Advancement of Small Molecule PSD95-nNOS Modulators as Novel Non-Opioid Analgesics

Background & Aims

Glutamate activation of N-methyl-D-aspartate (NMDA) receptors mediates central nervous system (CNS) sensitization, which is implicated in the development and maintenance of neuropathic pain. NMDA-mediated central sensitization depends on formation of a multi-protein complex at the receptor consisting of the NMDA receptor, the scaffolding protein, postsynaptic density protein 95 (PSD95), and neuronal nitric oxide synthase (nNOS). By bringing these proteins close together, multiple signaling cascades are activated leading to neural network reorganization (plasticity). Molecules that disrupt this complex act as effective analgesics in preclinical animal models with better side effect profiles than non-selective NMDA receptor antagonists and nNOS inhibitors. The aim of this program is to advance a new, orally available, small molecule targeting the PSD-nNOS complex through IND-enabling studies for the treatment of neuropathic pain.

Methods

Anagin, a preclinical stage company, in partnership with the BPN for small molecules program (https://neuroscienceblueprint.nih.gov/neurotherapeutics/bpn-small-molecules) and Anagin’s collaborators, has advanced compounds into the late lead optimization phase of small molecule drug development. Methods employed include traditional medicinal chemistry and computer-aided drug design approaches, evaluation of new compounds in binding and enzymatic assays, and evaluation of compounds for drug-like properties in in vitro ADME assays. Pharmacokinetic studies were conducted to understand plasma and brain levels after intravenous and oral administration. Target engagement assessment included in vitro profiling, cell-based co-immunoprecipitation studies in primary rat cortical neurons and evaluation of compound activity in the NMDA-induced thermal hyperalgesia model. Molecules were also evaluated in rodent models of neuropathic pain.

Results

Compound advancement through hit-to-lead and lead optimization studies was based on meeting a set of pre-defined acceptable and optimal criteria for potency, selectivity, and drug-like properties. BPN-36084, one of our lead molecules, progressed through a series of in vitro target assessments, binding and stability assays, in vitro ADME assays (permeability, metabolism, and safety) and in vivo studies. In vitro and in vivo studies have confirmed target engagement suggesting a tissue-based potency of 1 to 100 nM. BPN-36084 is well tolerated, well absorbed and brain penetrant in rats. BPN-36084 in vitro profiling and in vivo single dose studies in rats suggest a promising safety margin. BPN-36084, is orally efficacious in the rat spared nerve injury model during the neuropathic pain predominant phase (>14 post-operative days). BPN-36084 was modestly analgesic at the lowest dose tested (2.8 mg/kg, p.o.). New results will be presented for the lead molecules in this program.

Conclusions

Several molecules have entered late lead optimization evaluation for advancement to clinical candidacy. Our goal is to identify one molecule as a clinical candidate in Q2 or early Q3, 2024 for advancement into IND-enabling studies in 2024 and 2025. If successful, at the conclusion of the IND-enabling studies we will have a new clinical candidate thoroughly interrogated and poised for testing in clinical trials for chronic pain as the first orally available small molecule targeting PSD95-nNOS.

References

Andreasen JT, Bach A, Gynther M, Nasser A, Mogensen J, Strømgaard K, Pickering DS. UCCB01-125, a dimeric inhibitor of PSD-95, reduces inflammatory pain without disrupting cognitive or motor performance: comparison with the NMDA receptor antagonist MK-801. Neuropharmacology. 2013 Apr;67:193-200. doi: 10.1016/j.neuropharm.2012.11.006. Epub 2012 Nov 20. PMID: 23178182.

Andreasen JT, Nasser A, Caballero-Puntiverio M, Sahlholt M, Bach A, Gynther M, Strømgaard K, Pickering DS. Effects of the dimeric PSD-95 inhibitor UCCB01-144 in mouse models of pain, cognition and motor function. Eur J Pharmacol. 2016 Jun 5;780:166-73. doi: 10.1016/j.ejphar.2016.03.045. Epub 2016 Mar 28. PMID: 27032314.

