritoneal cysts, residual accessory ovary, subacute salpingo-oophoritis, tuberculous salpingitis; reproductive disorders (uterine) such as adenomyosis, chronic endometritis, atypical dysmenorrhea or ovulatory pain, cervical stenosis, endometrial or cervical polyps, leiomyomata, symptomatic pelvic relaxation (genital prolapse), intrauterine contraceptive device; psychological disorders such as bipolar personality disorders, depression, porphyria, sleep disturbances; and other conditions such as cardiovascular disease (eg, angina), peripheral vascular disease and chemotherapeutic, radiation, or surgical complications.

The modern concept of pain treatment emphasizes the significance of prophylactic prevention of pain, as pain is more easily prevented than it is relieved. Pain is generally controlled by the administration of short acting analgesic agents, steroids and non-steroidal anti- inflammatory drugs. Analgesic agents include opiates, agonistic-antagonistic agents, and antiinflammatory agents. However, all opiates have a wide variety of side effects that can decrease their clinical utility in certain situations. The side effects associated with the use of opiates include respiratory depression, reduced cough reflex, bronchial spasms, nausea, vomiting, release of histamine, peripheral vasodilation, orthostatic hypotension, alteration of vagal nerve activity of the heart, hyperexcitability of smooth muscles (sphincters), reduction of peristaltic motility in the gastrointestinal tract and urinary retention. Opiates also stimulate the release of adrenaline, anti-diuretic hormone, cause changes in the regulation of body temperature and sleep pattern, and are liable to promote the development of tolerance and addiction. In conclusion, there is still a need to provide novel analgesics. SUMMARY OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for treating pain in subject in need thereof

DETAILED DESCRIPTION OF THE INVENTION:

Mycobacterium ulcerans, the etiological agent of Buruli ulcer, causes extensive skin lesions, which despite their severity are not accompanied by pain. It was previously thought that this remarkable analgesia is ensured by direct nerve cell destruction. The inventors demonstrate here that M. ulcerans '-induced hypoesthesia is instead achieved through a specific neurological pathway triggered by the secreted mycobacterial polyketide mycolactone. They decipher this pathway at the molecular level, showing that mycolactone elicits signalling through type 2 angiotensin II receptors (AT2RS), leading to potassium-dependent hyperpolarization of neurons. The inventors further validate the physiological relevance of this mechanism with in vivo studies of pain sensitivity in mice infected with M. ulcerans, following the disruption of the identified pathway. These findings shed new light on molecular mechanisms evolved by natural systems for the induction of very effective analgesia, opening up the prospect of new families of analgesics derived from such systems. Accordingly a first object of the present invention relates to a method of treating pain in a subject thereof comprising administering the subject with a therapeutically effective amount of at least one agonist of the type 2 angiotensin II receptors (AT2Rs)- TWIK-related arachidonic acid stimulated K+ channel (TRAAK) pathway. Treatment may be for any purpose, including the therapeutic treatment of subjects suffering from pain, as well as the prophylactic treatment of subjects who do not suffer from pain (e.g., subjects identified as being at high risk pain). As used herein, the terms "treatment," "treat," and "treating" refer to reversing, alleviating, inhibiting the progress of a disease or disorder as described herein (i.e. pain), or delaying, eliminating or reducing the incidence or onset of a disorder or disease as described herein, as compared to that which would occur in the absence of the measure taken. The terms "prophylaxis" or "prophylactic use" and "prophylactic treatment" as used herein, refer to any medical or public health procedure whose purpose is to prevent the disease herein disclosed (i.e. pain). As used herein, the terms "prevent", "prevention" and "preventing" refer to the reduction in the risk of acquiring or developing a given condition (i.e. pain), or the reduction or inhibition of the recurrence or said condition (i.e. pain) in a subject who is not ill, but who has been or may be near a subject with the condition (i.e. pain). The method of the present invention is suitable in the treatment of a wide range of pain disorders, particularly acute pain, chronic pain, neuropathic pain, inflammatory pain, iatrogenic pain including cancer pain, infectious pain including herpetic pain visceral pain, central pain, dysfunctioning pain including fibromyalgia, nociceptive pain including post-surgical pain, and mixed pain types involving the viscera, gastrointestinal tract, cranial structures, musculoskeletal system, spine, urogenital system, cardiovascular system and CNS, including cancer pain, back and orofacial pain.

Pain is generally be classified as acute or chronic. Acute pain begins suddenly and is short-lived (usually twelve weeks or less). It is usually associated with a specific cause such as a specific injury and is often sharp and severe. It is the kind of pain that can occur after specific injuries resulting from surgery, dental work, a strain or a sprain. Acute pain does not generally result in any persistent psychological response. In contrast, chronic pain is long-term pain, typically persisting for more than three months and leading to significant psychological and emotional problems. Common examples of chronic pain are neuropathic pain (e.g. painful diabetic neuropathy, postherpetic neuralgia), carpal tunnel syndrome, back pain, headache, cancer pain, arthritic pain and chronic post-surgical pain. Clinical pain is present when discomfort and abnormal sensitivity feature among the patient's symptoms. Patients tend to be quite heterogeneous and may present with various pain symptoms. Such symptoms include: 1) spontaneous pain which may be dull, burning, or stabbing; 2) exaggerated pain responses to noxious stimuli (hyperalgesia); and 3) pain produced by normally innocuous stimuli. Although patients suffering from various forms of acute and chronic pain may have similar symptoms, the underlying mechanisms may be different and may, therefore, require different treatment strategies. Pain can also therefore be divided into a number of different subtypes according to differing pathophysiology, including nociceptive, inflammatory and neuropathic pain.

Nociceptive pain is induced by tissue injury or by intense stimuli with the potential to cause injury. Pain afferents are activated by transduction of stimuli by nociceptors at the site of injury and activate neurons in the spinal cord at the level of their termination. This is then relayed up the spinal tracts to the brain where pain is perceived (Meyer et al., 1994, Textbook of Pain, 13-44). The activation of nociceptors activates two types of afferent nerve fi res. Myelinated A-delta fihres transmit rapidly and are responsible for sharp and stabbing pain sensations, whilst unmyelinated C fibres transmit at a slower rate and convey a dull or aching pain. Moderate to severe acute nociceptive pain is a prominent feature of pain from central nervous system trauma, strains/sprains, burns, myocardial infarction and acute pancreatitis, postoperative pain (pain following any type of surgical procedure), posttraumatic pain, renal colic, cancer pain and back pain. Cancer pain may be chronic pain such as tumour related pain (e.g. bone pain, headache, facial pain or visceral pain) or pain associated with cancer therapy (e.g. postchemotherapy syndrome, chronic postsurgical pain syndrome or post radiation syndrome). Cancer pain may also occur in response to chemotherapy, immunotherapy, hormonal therapy or radiotherapy. Back pain may be due to herniated or ruptured intervertebral discs or abnormalities of the lumber facet joints, sacroiliac joints, paraspinal muscles or the posterior longitudinal ligament. Back pain may resolve naturally but in some patients, where it lasts over 12 weeks, it becomes a chronic condition which can be particularly debilitating.

Neuropathic pain is currently defined as pain initiated or caused by a primary lesion or dysfunction in the nervous system. Nerve damage can be caused by trauma and disease and thus the term 'neuropathic pain' encompasses many disorders with diverse aetiologies. These include, but are not limited to, peripheral neuropathy, diabetic neuropathy, post herpetic neuralgia, trigeminal neuralgia, back pain, cancer neuropathy, HIV neuropathy, phantom limb pain, carpal tunnel syndrome, central post- stroke pain and pain associated with chronic alcoholism, hypothyroidism, uremia, multiple sclerosis, spinal cord injury, Parkinson's disease, epilepsy and vitamin deficiency. Neuropathic pain is pathological as it has no protective role. It is often present well after the original cause has dissipated, commonly lasting for years, significantly decreasing a patient's quality of life (Woolf and Mannion, 1999, Lancet, 353, 1959-1964). The symptoms of neuropathic pain are difficult to treat, as they are often heterogeneous even between patients with the same disease (Woolf & Decosterd, 1999, Pain Supp., 6, S141-S147; Woolf and Mannion, 1999, Lancet, 353, 1959-1964). They include spontaneous pain, which can be continuous, and paroxysmal or abnormal evoked pain, such as hyperalgesia (increased sensitivity to a noxious stimulus) and allodynia (sensitivity to a normally innocuous stimulus).

The inflammatory process is a complex series of biochemical and cellular events, activated in response to tissue injury or the presence of foreign substances, which results in swelling and pain (Levine and Taiwo, 1994, Textbook of Pain, 45-56). Arthritic pain is the most common inflammatory pain. Rheumatoid disease is one of the commonest chronic inflammatory conditions in developed countries and rheumatoid arthritis is a common cause of disability. The exact aetiology of rheumatoid arthritis is unknown, but current hypotheses suggest that both genetic and microbiological factors may be important (Grennan & Jayson, 1994, Textbook of Pain, 397-407). It has been estimated that almost 16 million Americans have symptomatic osteoarthritis (OA) or degenerative joint disease, most of whom are over 60 years of age, and this is expected to increase to 40 million as the age of the population increases, making this a public health problem of enormous magnitude (Houge & Mersfelder, 2002, Ann Pharmacother., .36, 679-686; McCarthy et al., 1994, Textbook of Pain, 387-395). Most patients with osteoarthritis seek medical attention because of the associated pain. Arthritis has a significant impact on psychosocial and physical function and is known to be the leading cause of disability in later life. Ankylosing spondylitis is also a rheumatic disease that causes arthritis of the spine and sacroiliac joints. It varies from intermittent episodes of back pain that occur throughout life to a severe chronic disease that attacks the spine, peripheral joints and other body organs.

