Publications by authors named "Samantha Perez-Miller"

26 Publications

  • Page 1 of 1

Comparison of quinazoline and benzoylpyrazoline chemotypes targeting the CaVα-β interaction as antagonists of the N-type CaV2.2 channel.

Channels (Austin) 2021 Dec;15(1):128-135

Department of Pharmacology, College of Medicine, The University of Arizona , Tucson, AZ, USA.

Structural studies with an α subunit fragment of voltage-gated calcium (CaV) channels in complex with the CaVβ subunits revealed a high homology between the various CaVα-β subunits, predicting that targeting of this interface would result in nonselective compounds. Despite this likelihood, my laboratory initiated a rational structure-based screening campaign focusing on "hot spots" on the alpha interacting domain (AID) of the CaVβ2a subunits and identified the small molecule 2-(3,5-dimethylisoxazol-4-yl)-N-((4-((3-phenylpropyl)amino)quinazolin-2-yl)methyl)acetamide ( ) which selectively targeted the interface between the N-type calcium (CaV2.2) channel and CaVβ. (i) specifically bound to CaVβ2a; (ii) inhibited CaVβ2 's interaction with CaV.2-AID; (iii) inhibited CaV2.2 currents in sensory neurons; (iv) inhibited pre-synaptic localization of CaV2.2 ; and (v) inhibited spinal neurotransmission, which resulted in decreased neurotransmitter release. was anti-nociceptive in naïve rats and reversed mechanical allodynia and thermal hyperalgesia in rodent models of acute, neuropathic, and genetic pain. In structure-activity relationship (SAR) studies focused on improving binding affinity of , another compound (BTT-369), a benzoyl-3,4-dihydro-1'H,2 H-3,4'-bipyrazole class of compounds, was reported by Chen and colleagues, based on work conducted in my laboratory beginning in 2008. BTT-369 contains tetraaryldihydrobipyrazole scaffold - a chemotype featuring phenyl groups known to be significantly metabolized, lower the systemic half-life, and increase the potential for toxicity. Furthermore, the benzoylpyrazoline skeleton in BTT-369 is patented across multiple therapeutic indications. Prior to embarking on an extensive optimization campaign of , we performed a head-to-head comparison of the two compounds. We conclude that is superior to BTT-369 for on-target efficacy, setting the stage for SAR studies to improve on for the development of novel pain therapeutics.
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http://dx.doi.org/10.1080/19336950.2020.1863595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7808423PMC
December 2021

SARS-CoV-2 spike protein co-opts VEGF-A/neuropilin-1 receptor signaling to induce analgesia.

Pain 2021 01;162(1):243-252

Departments of Pharmacology, and.

Global spread of severe acute respiratory syndrome coronavirus 2 continues unabated. Binding of severe acute respiratory syndrome coronavirus 2's spike protein to host angiotensin-converting enzyme 2 triggers viral entry, but other proteins may participate, including the neuropilin-1 receptor (NRP-1). Because both spike protein and vascular endothelial growth factor-A (VEGF-A)-a pronociceptive and angiogenic factor, bind NRP-1, we tested whether spike could block VEGF-A/NRP-1 signaling. VEGF-A-triggered sensory neuron firing was blocked by spike protein and NRP-1 inhibitor EG00229. Pronociceptive behaviors of VEGF-A were similarly blocked through suppression of spontaneous spinal synaptic activity and reduction of electrogenic currents in sensory neurons. Remarkably, preventing VEGF-A/NRP-1 signaling was antiallodynic in a neuropathic pain model. A "silencing" of pain through subversion of VEGF-A/NRP-1 signaling may underlie increased disease transmission in asymptomatic individuals.
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http://dx.doi.org/10.1097/j.pain.0000000000002097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7737878PMC
January 2021

In silico identification and validation of inhibitors of the interaction between neuropilin receptor 1 and SARS-CoV-2 Spike protein.

bioRxiv 2020 Sep 23. Epub 2020 Sep 23.