Cai W, Wu S, Pan Z, Xiao J, Li F, Cao J, Zang W, Tao YX. Disrupting interaction of PSD-95 with nNOS attenuates hemorrhage-induced thalamic pain. Neuropharmacology. 2018 Oct;141:238-248. doi: 10.1016/j.neuropharm.2018.09.003. Epub 2018 Sep 5. PMID: 30193808; PMCID: PMC6191031.

Carey LM, Lee WH, Gutierrez T, Kulkarni PM, Thakur GA, Lai YY, Hohmann AG. Small molecule inhibitors of PSD95-nNOS protein-protein interactions suppress formalin-evoked Fos protein expression and nociceptive behavior in rats. Neuroscience. 2017;349:303-17. Epub 2017/03/14. doi: 10.1016/j.neuroscience.2017.02.055. PubMed PMID: 28285942.

Chen ZJ, Su CW, Xiong S, Li T, Liang HY, Lin YH, Chang L, Wu HY, Li F, Zhu DY, Luo CX. Enhanced AMPAR-dependent synaptic transmission by S-nitrosylation in the vmPFC contributes to chronic inflammatory pain-induced persistent anxiety in mice. Acta Pharmacol Sin. 2023 May;44(5):954-968. doi: 10.1038/s41401-022-01024-z. Epub 2022 Dec 2. PMID: 36460834; PMCID: PMC10104852.

Choi SR, Han HJ, Beitz AJ, Lee JH. nNOS-PSD95 interactions activate the PKC-? isoform leading to increased GluN1 phosphorylation and the development of neuropathic mechanical allodynia in mice. Neurosci Lett. 2019 Jun 11;703:156-161. doi: 10.1016/j.neulet.2019.03.043. Epub 2019 Mar 26. PMID: 30926374.

Chu YC, Guan Y, Skinner J, Raja SN, Johns RA, Tao YX. Effect of genetic knockout or pharmacologic inhibition of neuronal nitric oxide synthase on complete Freund’s adjuvant-induced persistent pain. Pain. 2005 Dec 15;119(1-3):113-123. doi: 10.1016/j.pain.2005.09.024. Epub 2005 Nov 16. PMID: 16297560.

Demir IE, Heinrich T, Carty DG, Saricaoglu ÖC, Klauss S, Teller S, Kehl T, Mota Reyes C, Tieftrunk E, Lazarou M, Bahceci DH, Gökcek B, Ucurum BE, Maak M, Diakopoulos KN, Lesina M, Schemann M, Erkan M, Krüger A, Algül H, Friess H, Ceyhan GO. Targeting nNOS ameliorates the severe neuropathic pain due to chronic pancreatitis. EBioMedicine. 2019 Aug;46:431-443. doi: 10.1016/j.ebiom.2019.07.055. Epub 2019 Aug 8. PMID: 31401195; PMCID: PMC6711864.

D’Mello R, Marchand F, Pezet S, McMahon SB, Dickenson AH. Perturbing PSD-95 interactions with NR2B-subtype receptors attenuates spinal nociceptive plasticity and neuropathic pain. Mol Ther. 2011 Oct;19(10):1780-92. doi: 10.1038/mt.2011.42. Epub 2011 Mar 22. PMID: 21427709; PMCID: PMC3188755.

Fairbanks CA, Schreiber KL, Brewer KL, Yu CG, Stone LS, Kitto KF, Nguyen HO, Grocholski BM, Shoeman DW, Kehl LJ, Regunathan S, Reis DJ, Yezierski RP, Wilcox GL. Agmatine reverses pain induced by inflammation, neuropathy, and spinal cord injury. Proc Natl Acad Sci U S A. 2000 Sep 12;97(19):10584-9. doi: 10.1073/pnas.97.19.10584. PMID: 10984543; PMCID: PMC27068.