Another type of inflammatory pain is visceral pain which includes pain associated with inflammatory bowel disease (IBD). Visceral pain is pain associated with the viscera, which encompass the organs of the abdominal cavity. These organs include the sex organs, spleen and part of the digestive system. Pain associated with the viscera can be divided into digestive visceral pain and non-digestive visceral pain. Commonly encountered gastrointestinal (Gl) disorders that cause pain include functional bowel disorder (FBD) and inflammatory bowel disease (IBD). These Gl disorders include a wide range of disease states that are currently only moderately controlled, including, in respect of FBD, gastro-esophageal reflux, dyspepsia, irritable bowel syndrome (IBS) and functional abdominal pain syndrome (FAPS), and, in respect of IBD, Crohn's disease, ileitis and ulcerative colitis, all of which regularly produce visceral pain. Other types of visceral pain include the pain associated with dysmenorrhea, cystitis and pancreatitis and pelvic pain.

In some embodiments, the method of the present invention is suitable for the treatment of pain resulting from musculo-skeletal disorders, including myalgia, fibromyalgia, spondylitis, sero-negative (non-rheumatoid) arthropathies, non-articular rheumatism, dystrophinopathy, glycogenosis, polymyositis and pyomyositis; - heart and vascular pain, including pain caused by angina, myocardical infarction, mitral stenosis, pericarditis, Raynaud's phenomenon, scleredoma and skeletal muscle ischemia.

In some embodiments, the method of the present invention is suitable for the treatment of head pain, such as migraine (including migraine with aura and migraine without aura), cluster headache, tension-type headache mixed headache and headache associated with vascular disorders.

In some embodiments, the method of the present invention is suitable for the treatment of orofacial pain, including dental pain, otic pain, burning mouth syndrome and temporomandibular myofascial pain.

In some embodiments, the method of the present invention is particularly suitable for the treatment of chronic pain which results from musculoskeletal disorders such as osteoarthritis/degenerative joint disease/spondylosis, rheumatoid arthritis, lyme disease, reiter syndrome, disk herniation facet osteoarthropathy, fractures/compression fracture of lumbar vertebrae, faulty or poor posture, fibromyalgia, polymyalgia rheumatica, mechanical low back pain, chronic coccygeal pain, muscular strains and sprains, pelvic floor myalgia (levator ani spasm), piriformis syndrome, rectus tendon strain, hernias (e.g. obturator, sciatic, inguinal, femoral, spigelian, perineal, umbilical), abdominal wall myofascial pain (trigger points), chronic overuse syndromes (e.g., tendinitis, bursitis); neurological disorders such as, brachial plexus traction injury, cervical radiculopathy, thoracic outlet syndrome, spinal stenosis, arachnoiditis syndrome, metabolic deficiency myalgias, polymyositis, neoplasia of spinal cord or sacral nerve, cutaneous nerve entrapment in surgical scar, postherpetic neuralgia (shingles), neuralgia (e.g., iliohypogastric, ilioinguinal, or genitofemoral nerves), polyneuropathies, polyradiculoneuropathies, mononeuritis multiplex, chronic daily headaches, muscle tension headaches, migraine headaches, temporomandibular joint dysfunction, temporalis tendonitis, sinusitis, atypical facial pain, trigeminal neuralgia, glossopharyngeal neuralgia, nervus intermedius neuralgia, sphenopalatine neuralgia, referred dental or temporomandibular joint pain, abdominal epilepsy, abdominal migraine, urologic disorders, bladder neoplasm, chronic urinary tract infection, interstitial cystitis, radiation cystitis, recurrent cystitis, recurrent urethritis, urolithiasis, uninhibited bladder contractions (detrusor-sphincter dyssynergia), urethral diverticulum, chronic urethral syndrome, urethral carbuncle, prostatitis, urethral stricture, testicular torsion, peyronie disease; gastrointestinal disorders such as chronic visceral pain syndrome, gastroesophageal reflux, peptic ulcer disease, pancreatitis, chronic intermittent bowel obstruction, colitis, chronic constipation, diverticular disease, inflammatory bowel disease, irritable bowel syndrome; reproductive disorders (extrauterine) such as endometriosis, adhesions, adnexal cysts, chronic ectopic pregnancy, chlamydial endometritis or salpingitis, endosalpingiosis, ovarian retention syndrome (residual ovary syndrome), ovarian remnant syndrome, ovarian dystrophy or ovulatory pain, pelvic congestion syndrome, postoperative peritoneal cysts, residual accessory ovary, subacute salpingo-oophoritis, tuberculous salpingitis; reproductive disorders (uterine) such as adenomyosis, chronic endometritis, atypical dysmenorrhea or ovulatory pain, cervical stenosis, endometrial or cervical polyps, leiomyomata, symptomatic pelvic relaxation (genital prolapse), intrauterine contraceptive device; psychological disorders such as bipolar personality disorders, depression, porphyria, sleep disturbances; and other conditions such as cardiovascular disease (eg, angina), peripheral vascular disease and chemotherapeutic, radiation, or surgical complications. In some embodiments the method of the present invention is particularly suitable for the treatment of pain which results from autoimmune diseases including multiple sclerosis, neurodegenerative disorders, neurological disorders including epilepsy and senso-motor pathologies, irritable bowel syndrome, osteoarthritis, rheumatoid arthritis, neuropathological disorders, functional bowel disorders, inflammatory bowel diseases, pain associated with dysmenorrhea, pelvic pain, cystitis, pancreatitis, migraine, cluster and tension headaches, diabetic neuropathy, peripheral neuropathic pain, sciatica, causalgia, and conditions of lower urinary tract dysfunction.

In some embodiments, the prophylactic methods of the invention are particularly suitable for subjects who are identified as at high risk for pain. Typically subject that are risk for pain include patient that will have a surgical operation.

As used herein the term "TRAAK" has its general meaning in the art and refers to TWIK-related arachidonic acid stimulated K+ channel. TRAAK is also known as Homo sapiens potassium channel, subfamily K, member 4 (KCNK4). This protein is a member of the superfamily of potassium channel proteins containing two pore-forming P domains. The encoded protein dimerizes and functions as an outwardly rectifying channel. It is expressed primarily in neural tissues and is stimulated by membrane stretch and polyunsaturated fatty acids. Accordingly, the term "TRAAK agonist" used herein relates to any compound binding specifically to, and thus stimulating, TRAAK.

As used herein the term "AT2R" has its general leaning in the art and refers to type 2 angiotensin II receptor. Accordingly, the term "angiotensin II type 2 receptor agonist" used herein relates to any compound binding specifically to, and thus stimulating, angiotensin II type 2 receptors.

As used herein the term "type 2 angiotensin II receptors (AT2Rs)- TWIK-related arachidonic acid stimulated K+ channel (TRAAK) pathway" refers to the pathway deciphered in the EXAMPLE. This novel pathway consists of the activation of TRAAK by the stimulation of AT2R receptors in neurons. This pathway leads to the hyperpolarization of the neurons. As used herein the term agonist of the type 2 angiotensin II receptors (AT2Rs)- TWIK-related arachidonic acid stimulated K+ channel (TRAAK) pathway refers to any compounds which activate this pathway. In some embodiments, the agonist of the type 2 angiotensin II receptors (AT2Rs)- TWIK-related arachidonic acid stimulated K+ channel (TRAAK) pathway is an AT2R agonist or a TRAKK agonist. The agonist used according to the invention may be any substance, derived from natural sources or from synthesis by chemical and/or genetic engineering methods. Agonists typically include but are not limited to small organic molecule, peptides, polypeptides (including antibodies), nucleic acids (e.g. aptamers).

AT2R agonists are well known in the art (Steckelings UM, Unger T. Angiotensin II type 2 receptor agonists—where should they be applied? Expert Opin Investig Drugs. 2012 Jun;21(6):763-6. doi: 10.1517/13543784.2012.681046. Epub 2012 Apr 21.; Steckelings UM, Paulis L, Unger T, Bader M. Emerging drugs which target the renin-angiotensin-aldosterone system. Expert Opin Emerg Drugs. 2011 Dec; 16(4):619-30. doi: 10.1517/14728214.2011.618495. Epub 2011 Sep 12. Review.; Steckelings UM, Larhed M, Hallberg A, Widdop RE, Jones ES, Wallinder C, Namsolleck P, Dahlof B, Unger T. Non- peptide AT2-receptor agonists. Curr Opin Pharmacol. 2011 Apr; 11(2): 187-92.).