Neuropilin-1 (NRP-1) is a multifunctional transmembrane receptor for ligands that affect developmental axonal growth and angiogenesis. In addition to a role in cancer, NRP-1 is a reported entry point for several viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19). The furin cleavage product of SARS-CoV-2 Spike protein takes advantage of the vascular endothelial growth factor A (VEGF-A) binding site on NRP-1 which accommodates a polybasic stretch ending in a C-terminal arginine. This site has long been a focus of drug discovery efforts for cancer therapeutics. We recently showed that interruption of the VEGF-A/NRP-1 signaling pathway ameliorates neuropathic pain and hypothesize that interference of this pathway by SARS-CoV-2 spike protein interferes with pain signaling. Here, we report hits from a small molecule and natural product screen of nearly 0.5 million compounds targeting the VEGF-A binding site on NRP-1. We identified nine chemical series with lead- or drug-like physico-chemical properties. Using an ELISA, we demonstrate that six compounds disrupt VEGF-A-NRP-1 binding more effectively than EG00229, a known NRP-1 inhibitor. Secondary validation in cells revealed that almost all tested compounds inhibited VEGF-A triggered VEGFR2 phosphorylation. Two compounds displayed robust inhibition of a recombinant vesicular stomatitis virus protein that utilizes the SARS-CoV-2 Spike for entry and fusion. These compounds represent a first step in a renewed effort to develop small molecule inhibitors of the VEGF-A/NRP-1 signaling for the treatment of neuropathic pain and cancer with the added potential of inhibiting SARS-CoV-2 virus entry.
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http://dx.doi.org/10.1101/2020.09.22.308783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523098PMC
September 2020

SARS-CoV-2 Spike protein co-opts VEGF-A/Neuropilin-1 receptor signaling to induce analgesia.

bioRxiv 2020 Aug 24. Epub 2020 Aug 24.

Department of Pharmacology, College of Medicine, The University of Arizona, Tucson, Arizona, 85724 United States of America.

Global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues unabated. Binding of SARS-CoV-2's Spike protein to host angiotensin converting enzyme 2 triggers viral entry, but other proteins may participate, including neuropilin-1 receptor (NRP-1). As both Spike protein and vascular endothelial growth factor-A (VEGF-A) - a pro-nociceptive and angiogenic factor, bind NRP-1, we tested if Spike could block VEGF-A/NRP-1 signaling. VEGF-A-triggered sensory neuronal firing was blocked by Spike protein and NRP-1 inhibitor EG00229. Pro-nociceptive behaviors of VEGF-A were similarly blocked via suppression of spontaneous spinal synaptic activity and reduction of electrogenic currents in sensory neurons. Remarkably, preventing VEGF-A/NRP-1 signaling was antiallodynic in a neuropathic pain model. A 'silencing' of pain via subversion of VEGF-A/NRP-1 signaling may underlie increased disease transmission in asymptomatic individuals.
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http://dx.doi.org/10.1101/2020.07.17.209288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7457601PMC
August 2020

The role of cyclin-dependent kinase 5 in neuropathic pain.

Pain 2020 12;161(12):2674-2689

Department of Pharmacology, College of Medicine, The University of Arizona, Tucson, AZ, United States.

The chronification of pain can be attributed to changes in membrane receptors and channels underlying neuronal plasticity and signal transduction largely within nociceptive neurons that initiate and maintain pathological pain states. These proteins are subject to dynamic modification by posttranslational modifications, creating a code that controls protein function in time and space. Phosphorylation is an important posttranslational modification that affects ∼30% of proteins in vivo. Increased phosphorylation of various nociceptive ion channels and of their modulators underlies sensitization of different pain states. Cyclin-dependent kinases are proline-directed serine/threonine kinases that impact various biological and cellular systems. Cyclin-dependent kinase 5 (Cdk5), one member of this kinase family, and its activators p35 and p39 are expressed in spinal nerves, dorsal root ganglia, and the dorsal horn of the spinal cord. In neuropathic pain conditions, expression and/or activity of Cdk5 is increased, implicating Cdk5 in nociception. Experimental evidence suggests that Cdk5 is regulated through its own phosphorylation, through increasing p35's interaction with Cdk5, and through cleavage of p35 into p25. This narrative review discusses the molecular mechanisms of Cdk5-mediated regulation of target proteins involved in neuropathic pain. We focus on Cdk5 substrates that have been linked to nociceptive pathways, including channels (eg, transient receptor potential cation channel and voltage-gated calcium channel), proteins involved in neurotransmitter release (eg, synaptophysin and collapsin response mediator protein 2), and receptors (eg, glutamate, purinergic, and opioid). By altering the phosphoregulatory "set point" of proteins involved in pain signaling, Cdk5 thus appears to be an attractive target for treating neuropathic pain conditions.
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http://dx.doi.org/10.1097/j.pain.0000000000002027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7669638PMC
December 2020