Florio SK, Loh C, Huang SM, Iwamaye AE, Kitto KF, Fowler KW, Treiberg JA, Hayflick JS, Walker JM, Fairbanks CA, Lai Y. Disruption of nNOS-PSD95 protein-protein interaction inhibits acute thermal hyperalgesia and chronic mechanical allodynia in rodents. British journal of pharmacology. 2009;158(2):494-506. Epub 2009/09/08. doi: 10.1111/j.1476-5381.2009.00300.x. PubMed PMID: 19732061.

Gu Y, Zhu D. nNOS-mediated protein-protein interactions: promising targets for treating neurological and neuropsychiatric disorders. J Biomed Res. 2020 Dec 10;35(1):1-10. doi: 10.7555/JBR.34.20200108. PMID: 33402546; PMCID: PMC7874267.

Guan Y, Yaster M, Raja SN, Tao YX. Genetic knockout and pharmacologic inhibition of neuronal nitric oxide synthase attenuate nerve injury-induced mechanical hypersensitivity in mice. Mol Pain. 2007 Oct 8;3:29. doi: 10.1186/1744-8069-3-29. PMID: 17922909; PMCID: PMC2089056.

Kitto KF, Haley JE, Wilcox GL. Involvement of nitric oxide in spinally mediated hyperalgesia in the mouse. Neurosci Lett. 1992 Dec 14;148(1-2):1-5. doi: 10.1016/0304-3940(92)90790-e. PMID: 1284437.

Lee WH, Xu Z, Ashpole NM, Hudmon A, Kulkarni PM, Thakur GA, Lai YY, Hohmann AG. Small molecule inhibitors of PSD95-nNOS protein-protein interactions as novel analgesics. Neuropharmacology. 2015;97:464-75. doi: 10.1016/j.neuropharm.2015.05.038. PubMed PMID: 26071110.

Li A, Huang CJ, Gu KP, Huang Y, Huang YQ, Zhang H, Lin JP, Liu YF, Yang Y, Yao YX. PSD-95 in the anterior cingulate cortex contributes to neuropathic pain by interdependent activation with NR2B. Sci Rep. 2022 Oct 12;12(1):17114. doi: 10.1038/s41598-022-21488-7. PMID: 36224339; PMCID: PMC9556829.

Li J, Zhang L, Xu C, Shen YY, Lin YH, Zhang Y, Wu HY, Chang L, Zhang YD, Chen R, Zhang ZP, Luo CX, Li F, Zhu DY. A pain killer without analgesic tolerance designed by co-targeting PSD-95-nNOS interaction and ?2-containning GABAARs. Theranostics. 2021 Apr 3;11(12):5970-5985. doi: 10.7150/thno.58364. PMID: 33897893; PMCID: PMC8058733.

Liu Y, Cui X, Sun YE, Yang X, Ni K, Zhou Y, Ma Z, Gu X. Intrathecal injection of the peptide myr-NR2B9c attenuates bone cancer pain via perturbing N-methyl-D-aspartate receptor-PSD-95 protein interactions in mice. Anesth Analg. 2014 Jun;118(6):1345-54. doi: 10.1213/ANE.0000000000000202. PMID: 24842180.

Oliva I, Saberi SA, Rangel-Barajas C, Iyer V, Bunner KD, Lai YY, Kulkarni PM, Garai S, Thakur GA, Crystal JD, Rebec GV, Hohmann AG. Inhibition of PSD95-nNOS protein-protein interactions decreases morphine reward and relapse vulnerability in rats. Addict Biol. 2022 Sep;27(5):e13220. doi: 10.1111/adb.13220. PMID: 36001441; PMCID: PMC9539577.

Peterson CD, Kitto KF, Verma H, Pflepsen K, Delpire E, Wilcox GL, Fairbanks CA. Agmatine requires GluN2B-containing NMDA receptors to inhibit the development of neuropathic pain. Mol Pain. 2021 Jan-Dec;17:17448069211029171. doi: 10.1177/17448069211029171. PMID: 34210178; PMCID: PMC8255568.