For example, AT2R agonists include compounds of formula I which are disclosed in US 20120035232, I

wherein

R¹ represents H;

R² and R³ independently represent H, Ci-β alkyl, Ci-β alkoxy, Ci-6 alkoxy-Ci-6-alkyl or halo;

Yi , Y2, Y3 and Y4 independently represent— CH— or— CF— ;

Zi represents— S— ;

Z2 represents— CH— or— N— ;

R⁴ represents— S(0)₂N(H)C(0)R⁶,— S(0)₂N(H)S(0)₂R⁶,— C(0)N(H)S(0)₂R⁶; R⁵ represents Ci-6 alkyl, C1-5 alkoxy, Ci-e alkoxy-Ci-6-alkyl or di-Ci-3-alkylamino-Ci-4- alkyl; and

R⁶ represents C 1-5 alkyl, C1-5 alkoxy, Ci-6 alkoxy-Ci-6-alkyl, C1-3 alkoxy-Ci-6-alkoxy, Ci- 6 alkylamino or di-Ci-6 alkylamino;

or a pharmaceutically-acceptable salt thereof,

In some embodiments, the AT2R agonist of the present invention is selected from the group consisting of:

N-Butyloxycarbonyl-3-(4-imidazol-l-ylmethylphenyl)-5-iso-butylthiophene-2- sulfonamide;

N-iso-butyloxycarbonyl-3-(4-imidazol- 1 -ylmethylphenyl)-5-iso-butyl-thiophene-2- sulfonamide;

N-iso-propyloxycarbonyl-3 -(4-imidazo 1- 1 -ylmethylphenyl)-5 -iso-butyl-thiophene-2- sulfonamide;

N-(butoxyacetyl)-3 -(4-imidazo 1- 1 -ylmethylphenyl)-5 -iso-butylthiophene-2- sulfonamide; N-butyloxycarbonyl-3 -(4-imidazo 1- 1 -ylmethylphenyl)-5 -butylthiophene-2- sulfonamide;

N-(butylamino)carbonyl-3 -(4-imidazo 1- 1 -ylmethylphenyl)-5 -iso-butyl-thiophene-2- sulfonamide;

N-butylsulfonyl-3-(4-imidazol-l-ylmethylphenyl)-5-iso-butylthiophene-2- sulfonamide;

N-butylsulfonyl-3-(4-imidazol-l-ylmethylphenyl)-5-iso-butylthiophene-2- carboxamide;

N-(2-methoxyethyloxy)carbonyl-3-(4-imidazol-l-ylmethylphenyl)-5-iso- butylthiophene-2-sulfonamide;

N-ethyloxycarbonyl-3-(4-imidazol-l-ylmethylphenyl)-5-iso-butylthiophene-2- sulfonamide;

N-tert-butylo ycarbonyl-3 -(4-imidazo 1- 1 -ylmetbylphenyl)-5 -iso-butyl-thiophene-2- sulfonamide;

N-butyloxycarbonyl-3-[4-(4-methylimidazol-l-ylmethyl)phenyl]-5-iso- butylthiophene-2-sulfonamide;

N-(N-butyl-N-methylamino)carbonyl-3-(4-imidazol-l-ylmethylphenyl)-5-iso- butylthiophene-2-sulfonamide; and

N-butylo ycarbonyl-3 -(4-imidazo 1- 1 -ylmetbylphenyl)-5 -(2-methoxyethyl)-thiophene- 2-sulfonamide.

Other examples of AT2R agonists include those described in WO1999043339, and WO1996039164, WO2012070936. In some embodiments, the AT2R agonist is a peptide or a peptide mimetic with high selectivity for the AT2R. Examples of peptides functioning as AT2R agonist and thus suitable for use according to the present invention are p-aminophenylalanine6- angiotensin II or N- - nicotinoyl-Tyr-(N-a-CBZ-Arg)-Lys-His-Pro-Ile-OH. When p-aminophenylalanine6- angiotensin II is used, it is typically administered to the patient directly into the blood. A peptide mimetic may contain elements that enforce steric constraints of a peptide and a peptide mimetic may retain some peptidic character. A peptide mimetic may alternatively be lacking peptidic fragments and consist of an organic molecule. A peptide mimetic can be an organic molecule comprising biaryl, arylheteroaryl, or biheteroaryl fragments that can be attached to a nitrogen containing monocyclic or bicyclic heterocycle by a one, two or three atom linker. A selective AT2R agonist may be an analogue of the non- selective angiotensin II type 2 receptor ligand 5,7- dimethyl-2-ethyl-3- [ [4- [2 (n-butyloxycarbonylsulfonamido) - 5-isobutyl-3-thienyl] phenyl] methyl] -imidazo [4 , 5-b] - pyridine. According to the invention, the compound of the present invention is administered to the patient with a therapeutically effective amount. By a "therapeutically effective amount" is meant a sufficient amount of the compound of the present invention to treat the disease (i.e. pain) at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1 , 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

The compound of the present invention is typically combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum- drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. A further object of the present invention relates to a method for screening a plurality of test compounds useful for treatment of pain comprising the steps consisting of (a) testing each of the test compounds for its ability to activate the angiotensin II receptors (AT2Rs)- TWIK- related arachidonic acid stimulated K+ channel (TRAAK) pathway and (b) positively selecting the test compounds capable of said activation. In some embodiments, the screening method of the invention comprises a first step of determining whether the test compound is able to bind to TRAAK or AT2R. Methods for determining whether a test compound binds to a protein are well known in the art. Binding of the test compound to the amyloid channel protein(s) can be detected by any of a number of methods known to those of skill in the art. For example, in some embodiments, the test compounds are labeled with a detectable label (e.g., a fluorescent label, a colorimetric label, a radioactive label, a spin (spin resonance) label, a radioopaque label, etc.). The membrane comprising the protein of interest (i.e. AT2R or TRAAK) is contacted with the test compound, typically washed, and then the membrane is screened for the detectable label indicating association of the test agent with the protein of interest. In some embodiments a secondary binding moiety (e.g. bearing a label) is used to bind and thereby label the bound test agents, or to bind the protein in which case association of the label on the secondary agent with the label on the test agent indicates binding of the test agent to the protein. In the latter case, in some embodiments, the label on the test agent and the label on the secondary agent can be labels selected that undergo fluorescent resonance energy transfer (FRET) so that excitation of one label results in emission from the second label thereby providing an efficient means of detecting association of the labels. In some embodiments, a competitive binding assay as described in the EXAMPLE is used. In such assays, a "competitive" agent known to bind to the protein of interest is also utilized. The competitive agent can be labeled and the amount of such agent displaced when the bilayer containing the protein of interest is contacted with a test agent provides a measure of the biding of the test agent. Methods of detecting specific binding are well known and commonly used, e.g. in various immunoassays. Any of a number of well recognized immunological binding assays (see, e.g., U.S. Patents 4,366,241 ; 4,376,110; 4,517,288; and 4,837, 168) are well suited to detection of test agent binding to proteins in a lipid bilayer. For a review of the general immunoassays, see also Asai (1993) Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Academic Press, Inc. New York; Stites & Terr (1991) Basic and Clinical Immunology 7th Edition.

In some embodiments, the screening method of the present invention is performed in screening cells. Typically, said screening cells are neurons, such as neurons derived from PC 12 cells or are macrophages, such as Raw267.4 as described in the EXAMPLE. In some embodiments, the screening cells express a first DNA that encodes a TRAAK channel and optionally a second DNA encoding for AT2R. In some embodiments, the screening cells express a first DNA that encodes or express a TRAAK channel and a second DNA encoding for AT2R. Method for introducing a nucleic acid sequence encoding for a protein of interest are well known in the art.

In some embodiments, the screening method consists in determining whether the test compound is able to induce hyperpolarisation of the screening cells. The step of contacting the screening cells with a test compound is accomplished for example by adding the test compound to the saline solution used for the patch clamp assay. The step of contacting a screening cell with a test compound also includes contacting the cell with a plurality of test compounds simultaneously, such as two or more test compounds, or three or more test compounds, etc.