Druggability of CRMP2 for Neurodegenerative Diseases.

ACS Chem Neurosci 2020 09 4;11(17):2492-2505. Epub 2020 Aug 4.

BrightRock Path, LLC, Tucson, Arizona 85704, United States.

Collapsin response mediator proteins (CRMPs) are ubiquitously expressed phosphoproteins that coordinate cytoskeletal formation and regulate cellular division, migration, polarity, and synaptic connection. CRMP2, the most studied of the five family members, is best known for its affinity for tubulin heterodimers and function in regulating the microtubule network. Accumulating evidence has also demonstrated a key role for CRMP2 in trafficking of voltage- and ligand-gated ion channels. These functions are tightly regulated by post-translational modifications including phosphorylation and SUMOylation (addition of a small ubiquitin like modifier). Over the past decade, it has become increasingly clear that dysregulated post-translational modifications of CRMP2 contribute to the pathomechanisms of diverse diseases, including cancer, neurodegenerative diseases, chronic pain, and bipolar disorder. Here, we review the discovery, functions, and current putative preclinical and clinical therapeutics targeting CRMP2. These potential therapeutics include CRMP2-based peptides that inhibit protein-protein interactions and small-molecule compounds. Capitalizing on the availability of structural information, we identify druggable pockets on CRMP2 and predict binding modes for five known CRMP2-targeting compounds, setting the stage for optimization and de novo drug discovery targeting this multifunctional protein.
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http://dx.doi.org/10.1021/acschemneuro.0c00307DOI Listing
September 2020

A modulator of the low-voltage-activated T-type calcium channel that reverses HIV glycoprotein 120-, paclitaxel-, and spinal nerve ligation-induced peripheral neuropathies.

Pain 2020 11;161(11):2551-2570

Department of Pharmacology, College of Medicine, The University of Arizona, Tucson, AZ, United States.

The voltage-gated calcium channels CaV3.1-3.3 constitute the T-type subfamily, whose dysfunctions are associated with epilepsy, psychiatric disorders, and chronic pain. The unique properties of low-voltage-activation, faster inactivation, and slower deactivation of these channels support their role in modulation of cellular excitability and low-threshold firing. Thus, selective T-type calcium channel antagonists are highly sought after. Here, we explored Ugi-azide multicomponent reaction products to identify compounds targeting T-type calcium channel. Of the 46 compounds tested, an analog of benzimidazolonepiperidine-5bk (1-{1-[(R)-{1-[(1S)-1-phenylethyl]-1H-1,2,3,4-tetrazol-5-yl}(thiophen-3-yl)methyl]piperidin-4-yl}-2,3-dihydro-1H-1,3-benzodiazol-2-one) modulated depolarization-induced calcium influx in rat sensory neurons. Modulation of T-type calcium channels by 5bk was further confirmed in whole-cell patch clamp assays in dorsal root ganglion (DRG) neurons, where pharmacological isolation of T-type currents led to a time- and concentration-dependent regulation with a low micromolar IC50. Lack of an acute effect of 5bk argues against a direct action on T-type channels. Genetic knockdown revealed CaV3.2 to be the isoform preferentially modulated by 5bk. High voltage-gated calcium, as well as tetrodotoxin-sensitive and -resistant sodium, channels were unaffected by 5bk. 5bk inhibited spontaneous excitatory postsynaptic currents and depolarization-evoked release of calcitonin gene-related peptide from lumbar spinal cord slices. Notably, 5bk did not bind human mu, delta, or kappa opioid receptors. 5bk reversed mechanical allodynia in rat models of HIV-associated neuropathy, chemotherapy-induced peripheral neuropathy, and spinal nerve ligation-induced neuropathy, without effects on locomotion or anxiety. Thus, 5bk represents a novel T-type modulator that could be used to develop nonaddictive pain therapeutics.
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http://dx.doi.org/10.1097/j.pain.0000000000001955DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572723PMC
November 2020

Corrigendum: Structural Insights Into TDP-43 and Effects of Post-translational Modifications.