Peterson CD, Waataja JJ, Kitto KF, Erb SJ, Verma H, Schuster DJ, Churchill CC, Riedl MS, Belur LR, Wolf DA, McIvor RS, Vulchanova L, Wilcox GL, Fairbanks CA. Long-term reversal of chronic pain behavior in rodents through elevation of spinal agmatine. Mol Ther. 2023 Apr 5;31(4):1123-1135. doi: 10.1016/j.ymthe.2023.01.022. Epub 2023 Jan 30. PMID: 36710491; PMCID: PMC10124077.

Rosenbaum MI, Clemmensen LS, Bredt DS, Bettler B, Strømgaard K. Targeting receptor complexes: a new dimension in drug discovery. Nat Rev Drug Discov. 2020 Dec;19(12):884-901. doi: 10.1038/s41573-020-0086-4. Epub 2020 Nov 11. PMID: 33177699.

Smith AE, Xu Z, Lai YY, Kulkarni PM, Thakur GA, Hohmann AG, Crystal JD. Source memory in rats is impaired by an NMDA receptor antagonist but not by PSD95-nNOS protein-protein interaction inhibitors. Behav Brain Res. 2016;305:23-9. Epub 2016/02/26. doi: 10.1016/j.bbr.2016.02.021. PubMed PMID: 26909849.

Tao F, Tao YX, Gonzalez JA, Fang M, Mao P, Johns RA. Knockdown of PSD-95/SAP90 delays the development of neuropathic pain in rats. Neuroreport. 2001 Oct 29;12(15):3251-5. doi: 10.1097/00001756-200110290-00022. PMID: 11711866.

Wei W, Liu W, Du S, Govindarajalu G, Irungu A, Bekker A, Tao YX. A Compound Mitigates Cancer Pain and Chemotherapy-Induced Neuropathic Pain by Dually Targeting nNOS-PSD-95 Interaction and GABAA Receptor. Neurotherapeutics. 2021 Oct;18(4):2436-2448. doi: 10.1007/s13311-021-01158-8. Epub 2021 Nov 18. PMID: 34796458; PMCID: PMC8804143.

Xu F, Zhao X, Liu L, Song J, Zhu Y, Chu S, Shao X, Li X, Ma Z, Gu X. Perturbing NR2B-PSD-95 interaction relieves neuropathic pain by inactivating CaMKII-CREB signaling. Neuroreport. 2017 Sep 6;28(13):856-863. doi: 10.1097/WNR.0000000000000849. PMID: 28746067.

Zhou L, Li F, Xu HB, Luo CX, Wu HY, Zhu MM, Lu W, Ji X, Zhou QG, Zhu DY. Treatment of cerebral ischemia by disrupting ischemia-induced interaction of nNOS with PSD-95. Nature medicine. 2010;16(12):1439-43. Epub 2010/11/26. doi: 10.1038/nm.2245. PubMed PMID: 21102461.

Presenting Author

Stephanie K. Florio

Poster Authors

Stephanie Florio

PhD

Anagin, Inc.

Lead Author

Advancement of Small Molecule PSD95-nNOS Modulators as Novel Non-Opioid Analgesics

Background & Aims

Methods

Results

Conclusions

References

Presenting Author

Stephanie K. Florio

Poster Authors

Stephanie Florio

Matthew Surman

Carlos Alves Jesus

Whitney Childress

Patrick Harrington

Robert Lewis

Kerry W. Fowler

Jheel Patel

Ubongabasi Asuquo

Kelsey Guenther

Cristina Peterson

Kelley F. Kitto

Ronald B. Franklin

William H. Martin

Carolyn A. Fairbanks

Andrea G. Hohmann

Topics

IASP 2024 World Congress on Pain