Methods for analysing hyperpolarization of a screening cell are well known in the art and typically involve patch-clamp technique as described in the EXAMPLE. Indeed, single channel ion currents are studied using the patch-clamp technique (see, e.g., Neher and Sakmann (1976) Nature, 260: 799-802; Sakmann and Neher (1983) Single Channel Recording, Plenum New York), in which a glass pipette filled with electrolyte (i.e. saline solution) is used to contact the membrane surface and measure ionic current. Various chip-based patch clamping methods are also known (see, e.g., Fertig et al. (2002) Appl. Phys. Letts., 81: 4865-4867). In some embodiments the use of an ex vivo system comprising two chambers separated by a lipid bilayer, that contains channel of the present invention (i.e. TRAAK). The conductance across the lipid bilayer is monitor continuously. Typically, the TRAKK channel is activated so that the resting membrane potential becomes deeper in the negative direction, or in other words, so that the negative potential increases. The resting membrane potential is preferably deepened in the negative direction to a degree that does not affect cell viability. The induced hyperpolarization is -5; -6; -7; -8; -9; -10; -11; -12; -13; -14; -15; -16; -17; -18; -19; -20; -21; - 22; -23; -24; -25; -26; -27; -28; -29; -30; -31; -32; -33; -34; -35; -36; -37; -38; -39; -40; -41; - 42; -43; -44; -45; -46; -47; -48; -49; or -50 mV. In some embodiments, the induced hyperpolarization is compared to a predetermined reference value and when the hyperpolarization induced by the test compound is higher than the predetermined reference value then the test compound is selected. Typically the predetermined reference value represents the hyperpolarization determined in the absence of the test compound or the hyperpolarization induced by a reference compound (i.e. a compound which is known to activate the pathway of the present invention). Said reference compound is for example the myco lactone toxin as described in the EXAMPLE. In some embodiments, a phenotypic assay could be used for analysing hyperpolarization of a screening cell. Such a phenotypic assay may consist in the cell-based fluorescence assay coupled with image acquisition by automated confocal microscopy as described in the EXAMPLE. Briefly this assay consists of measuring changes in the fluorescent intensity of a potential-sensitive fluorochrome when the screening cells are contacted with the test compound. The potential-sensitive fluorochrome as used herein may be any of the types that are generally available in the art concerned and a suitable one may be selected from among the following: styryl-based potential-sensitive fluorochromes comprising ANEPPSs, ANRPEQs and RHs; cyanine- or oxonol-based potential- sensitive fluorochromes comprising DiSC's, DiOC's, DilC's, DiBAC's, and DiSBAC's; and rhodamine-derived potential-sensitive fluorochrome such as Rh 123, TMRM, and TMRE. In the present invention, it is more preferred to use styryl-based potential-sensitive fluorochromes comprising ANEPPSs, ANRPEQs and RHs, which are specifically exemplified by di-8-ANEPPS, di-4-ANEPPS, RH-237, RH-1691 , di-5-ASP, RH-160, RH-421, RH-795, di-4-ANEPPDHQ, ΑΝΝΓΝΕ-5, and ΑΝΝΓΝΕ-6, and a preferred potential-sensitive fluorochrome may be selected from among these. For fluorescence assay, any type of fluorescent microscope that can be used in the art concerned may be applied in the present invention and a typical example is 1X71 (OLYMPUS Corporation). In the Examples that follow, 1X71 (OLYMPUS Corporation) was used as a fluorescent microscope and combined with a suitable light source unit such as a mercury lamp (OLYMPUS Corporation) or an LED assembly (OLYMPUS Corporation). For the purposes of capturing fluorescent images, imaging and numerical calculations, any models of analysis software for imaging and numerical calculations that can be used in the art concerned may be applied in the present invention. In some embodiments, confocal images are recorded on an automated fluorescent confocal microscope Opera™ (Evotec) as described in the EXAMPLE. Each image are then processed using a dedicated image analysis software. In some embodiments, the screening cells (e.g. Raw267.4 cells) are stained with the potential-sensitive fluorochrome for a sufficient time. Then the extracellular dye is then washed away and image acquisition is performed (Tl). The test compound is then added to the assay plate, which is incubated for an additional sufficient time. A second image acquisition is then performed (T2). Automated image analysis determined the average intensities for each well and the intensity ratio is calculated ([T2int] / [Tlint]). Accordingly, in some embodiments, the screening method of the present invention comprises the steps of i) labeling the screening cells with the potential- sensitive fluorochrome into contact with screening cells of the present invention previously, ii) determining the fluorescence intensity [Tlint] of the screening cells, iii) bringing the screening cells into contact with the test compound, iv) determining the fluorescence intensity [T2int] of the screening cells, v) calculating the intensity ratio [T2int] / [Tlint] and vi) selecting the test compound when the intensity ratio is inferior to 1 (<1). In some embodiments, the intensity ratio is compared with the intensity ratio determined for a compound of reference (e.g. myco lactone), wherein when the intensity ratio determined for the test compound is about the same or inferior to the intensity ratio determined for the compound of reference, then the test compound is selected. In some embodiments, step i) is performed in presence of a blocker of AT2R or TRAA well known in the art (e.g. a siRNA specific for AT2R or TRAAK) and when the intensity ratio is equal or superior to 1 then it is concluded that the test compound (for which a decrease in the intensity ratio was previously determined) is a specific agonist of angiotensin II receptors (AT2Rs)- TWIK-related arachidonic acid stimulated K+ channel (TRAAK) pathway.

Typically, the test compound of may be selected from the group consisting of peptides, peptidomimetics, small organic molecules, antibodies, aptamers or nucleic acids. For example the test compound according to the invention may be selected from a library of compounds previously synthesized, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesized de novo. In some embodiments, the test compound may be selected form small organic molecules. As used herein, the term "small organic molecule" refers to a molecule of size comparable to those organic molecules generally sued in pharmaceuticals. The term excludes biological macromolecules (e.g.; proteins, nucleic acids, etc.); particular small organic molecules range in size up to 2000 Da, and most typically up to about 1000 Da. The screening methods of the invention are very simple. It can be performed with a large number oftest compounds, serially or in parallel. The method can be readily adapted to robotics. For example, the above assays may be performed using high throughput screening techniques for identifying test compounds for developing drugs that may be useful to the treatment or prevention of pain. High throughput screening techniques may be carried out using multi-well plates (e.g., 96-, 384-, or 1536-well plates), in order to carry out multiple assays using an automated robotic system. Thus, large libraries of test compounds may be assayed in a highly efficient manner. More particularly, stably-transfected cells growing in wells of micro-titer plates (96 well or 384 well) can be adapted to high through-put screening of libraries of compounds. Compounds in the library will be applied one at a time in an automated fashion to the wells of the microtitre dishes containing the transgenic cells described above.

In some embodiments, the test compounds that have been positively selected may be subjected to further selection steps in view of further assaying its properties in vitro assays or in an animal model organism, such as a rodent animal model system, for the desired therapeutic activity prior to use in humans. Any well-known animal model may be used for exploring the in vivo therapeutic effects of the screened test compounds. For example, the therapeutic activity of the screened test compounds can be determined by using various experimental animal models of pain known in the art such as those described in the EXAMPLE (e.g. Pain Receptive Assay). Once again the screened test compound may be compared to a reference compound such as a well-known analgesic or the myco lactone toxin. If the screened test compound provide the same effects or even better effects than the reference compound, said test compound could be typically selected for clinical investigation.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES:

Figure 1. Absence of Pain in Burn I i Ulcer Lesions Results from a Direct Anaesthetic Effect of Mycolactone. (A) The nociceptive reflex was quantified using a tail- flick Unit. Mice infected with ulcerans (Mu) showed redness at day 35 and oedema at day 45. NI: non-infected group [***P < 0.001, t test; n = 5 redness stage mice, n = 5 oedema mice]. (B) Histological analysis of cutaneous tissue from the footpad inoculated with M. ulcerans- GFP (green). Blue, DAPI; red, β3 tubulin staining with Texas red-conjugated anti- 3 tubulin antibody. Representative image from a set of slides of tissues from 5 mice (30 slides per tissue) that were analysed at days 35 and 45 after ulcerans-GFP inoculation. Scale bars: 100 μηι. Inset: Picture of the corresponding mouse footpad. (C) Observation at the optical microscope and (D) ultrastructural (electron microscope) levels of sciatic nerves innervating the infected (left) and non-infected (right) footpads of animals at either the redness or the oedema stage of injury. The black dotted line circumscribes a Remak bundle. Ax: axon; Mi: mitochondria; Sch N: Schwann cell nucleus; Mt: microtubule; Mf: microfilament; My: myelin; CF: C-Fibre; SLI: Schmidt-Lanterman incisures. The scale bars in C are 40 μηι, and those in D are 5 μηι. (E) The nociceptive reflex was measured in mice after injection of 5 μg of purified myco lactone (black bars), synthetic myco lactone (hatched bars), lidocaine and prilocaine anaesthetics (grey bars) or absolute ethanol alone (vehicle control) (white bars).

Figure 2. Mycolactone Provokes Hyperpolarization via K+ Channels in Murine Primary Neurons. (A) Representative chart recordings of the membrane potential of PC 12 cells challenged either by ethanol (vehicle), purified mycolactone or synthetic mycolactone. (B) Pooled data illustrating a 10 mV-hyperpolarization triggered by continuous application of purified mycolactone or synthetic mycolactone for 10-20 min. (C) Photomicrographs of hippocampal neurons loaded with bis-oxonol (DiSBAC2(3)) and inoculated with mycolactone at 350 ng mL or with ethanol (vehicle) at various times. Scale bars: 100 μηι. (D) Quantification of the fluorescence intensity expressed as the ratio of bis-oxonol (DiSBAC2(3)) signal intensity at 20 min (T2int) to that at 0 min (Tlint). (E, F) Effect of two + channels blockers, tetraethylammonium (TEA) and barium chloride (BaCb) on the cell hyperpolarization caused by mycolactone.

Figure 3. AT2R is Targeted by Mycolactone. (A) Fluorescence ratio of Raw267.4 cells treated either with siRNA targeting AT₂R or with scrambled siRNA, and further loaded with bis-oxonol (DiSBAC₂(3)) before incubation with mycolactone. The ratio is normalised to that of the scrambled siRNA control. (B) Fluorescence ratio of neurons from AT2R deficient (AT2R KO) or wild-type (WT) mice; the neurons were loaded with bis-oxonol (DiSBAC2(3)) before incubation with mycolactone. (C) Competitive binding of mycolactone to human AT2R. (D) Fluorescence ratio of neurons incubated with PD123,319, a selective inhibitor of AT2R and loaded with bis-oxonol (DiSBAC2(3)), before addition of mycolactone. (E) Fluorescence ratio of neurons incubated with Ga inhibitor pertussis toxin (PTX) and loaded with bis-oxonol (DiSBAC2(3)) before addition of mycolactone. (F) Fluorescence ratios of Raw267.4 cells treated with various siRNAs or scrambled siRNA and loaded with bis-oxonol DiSBAC2(3) before incubation with mycolactone. The ratios are normalised to that of the scrambled siRNA control. Pla2g2f, Pla2glbr and Pla2gl0 are members of three different groups of PLA2- encoding genes. Ptgsl : COX-1 ; Ptges2: prostaglandin E synthase 2 Kcnk4: potassium channel subfamily K member 4; cnkl3 : potassium channel subfamily K member 13. (G) Fluorescence ratio of neurons incubated with FR122047, a selective inhibitor of Ptgsl, before addition of myco lactone. (H) Current increase in inside-out patches containing TRAA after application of 10 μΜ ΡΟΕ2.

Figure 4. M. ulcerans (Mu) and Mycolactone-mediated Hypoesthesia Impaired in vivo Upon Blockage of the AT2R Signalling Cascade. (A) Detection of AT2R by western blot in neurons of wild-type (WT) or AT2R-deficient mice (AT2R KO). PC12 cells, strongly expressing AT2R were used as a positive control. (B) The nociceptive reflex was measured in AT2R-KO (grey bars) or wild-type (white bars) mice, which had been inoculated with 5 μg of mycolactone. (C) The nociceptive reflex was measured in AT2R- O (grey bars) or wild-type (white bars) mice, which had been inoculated with M. ulcerans. (D) The nociceptive reflex was measured in wild-type mice with M. ulcerans-induced non-ulcerative lesions, subsequently treated with PD123,319 (white bars) or water vehicle (grey bars). (E) The nociceptive reflex was measured in wild-type mice with M. ulcerans-induced non-ulcerative lesions, subsequently treated with piroxicam (white bars) or gelosed water only (grey bars).