Front Mol Neurosci 2020;13:45. Epub 2020 Apr 2.

Department of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ, United States.

[This corrects the article DOI: 10.3389/fnmol.2019.00301.].
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http://dx.doi.org/10.3389/fnmol.2020.00045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7143466PMC
April 2020

Structural Insights Into TDP-43 and Effects of Post-translational Modifications.

Front Mol Neurosci 2019 17;12:301. Epub 2019 Dec 17.

Department of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ, United States.

Transactive response DNA binding protein (TDP-43) is a key player in neurodegenerative diseases. In this review, we have gathered and presented structural information on the different regions of TDP-43 with high resolution structures available. A thorough understanding of TDP-43 structure, effect of modifications, aggregation and sites of localization is necessary as we develop therapeutic strategies targeting TDP-43 for neurodegenerative diseases. We discuss how different domains as well as post-translational modification may influence TDP-43 overall structure, aggregation and droplet formation. The primary aim of the review is to utilize structural insights as we develop an understanding of the deleterious behavior of TDP-43 and highlight locations of established and proposed post-translation modifications. TDP-43 structure and effect on localization is paralleled by many RNA-binding proteins and this review serves as an example of how structure may be modulated by numerous compounding elements.
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http://dx.doi.org/10.3389/fnmol.2019.00301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934062PMC
December 2019

The Natural Flavonoid Naringenin Elicits Analgesia through Inhibition of NaV1.8 Voltage-Gated Sodium Channels.

ACS Chem Neurosci 2019 12 21;10(12):4834-4846. Epub 2019 Nov 21.

Department of Pharmacology, College of Medicine , The University of Arizona , Tucson 85724-5050 , Arizona , United States.

Naringenin (2S)-5,7-dihydroxy-2-(4-hydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-4-one is a natural flavonoid found in fruits from the citrus family. Because (2S)-naringenin is known to racemize, its bioactivity might be related to one or both enantiomers. Computational studies predicted that (2R)-naringenin may act on voltage-gated ion channels, particularly the N-type calcium channel (CaV2.2) and the NaV1.7 sodium channel-both of which are key for pain signaling. Here we set out to identify the possible mechanism of action of naringenin. Naringenin inhibited depolarization-evoked Ca influx in acetylcholine-, ATP-, and capsaicin-responding rat dorsal root ganglion (DRG) neurons. This was corroborated in electrophysiological recordings from DRG neurons. Pharmacological dissection of each of the voltage-gated Ca channels subtypes could not pinpoint any selectivity of naringenin. Instead, naringenin inhibited NaV1.8-dependent and tetrodotoxin (TTX)-resistant while sparing tetrodotoxin sensitive (TTX-S) voltage-gated Na channels as evidenced by the lack of further inhibition by the NaV1.8 blocker A-803467. The effects of the natural flavonoid were validated ex vivo in spinal cord slices where naringenin decreased both the frequency and amplitude of sEPSC recorded in neurons within the substantia gelatinosa. The antinociceptive potential of naringenin was evaluated in male and female mice. Naringenin had no effect on the nociceptive thresholds evoked by heat. Naringenin's reversed allodynia was in mouse models of postsurgical and neuropathic pain. Here, driven by a call by the National Center for Complementary and Integrative Health's strategic plan to advance fundamental research into basic biological mechanisms of the action of natural products, we advance the antinociceptive potential of the flavonoid naringenin.
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http://dx.doi.org/10.1021/acschemneuro.9b00547DOI Listing
December 2019

Small Molecule Targeting TDP-43's RNA Recognition Motifs Reduces Locomotor Defects in a Model of Amyotrophic Lateral Sclerosis (ALS).