Figure 5. Model of the Signalling Cascade triggered by Mycolactone. Upon binding to the AT2R receptor, coupled to God, mycolactone triggers a signalling cascade leading to PLA2 activation and inducing the synthesis of arachidonic acid. Arachidonic acid is then metabolized by COX-1 into PGE2, leading to the opening of TRAAK channels. AA: arachidonic acid; PTGS1 : COX-1 ; PGE2: prostaglandin E2; TRAAK:potassium channel subfamily K member 4.

Figure 6. Mycolactone application increases TRAAK current when AT2R is co- expressed. TRAAK current densities before and after mycolactone application. Representative TRAAK current before and after mycolactone application (5μΜ) Current was elicited by voltage-ramps (from -100 to 50 mV, Is in duration). Student's t test (**P < 0.01). The numbers of cells tested are indicated in parentheses.

Figure 7. C21 inhibits TRAAK current when AT2R is co-expressed

TRAAK current densities before and after C21 application (Fig 7A). Representative TRAAK current before and after C21 application (5μΜ) (Fig 7B). Current was elicited by voltage-ramps (from -100 to 50 mV, Is in duration). Student's t test (*P < 0.05). The numbers of cells tested are indicated in parentheses. Figure 8. TREK1 is not sensitive to AT2R activation by Mycolactone. TREK1 current density before and after mycolactone application. Representative TREK1 current before and after mycolactone application (5 μΜ) Current was elicited by voltage-ramps (from -100 to 50 mV, Is in duration). The numbers of cells tested are indicated in parentheses.

EXAMPLE: Material & Methods Bacterium

M. ulcerans (strain no. 1615) was originally isolated from a skin biopsy of a hu man patient from Malaysia (George et al., 1999) The recombinant M. ulcerans-GFF bacterium (strain no. JKD8083) was derived from an Australian strain in which green fluorescent protein (GFP) expression is controlled by the mis SigA-like promoter (Tobias et al., 2009).

Mycolactones

Mycolactones were purified from M. ulcerans (strain 1615) extracts as previously described (George et al., 1999). Mycolactone purity was estimated to be better than 98%, based on HPLC profiles. Mycolactone was diluted to 4 mg/mL in absolute ethanol and stored in the dark in amber glass tubes. Synthesis of mycolactone was described elsewhere (Gersbach et al, 201 1; Scherr et al, 2013). Synthetic mycolactone was diluted to 500 ng/niL.

Animal Experimentation

AT2Rknockout mice were generated and provided by L. Hein (Hein et al, 1995). Six- week-old female FV BN wild-type (Charles River France, http://www.criver.com/ico) and AT2Rknockout (Hein et al., 1 95) mice were maintained under conventional conditions in the animal house facility of the Centre Hospitalier Universitaire, Angers, France (Agreement A 49 007 002), adhering to the institution's guidelines for animal husbandry. Mouse Models

For inoculation with M. ulcerans, fifty microlitres of a suspension containing 5 ^x 10⁴ bacteria were injected subcutaneously into the left footpads of mice. For inoculation with mycolactone, five micrograms of mycolactone diluted in 10 μΐ of ethanol were injected subcutaneously into the left footpads of mice. In controls, 10 μΐ of absolute ethanol alone were injected into the left footpads.

Pain Receptive Assay

The pain receptive assay was performed by adapting the tail- flick procedure, using the tail- flick Unit (UGO BASILE). The sensitivity of the tissue to a noxious thermal stimulus was measured. The radiant heat stimulus was focused on the left footpad. The assay was performed twice each week on mice that had been previously anesthetized. This assay was validated by local application of Emla® analgesic cream containing 5% lidocaine/prilocaine (Astra Zeneca).

Immunochemistry

After sacrifice, footpads were removed, frozen in isopentane cooled to -30°C, and stored at -80°C. Frozen tissues were serially sectioned using a Leica cryostat. Sections (12 μητ) were fixed in cold methanol for 10 min and washed 3 times in PBS before blocking at room temperature for 1 hour in BSA 5%. Sections were incubated overnight with Texas red- conjugated anti-B3 tubulin antibody (Sigma) diluted 1/200 in PBS with 5% BSA and then rinsed with PBS (3 x 5 minutes). The preparations were counterstained with 3 μΜ 4'6- diamidino-2-phenylindole (DAPI; Sigma) for 5 min and washed twice with PBS. Slides were mounted with an anti-fading solution and observed with a confocal microscope (LSM, Leica). Tissues from 7 mice per stage were analysed, with 50 slides per mouse.

Mouse Sciatic Nerve Sections for Transmission Electron Microscopy

After deep anaesthesia, a perfusion with phosphate buffer was performed after section of the femoral artery. Fixation of the tissue was then performed with a second perfusion with 4% paraformaldehyde and 0.1 % glutaraldehyde in phosphate buffer fixation. The sciatic nerve was subsequently isolated by dissection. The samples were fixed for 30 min in 0.1 M cacodylate buffer (pH 7.2) containing 2.5% glutaraldehyde for 1 h at 4°C and left to stand for 12 h at 20°C in cacodylate buffer. Specimens were progressively dehydrated and embedded in Araldite (Fluka). After dehydration, thin sections were stained with uranyl acetate and Reynold's lead citrate and then examined on a JEOL 120 EX electron microscope.

Cell Culture

Macrophage cells (Raw264.7, ATCC TIB-71) were cultured in RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated foetal calf serum (Invitrogen). Pheochromocytoma cell 12 (PC 12) cells were plated on collagen IV-coated culture dishes in DMEM supplemented with 10% horse serum and 5% foetal bovine serum. After 6 days, differentiation to a neuronal phenotype was induced by incubation in DMEM supplemented with 2% horse serum, 1% foetal bovine serum, and 100 ng/mL nerve growth factor (Invitrogen). The culture medium was changed every two days until differentiation was complete (6-7 days). Primary cultures of hippocampal neurons were prepared from neonatal (P0 to P2) BALB/c and FVB N mice according to an established protocol (Banker and Cowan, 1977) with minor modifications.

Electrophysiology

The membrane potential of PC- 12 cells was recorded using the whole-cell configuration of the patch-clamp technique in the current-clamp mode. PC-12 cells were grown for 7 days on glass coverslips before being harvested. After an overnight incubation, the glass coverslips were transferred into a recording chamber and continuously superfused with Ringer's saline buffer containing 125 mM NaCl, 2.5 mM KC1, 2 mM CaCl₂, 1 mM MgCb, 1.25 mM NaH₂P0₄, 26 mM NaHCOa, 12 mM glucose, buffered to pH 7.4, at 30-32°C and bubbled with 95% 0₂ and 5% CO2. Recording pipettes (3-5 MW resistance) were filled with 140 mM potassium gluconate, 5 mM KC1, 2 mM MgCl₂, 1.1 mM EGTA, 5 mM HEPES, 4 mM MgATP, 0.3 mM NaGTP and titrated to pH 7.2 with KOH. Membrane potential changes were monitored using an EPC10 quadra USB patch-clamp amplifier and PATCHMASTER software (HEKA Electronik, Germany). Signals were sampled at 10 Hz, filtered at 3 kHz and analysed with FITMASTER software (HEKA Electronik). Cells were current-clamped close to their resting potential (-55 to -65 mV in average). Membrane potential was continuously monitored for 10- 20 min before and 10-20 min after my co lactone application at 1.4 g/mL using a pneumatic drug ejection system (PDES, npi electronic GmbH, Germany). Control cells were stimulated with 2% ethanol as vehicle. For IC50 determinations for TRAAK, defolliculated Xenopus oocytes were injected with 50 nL of cRNA at 0.02-0.4 μg/μL encoding either mTRAAK and recorded 2-4 days later. For electrophysiology, single oocytes were placed in a 0.3-mL perfusion chamber and impaled with two standard microelectrodes (1-2.5 ΜΩ resistance) filled with 3 M KC1 and voltage clamped with a Dagan CA-1 amplifier, in ND96 solution (96 mM NaCl, 2 mM KC1, 1.8 mM CaCl₂, 2 mM MgCl₂, 5 mM Hepes, pH 7.4 with NaOH). Stimulation of the preparation, data acquisition, and analysis were performed using pClamp software (Axon Instruments). Cell-based Fluorescence Assay, Image Acquisition by Automated Confocal Microscopy and Data Analysis

aw264.7 macrophages were harvested with Versene (Gibco™) and seeded at a density of 10⁴ cells per well in 384-well plates (Evotec, Hamburg, Germany) in 50 μΐ RPMI 1640 supplemented with 10% heat-inactivated foetal calf serum (FCS) (all from Invitrogen). After overnight incubation, the medium was removed, and the cells were labelled for 2 hours at 37°C with bis-(l ,3-diethylthiobarbituric acid) trimethine oxonol (DiSBAC2(3), Invitrogen, B413) at 62.5 g mL. The cells were then washed three times with imaging buffer containing 150 mM NaCl, 5 mM C1, 10 mM HEPES, 2 mM CaCk, 2 mM MgCl₂, 5.5 mM glucose, 2.9 mM sucrose. A first series of image acquisition was conducted to ascertain the proper labelling of the cells (Tlint); myco lactone was then added to the cells. After 20 minutes incubation at room temperature under mild shaking (500 rpm), a second scan was performed to measure the intensity of the fluorescence of the cells (T2int). Confocal images were recorded on an automated fluorescent confocal microscope Opera™ (Evotec) using a 20X water objective (NA 0.70), 532-nm laser and 600-nm camera. Four fields per well were recorded. Each image was then processed using dedicated in-house image analysis software (IM). The image dynamics were checked through a series of statistical tests (assessing notably average intensities and standard deviations), designed to allow the removal of black or out-of-focus images. DiSBAC2(3) staining was then segmented in the "DiSBAC stain" band using an in-house method (Fenistein et al., 2008), and the ratio of the surface intensity of the "DiSBAC stain" before and after myco lactone addition was determined for each well. This ratio is referred to as the Ratio [T2int/Tlint].

Competitive Binding of Mycolactone to the Human AT2R

[¹²⁵I]CGP 42,112A at 0.01 nM and were used as agonist radioligand. Radioactivity was detected by scintillation counting (Cerep Inc, Seattle, WA, Assay Reference 0026). The radioligand was first incubated with membranes from human recombinant HE -293 cells and the bound radioactivity was measured. The membranes were then incubated with a high concentration of angiotensin II (1 μΜ) and with [¹²⁵I]CGP 42,1 12A radioactive ligand to determine non-specific binding of the ligand to the AT2R receptor. The radioligand and the mycolactone (8 different concentrations) were incubated with the cell membranes for 4 h at 37°C to determine the IC50 of the mycolactone. Chemical Reagents

Tetraethylammonium (TEA), an inhibitor of various potassium channels and Piroxicam, a highly active inhibitor of prostaglandin were from Sigma. EMLA cream containing 5% lidocaine and prilocaine anaesthetics was from Astra Zeneca. FR122047, a selective inhibitor of COX-1 signalling and PD123,319, a selective inhibitor of AT2R signalling were from Tocris Bioscience. For the mouse study, 200 (daily renewed) solution of piroxicam at 0.5 mg/mL in 1 % gelosed water was administered by gavage every day starting 30 days after M. ulcer cms inoculation. Osmotic mini-pumps (AlzetVR model 2002, Palo Alto, CA) containing PD123,319 were implanted subcutaneously under anaesthesia in the back of the neck of mice 30 days after M. ulcer ans inoculation, with a delivery rate of 20 mg/kg/day in water.

Results

M. ulcerans Induces Hypoesthesia in Early Stages of Oedema

We developed a mouse model to assess the relationship between hypoesthesia and nerve degeneration under low dose conditions of ulcerans infection. M. ulcerans was injected into the footpads of mice, and hypoesthesia was monitored with the pain receptive tail- flick assay at different time points. Under such conditions, mice exhibited redness at day 35 post-infection and oedema at day 4 . At both time points, hypoesthesia was observed in M. ulcerans- fected animals, with a significant tail- flick latency of 4 seconds, as compared to non-infected controls (Figure 1A). Histological analysis of mice inoculated with M. ulcerans expressing green fluorescent protein (GFP) showed the presence of bacilli around the inoculation site, both in the stages of redness and oedema (Figure IB). On the other hand, no GFP bacilli were found in nervous tissue, either in glial cells or in axons. Macroscopic and ultrastructural inspection revealed no signs of nerve degeneration (Figure 1C, ID). Similarly, extensive examination of more than 480 Remak fibres showed no differences between M. ulcerans -infected footpads and non-infected controls at the level of cytoskeletal cell composition, organisation of microtubules and filaments, structure of unmyelinated axons or size of Schwann cell nuclei (Figure 1C, ID). Finally, no obvious anomalies were observed in the infected footpads at the level of myelinated or C-fibres involved in sensory information transmission (Besson, 1999; Griffin and Thompson, 2008). Taken together, these results demonstrate that hypoesthesia in M. ulcerans infection is not attributable to nerve degeneration, at least in this mouse model.

We next investigated the effect of direct inoculation of purified mycolactone on hypoesthesia and nerve damage. Used as control, lidocaine induced a transient hypoesthesia in our model shortly after injection. Low doses of mycolactone produced sustained analgesia for up to 2 days (Figure IE). Noticeably, nociceptive abilities were recovered after 8 days. Under these conditions, as for M. lcerans-mfected samples, we performed a close inspection of emak fibres observing again that the Schwann cells were intact (Besson, 1 99; Hoeijmakers et al., 2012). Thus, mycolactone injection alone is sufficient to produce hypoesthesia without nerve damage. Further, we also tested chemically synthesized mycolactone, to rule out possible effects of contaminants in our preparation of purified mycolactone. The synthetic mycolactone induced the same level of reversible hypoesthesia as the purified toxin, with the same concentrations and application durations. This result clearly demonstrates that the observed analgesic effect is attributable to the mycolactone toxin (Figure IE).

Mycolactone Induces Hyperpolarization of Neurons

With this hypothesis of nerve degeneration to account for hypoesthesia ruled out, it was natural to ask whether mycolactone could instead produce hypoesthesia by interfering with relevant neural transmission pathways. To this end, we examined the effect of mycolactone on membrane potential in neurons derived from PC 12 cells using the patch-clamp technique in the current-clamp mode. Upon local and continuous application, mycolactone induced a slowly developing cell hyperpolarization. As compared to vehicle-treated neurons, an average decrease of - 10 mV in the membrane potential was observed after 20 minutes of incubation with purified mycolactone at sub toxic doses (Figure 2A, 2B). The same membrane potential change was also observed with the synthetic mycolactone, at the same concentration. After removal of the toxin, the cells recovered their resting membrane potential, displaying signal intensities similar to those before addition of toxin. As a matter of fact, such reversibility ofthe effect of mycolactone also supports the conclusion relative to the absence of neuronal damage (Figure 2C). For enhanced characterization, we next developed an image-based phenotypic assay based on the monitoring of mycolactone-induced hyperpolarization. More specifically, neurons were loaded with the voltage-sensitive fluorescent bis-oxonol probe DiSBAC2(3) (DallAsta et al, 1997) and exposed to mycolactone. In this assay the cell fluorescence decreased within 20 minutes (Figure 2C), with the cells exhibiting the same type of slowly developed hyperpolarization as the one observed by electrophysiological recordings of membrane potentials. Accordingly we decided to monitor the effect of toxin at 20 minutes in the following experiments. For image- based quantification of the samples, the DiSBAC2(3) signal was recorded before addition of toxin and 20 minutes after ([Tlint] and after [T2int], respectively), and the intensity ratio [T2int/Tlint] was calculated. Such quantification of the samples showed that 40% of the signal intensity was lost in the presence of 0.175 ng/niL mycolactone (Figure 2D). Hyperpolarization is typically associated with K+ channels, and we checked that such channels were involved in the mycolactone-induced hyperpolarization. To this end we treated cells with the two + channel blockers teftaemylammonium (TEA) and barium chloride (BaCh). As shown in Figures 2E and 2F, TEA and BaCb abolished the mycolactone-mediated hyperpolarization demonstrating that it is mediated by K+ efflux. At this level, the analgesic action of mycolactone appears to share common features with the non-opioid analgesic flupirtine, which also hyperpolarises neurons by activation of ⁺ channels (Szelenyi, 2013). Furthermore, the + channels involved in the mycolactone-mediated hyperpolarization appear to be sensitive to TEA and BaCb. only at relatively high concentrations (above 10 mM and 1 mM, respectively), suggesting that they belong to the K2P channel family (Noel et al., 2011; Sandoz et al, 2012).

Identification of AT2R as the Target Receptor for Mycolactone Involved in Cell Hyperpolarization

To further elucidate the cellular and molecular mechanisms underlying the action of mycolactone we seeked to identify its target receptor involved in the K+ mediated hyperpolarization. To this end we adapted the assay above to the large-scale screening of a siRNA library targeting 8,000 different host genes. Macrophage-like cells of the Raw267.4 cell line were used in this screening as they are efficiently silenced by siRNAs (Carralot et al., 2009). We first checked that the effect of mycolactone on hyperpolarization in Raw267.4 cells is the same as in neurons. For the screening experiments cells were first incubated with siRNA and then loaded with DiSBAC2(3) immediately before the addition of mycolactone. A decreased intensity ratio was observed after the addition of toxin to cells that had been incubated with non-target (scrambled) siRNAs. The most significant hits (roughly 1% of the total) were selected from the primary screen, leading to 34 siRNA-targeted genes upon retesting. In this set only three genes appeared to be associated with receptors, namely IL23R, ICAM1 and AGTR2. With this respect, it is noticeable that no genes encoding opioid receptors, common target of analgesics, were found among the most significant hits from the siRNA screen. Whereas and ICAM1 and IL23R ICAM1 , belonging respectively to the immunoglobulin and interleukin receptor families, do not appear to be associated in any -direct- known way with nociceptive pathways, the angiotensin II type 2 receptor (AGTR2 or AT2R) represented a favourable candidate to account for the observed analgesia effects. This receptor has been reported recently, in a completely different context, to be specifically involved in nociception in the course of experiments testing the effects of the AT2R chemical antagonist PD 123,319 (Anand et al, 2012). Accordingly we focused our subsequent investigations on AT2R receptors. First, we checked that silencing AT2R led to a high ratio value comparable to that of the scramble control in the absence of toxin (Figure 3A). We then proceeded to investigate in detail the potential role of AT₂R as a mediator of mycolactone-mediated hyperpolarization, seeking to fully characterize and decipher the associated pathway. As a first direct characterization, we showed that neurons isolated from AT2R knockout (AT2R- O) mice (Hein et al., 1995) were not hyperpolarized upon addition of mycolactone, with no observed decrease in DiSBAC2(3) signal (Figure 3B). Furthermore, we assessed in vitro the binding affinity of the mycolactone toxin to human AT2R, which displays 95% amino acid sequence identity with murine AT2R. Binding affinity of mycolactone to AT2R was determined in a radioligand competition assay using the agonist [¹²⁵I] CGP42,112A (Pelegrini-da-Silva et al., 2005). Mycolactone was able to inhibit the AT2R agonist with an IC50 value of 3 μg/mL (Figure 3C). We further analysed the expression of AT2R in our settings and found that AT2R was expressed in mouse footpad. In this assay, infection with M.ulcerans did not change any significantly the level of expression of AT2R, showing that AT2R is not upregulated after infection. To further pinpoint the causal role of mycolactone in cell hyperpolarization via AT2R receptors we performed two additional assays. First we showed that the AT2R-selective antagonist PD 123,319 inhibits mycolactone- induced hyperpolarization in neuronal cells (Figure 3D). In addition, it is known that AT2R is a G-protein-coupled receptor, which couples selectively to pertussis toxin (PTX)-sensitive Gia2 and Gia3 proteins (Kang et al, 1994; Sumners and Gelband, 1998). Upon pre-incubation of the cells with the G-cti inhibitor PTX (Figure 3E) we showed that mycolactone-mediated hyperpolarization was abolished, thus demonstrating that G-ai proteins are involved in mycolactone-induced AT₂R signalling. In conclusion, inhibition of mycolactone signalling in absence of AT2R was demonstrated through three different approaches, relying on chemical inhibition, genetic knock-down as well as genetic knock-out. We further checked whether mycolactone could also signal through ATiR, as is the case for angiotensin II. As a matter of fact mycolactone displayed binding affinity to the human ATiR similar to that measured for AT2R. Accordingly, we tested the effect of mycolactone in an ATiR functional assay whereby cytosolic Ca²⁺ ion mobilization is measured in HE -293 cells expressing ATiR. No effect of mycolactone was observed in this assay, up to 20 μg/mL, showing that despite its binding on the receptor mycolactone does not trigger ATiR activation. Confirming this result, we showed that mycolactone signaling was abolished in neurons from mice KO for AT2R (Clere et al., 2010; Hein et al., 1995) not impaired for ATiR expression, thus excluding a role for ATiR in myco lactone-induced hyperpolarization. AT2R Induces Cell Hyperpolarization via Cyclooxygenase Pathway

To dissect the mycolactone-induced pathway at the molecular level, we performed a series of additional analyses of our siRNA data, complemented by the screening of a chemical library of inhibitors with known targets. Based on the identification of the AT2R target above, the rationale in this two-pass screening approach was to reanalyse the siRNA data, with a significantly relaxed threshold, in order to further identify relevant genes involved in the underlying pathway seeking notably to bridge the gap between the AT₂R-input and + channels-output levels. In this second-pass screening the analyses were conducted in the light of information available in the literature, concerning various alternative pathways associated with the activation of AT2R. As a matter of fact it was documented in the literature that modulation of + currents by AT2R signalling can follow several different intracellular pathways (Nouet and Nahmias, 2000). The primary reported pathway involves the protein PTPN6-SHP1, which becomes phosphorylated upon AT2R activation (Bedecs et al, 1997). In our cellular model, it was possible to exclude this pathway for AT2R mediated signalling because addition of mycolactone did not result in any notable variation in the amount of phosphorylated SHP 1. Another reported pathway for AT2R modulation of K+ currents involves signalling through activation of phospho lipase A2 (PLA2) and release of arachidonic acid (AA) (Lauritzen et al., 2000). We showed that the AT2R-mediated signalling of mycolactone is consistent with this pathway. More precisely, we were able to dissect in detail the triggering of this pathway by mycolactone and to assess the effects of the enzymes involved in the biosynthesis and metabolism of AA. We found that mycolactone-induced hyperpolarization was inhibited upon silencing of several PLA2 homologues that mediate the release of AA from membrane phospholipids (Figure 3F). Mycolactone signalling was also impaired upon silencing of COX-1 (PTGS1), which converts AA into prostaglandins (Fletcher et al., 2010) (Figure 3F). We showed that inhibition of PTGS1 by the chemical inhibitors FR122047 and Piroxicam abolished mycolactone-mediated hyperpolarization (Figure 3G). As Piroxicam is a nonselective COX inhibitor, we further wanted to investigate the possible involvement of COX-2 in this pathway. Accordingly, we tested COX-2 inhibition either by silencing the expression of PTGS2 or by using COX-2 selective inhibitors (DuP-697 and NS398). In both cases we observed no effects in our assay, thus ruling out a putative role of COX-2 signaling in our scheme. Refined Characterization of the K+ Channels Subfamily

Beyond the detailed characterization of the molecular pathway between AT2R-input and + channels-output, the second-pass reanalysis of our siRNA primary screen further allowed refining the characterization of the K+ channels in terms of subfamily assignment. More precisely such reanalysis allowed to pinpoint two possible candidate subfamilies to which the potassium channels involved in the mycolactone induced hyperpolarization belong, namely CNK4 (TRAAK) and KCNK13 (THIK1) from the K2P channels family (Figure 3F). Channels from the K2P family function as regulatory hubs for the generation of negative resting membrane potentials and therefore they constitute appropriate final targets of the signaling pathway involved in the mycolactone-induced hyperpolarization. As high concentrations of either Barium or TEA reversed the mycolactone-induced hyperpolarization, we wanted to investigate the ability of BaCb and TEA to block the activity of TRAAK. We showed that TRAAK is sensitive to TEA and BaCb with an IC50 of 7 ± 2 mM and 0.5 ± 0.1 mM respectively. These IC50 values are in good agreement with the concentration that provokes an inhibition of the mycolactone-induced hyperpolarization (Figure 2E and 2F). Accordingly we asked the question as to which mediator was involved in TRAAK activation. Based on the evidence above concerning the involvement of the cyclooxygenase pathway in the AT2R- induced cell hyperpolarization, it was natural to consider first a possible direct role for arachidonic acid, known to activate TRAAK channels. However we could rule out this possibility, as PTGS1 inhibitors abolished mycolactone-induced hyperpolarization. Accordingly we reasoned that the mediator responsible for the activation of TRAAK channels could correspond to one of the metabolites of arachidonic acid. With this respect, the analysis of our screen data identified only one relevant candidate, namely the enzyme prostaglandin E synthase 2 (PTGES2) (Figure 3F). Application of PGE2, the synthesis of which is catalyzed by Ptges2, induced a 4.1 ± 0.8 fold increase of the TRAAK current showing that release of potassium via TRAAK can be mediated by PGE2 (Figure 3H). Altogether these results are consistent with a model in which the mycolactone-induced pathway, via AT2R at the input level, results in an increased activity of TRAAK potassium channels at the output level, with the intermediate pathway as detailed above.

Blockage of the AT2R Signalling Cascade Results in Inhibition of Hypoesthesia caused by Mycolactone

Completing the rationale for this molecular characterisation, we further demonstrated its physiological relevance by investigating in vivo the causal role of AT2R in hypoesthesia. We first verified that AT2RS are not expressed in neurons in our KO model (Hein et al., 1995) (Figure 4A). When mycolactone was injected into the footpads of AT2R KO mice, the animals exhibited unaltered pain sensitivity in the tail-flick test, with an average response time of 8 seconds, which contrasted with a delayed response by an average of 12 seconds in wild-type animals (Figure 4B). Thus, AT2R expression is critical for the occurrence of mycolactone- mediated hypoesthesia under physiological conditions. This result was substantiated by the tail- flick test on wild-type and AT2R-KO mice that presented non-ulcerative lesions after infection with M. ulcerans (Figure 4C). In addition, upon treating M.ulcerans-infected mice exhibiting oedema with the selective AT2R blocker PD123,319 or with piroxicam, an inhibitor of prostaglandin synthesis by PTGS 1 , we observed the same pain-sensitive phenotype as in the AT2R-KO animals (Figure 4D, 4E). Taken together our results demonstrate the pivotal role of AT2R signalling cascade in buruli ulcer induced hypoesthesia.

Mycolactone triggers TRAAK activation in heterologous system

As shown in Figure 6A and B, Mycolactone is able to induce a 3 fold increase of the TRAAK current obtained from HEK cells expressing AT2R and TRAAK channel. It is worth to note that Mycolactone as no effect on TRAAK current in the absence of AT2R. In addition, the TRAAK current density shows no modification induced by AT2R expression showing that the AT2R has no basal activity for the AT2R- TRAAK pathway.

Mycolactone triggers a very specific signaling to activate TRAAK

Importantly, we showed that mycolactone-dependent activation of AT2R receptor induces a K+-TRAAK dependent hyperpolarization, as a difference from the action of the compound C21 for which we did not observe TRAAK activation (Figure 7A&B) but we observed a diminution of the TRAAK current which is translated in a depolarization of the membrane potential of the cell. These results demonstrate that AT2R activation by Mycolactone triggers a very specific signaling involving TRAAK.

TREKl is not sensitive to AT2R activation by Mycolactone.

We further asked the question whether TREKl, which belongs to the same channel subfamily as TRAAK, sharing a high degree of homology with TRAAK, could be activated by Mycolactone. We showed that TREKl is not sensitive to AT2R activation by Mycolactone (Figure 8, P>0.8), thus the role of TREKl in our system will not be investigated any further. In addition, as TREKl is known to be activated through a canonical GiPCR pathway, the absence of regulation in our system provides an additional argument to rule out a major contribution of a classical G protein pathway.

Thus these recent results taken together further give weight to our working model concerning a very specific complex formed by AT2R and TRAAK. In such context it appears reasonable to hypothesize that the AT2R-TRAAK signaling pathway may require a particular spatial organization localized at the plasma membrane that we will further investigate.

Discussion:

The focus of the work here is the molecular dissection of a previously unanticipated infection strategy employed by a bacterium that uses the mycolactone toxin as an effective analgesic to annul the pain of the lesions it causes. It was previously believed that absence of pain in Buruli ulcer disease could be attributed to nerve damage, caused by the action of the toxin mycolactone secreted by M. ulcerans. Accordingly we first revisited this hypothesis to test it in detail. We demonstrated, with purified as well as synthetic mycolactone, that the analgesic effect of the toxin was not accompanied by nerve degeneration. In addition, it is clear that M. ulcerans does not associate with nerve fibres and it is most likely that the toxin reaches neurons through diffusion. Indeed we have previously shown that the toxin mycolactone can be shuttled within vesicles in human lesions (Marsollier et al., 2007). In this background we endeavoured to explore alternative mechanisms, other than nerve damage, accounting for the analgesic effect of mycolactone. Instead we sought to investigate possible interference of the toxin with neural processes which could be involved in pain perception and we addressed the question of painlessness for early lesions that are not yet necrotic. It is likely that the effect of mycolactone on necrosis is relevant to yet another phenomenon (Guenin-Mace et al, 2013; Sarfo et al., 2013).

At a global level, we first demonstrated with electrophysiology that mycolactone, purified as well as synthetic, exerts a clear-cut effect on membrane potential, inducing hyperpolarization of neurons. Based on this result we then deployed a coherent, multifaceted approach, to unravel the detailed molecular pathway underlying this effect. In this approach we identified, at the input level, AT2R receptor as the target of mycolactone. We further confirmed this result by showing that mycolactone- induced hyperpolarization is inhibited upon blocking AT₂R expression. Similarly, we showed that at the output level hyperpolarization was mediated by + channels, and we further refined this characterization showing the specific involvement of TRAAK potassium channels. In order to gain a complete molecular picture of the effect of myco lactone, we further dissected the intermediate pathway between the input and output levels, showing that the intracellular pathway involved i) activation of phospholipase A2, ii) release of arachidonic acid by COX-1 and iii) activation of TRAAK by PGE2. -Finally to firmly validate these findings on physiological grounds, we demonstrated the causal role in vivo of AT2 in hypoesthesia.

In a global context, it will be interesting in future studies to consider in more detail several aspects of the pathway underlying the hypoesthesia model above. Thus, concerning the binding of mycolactone to AT2R, the observed difference between affinities measured in vitro and quantifications in tissues can seem at first surprising. However very little is known about the stability and the metabolism of the toxin in vivo, with the possibility that metabolites derived from the toxin being active on AT2R (Scherr et al., 2013). The investigation of such possibility can then be of potential pharmaceutical relevance. With this respect it is also significant that a similar range of difference in concentrations between in vitro binding and cellular activity assays was previously reported for mycolactone in a completely different context (Guenin- Mace et al., 2013). Another intriguing aspect in our hypoesthesia model concerns the involvement of COX-1, whereas analgesic effect of NSAIDs was shown to be associated with the inhibition of COX-1. Such observations clearly identify COX-1 as central, multifaceted, player in the control of pain. Of course it will be highly informative to determine whether such different mechanisms could be associated with different types of pain. Also COX-1 is known to mediate anti-inflammatory effect in immune cells, raising the question whether the AT2R- mediated analgesic effect could also involve other immune cells. As a matter of fact, our demonstration here of the effect of mycolactone on macrophages, again via AT2R, raises the possibility that the toxin acts as a multi-target weapon. Even though the scope of the study here concerned neuron targets for hypoesthesia, future studies concerning macrophages should identify the underlying effects (such as anti-inflammatory) in the case of such targets.

Beyond the molecular and physiological characterizations, the findings here can be cast in more general perspectives relevant to the theme of counteracting pain on health-oriented grounds. It is particularly significant to notice that the pathway dissected here involves a natural effector that does not belong to the different classes of analgesics of common use today, such as paracetamol (or acetaminophen), opiate molecules such as morphine or non-steroidal antiinflammatory drugs (NSAID) such as salicylates. Notably because of series of, more or less severe, secondary effects it is rather unanimously recognized that new potent analgesics, showing less adverse effects, are highly desirable. With this respect the perspective in connecting the understanding of the implementation of analgesia in natural systems, such as the one described here, to the rational development of medical pain-killing products does not appear to be unrealistic. Emerging, but as yet disparate, results of genetic as well as pharmacological studies in humans and rodents, point towards the critical involvement of various components of the pathway deciphered here in different steps of pain perception and alleviation. Thus, it is highly significant that recent studies aiming to identify rare genetic variants associated with pain sensation precisely pinpointed angiotensin pathways as being of critical importance in trait heritability for pain sensitivity (Williams et al., 2012). On the other hand AT₂R receptors have been pinpointed recently, in completely independent contexts, as important potential targets for several synthetic analgesic drugs. Thus AT2R antagonist PD 123,319 (also called EMA200) was reported to be effective in a rat model of prostate cancer-induced bone pain (Anand et al., 2012; Muralidharan et al, 2014). This is further corroborated by the fact that other EMA derivatives, EMA300 and EMA401 alleviate neuropathic pain through AT₂R (Rice et al., 2014; Smith et al., 2013).

It is then well possible that various such results may fit into a coherent yet multifaceted model, inspired by the one developed here (Figure 5), with the perspective of developing a rational basis for the design and selection of efficient analgesic molecules, directly inspired by natural ones. On more immediate grounds and in terms of health policies, unmasking the pathogenesis strategies of the M. ulcerans bacillus should contribute to a more knowledgeable attitude towards the disease and encourage patients to seek care in the early stages of infection.

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Claims

CLAIMS:

1. A method for screening a plurality of test compounds useful for treatment of pain comprising the steps consisting of (a) testing each of the test compounds for its ability to activate the angiotensin II receptors (AT2Rs)- TWIK-related arachidonic acid stimulated K+ channel (TRAAK) pathway and (b) positively selecting the test compounds capable of said activation.

2. The method of claim 1 which comprises a first step of determining whether the test compound is able to bind to TRAAK or AT2R.

3. The method of any of claims 1 or 2 wherein the test compounds are labeled with a detectable label.

4. The method of claim 3 wherein the detectable label is a fluorescent label, a colorimetric label, a radioactive label, a spin label, or a radioopaque label.

5. The method of any of claims 3 or 4 wherein a membrane comprising TRAAK or AT2R is contacted with the test compound, typically washed, and then the membrane is screened for the detectable label indicating association of the test compound with the protein of interest.

6. The method of any of claims 1 to 4 which is performed in screening cells.

7. The method of claim 6 which is performed in a screening cell that expresses a first DNA that encodes a TRAAK channel and optionally a second DNA encoding for AT2R.

8. The method of claim 6 which is performed in a screening cell that expresses a first DNA that encodes a TRAAK channel and a second DNA encoding for AT2R.

9. The method of any of claims 6 to 8 which consists in determining whether the test compound is able to induce hyperpolarisation of the screening cells.

10. The method of claim 9 wherein analysing hyperpolarization of the screening cell involves the patch-clamp technique.

11. The method of claim 9 wherein analysing hyperpolarization of the screening cell is determined with a phenotypic assay.

12. The method of claim 11 wherein the phenotypic assay consists in the cell-based fluorescence assay coupled with image acquisition by automated confocal microscopy.

13. The method of claim 12 wherein the phenotypic assay consists of measuring changes in the fluorescent intensity of a potential-sensitive fluorochrome when the screening cells are contacted with the test compound.

14. The method of claim 13 wherein the potential-sensitive fluorochrome is selected from styryl-based potential-sensitive fluorochromes comprising ANEPPSs, ANRPEQs and RHs; cyanine- or oxonol-based potential-sensitive fluorochromes comprising DiSC's, DiOC's, DilC's, DiBAC's, and DiSBAC's; and rhodamine-derived potential-sensitive fluorochrome such as Rh 123, TMRM, and TMRE.

15. The method of any of claims 13 or 14 which comprises the steps of i) labeling the screening cells with the potential-sensitive fluorochrome into contact with screening cells, ii) determining the fluorescence intensity [Tlint] of the screening cells, iii) bringing the screening cells into contact with the test compound, iv) determining the fluorescence intensity [T2int] of the screening cells, v) calculating the intensity ratio [T2int] / [Tlint] and vi) selecting the test compound when the intensity ratio is inferior to 1 (<1).

16. The method of claim 15 wherein the intensity ratio is compared with the intensity ratio determined for a compound of reference such as mycolactone, wherein when the intensity ratio determined for the test compound is about the same or inferior to the intensity ratio determined for the compound of reference, then the test compound is selected.

17. The method of claim 1 wherein the step i) is performed in presence of a blocker of AT2R or TRAAK such as a siRNA specific for AT2R or TRAAK, and when the intensity ratio is equal or superior to 1 then it is concluded that the test compound for which a decrease in the intensity ratio was previously determined is a specific agonist of angiotensin II receptors (AT2Rs)- TWI -related arachido ic acid stimulated K+ channel (TRAAK).

18. The method of any of claims 15 to 17 wherein the test compounds that have been positively selected may be subjected to further selection steps in view of further assaying its properties in vitro assays or in an animal model organism, such as a rodent animal model system, for the desired therapeutic activity prior to use in humans pathway.