ACS Chem Biol 2019 09 27;14(9):2006-2013. Epub 2019 Aug 27.

Department of Pharmacology, College of Medicine , University of Arizona , Tucson , Arizona 85724 , United States.

RNA dysregulation likely contributes to disease pathogenesis of amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. A pathological form of the transactive response (TAR) DNA binding protein (TDP-43) binds to RNA in stress granules and forms membraneless, amyloid-like TDP-43 aggregates in the cytoplasm of ALS motor neurons. In this study, we hypothesized that by targeting the RNA recognition motif (RRM) domains of TDP-43 that confer a pathogenic interaction between TDP-43 and RNA, motor neuron toxicity could be reduced. docking of 50000 compounds to the RRM domains of TDP-43 identified a small molecule (rTRD01) that (i) bound to TDP-43's RRM1 and RRM2 domains, (ii) partially disrupted TDP-43's interaction with the hexanucleotide RNA repeat of the disease-linked gene, but not with (UG) canonical binding sequence of TDP-43, and (iii) improved larval turning, an assay measuring neuromuscular coordination and strength, in an ALS fly model based on the overexpression of mutant TDP-43. Our findings provide an instructive example of a chemical biology approach pivoted to discover small molecules targeting RNA-protein interactions in neurodegenerative diseases.
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http://dx.doi.org/10.1021/acschembio.9b00481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6911355PMC
September 2019

Targeting the CaVα-CaVβ interaction yields an antagonist of the N-type CaV2.2 channel with broad antinociceptive efficacy.

Pain 2019 07;160(7):1644-1661

Department of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ, United States.

Inhibition of voltage-gated calcium (CaV) channels is a potential therapy for many neurological diseases including chronic pain. Neuronal CaV1/CaV2 channels are composed of α, β, γ and α2δ subunits. The β subunits of CaV channels are cytoplasmic proteins that increase the surface expression of the pore-forming α subunit of CaV. We targeted the high-affinity protein-protein interface of CaVβ's pocket within the CaVα subunit. Structure-based virtual screening of 50,000 small molecule library docked to the β subunit led to the identification of 2-(3,5-dimethylisoxazol-4-yl)-N-((4-((3-phenylpropyl)amino)quinazolin-2-yl)methyl)acetamide (IPPQ). This small molecule bound to CaVβ and inhibited its coupling with N-type voltage-gated calcium (CaV2.2) channels, leading to a reduction in CaV2.2 currents in rat dorsal root ganglion sensory neurons, decreased presynaptic localization of CaV2.2 in vivo, decreased frequency of spontaneous excitatory postsynaptic potentials and miniature excitatory postsynaptic potentials, and inhibited release of the nociceptive neurotransmitter calcitonin gene-related peptide from spinal cord. IPPQ did not target opioid receptors nor did it engage inhibitory G protein-coupled receptor signaling. IPPQ was antinociceptive in naive animals and reversed allodynia and hyperalgesia in models of acute (postsurgical) and neuropathic (spinal nerve ligation, chemotherapy- and gp120-induced peripheral neuropathy, and genome-edited neuropathy) pain. IPPQ did not cause akinesia or motor impairment, a common adverse effect of CaV2.2 targeting drugs, when injected into the brain. IPPQ, a quinazoline analog, represents a novel class of CaV2.2-targeting compounds that may serve as probes to interrogate CaVα-CaVβ function and ultimately be developed as a nonopioid therapeutic for chronic pain.
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http://dx.doi.org/10.1097/j.pain.0000000000001524DOI Listing
July 2019

A Chemical Biology Approach to Model Pontocerebellar Hypoplasia Type 1B (PCH1B).

ACS Chem Biol 2018 10 6;13(10):3000-3010. Epub 2018 Sep 6.

Department of Pharmacology , College of Medicine, University of Arizona , Tucson , Arizona 85724 , United States.

Mutations of EXOSC3 have been linked to the rare neurological disorder known as Pontocerebellar Hypoplasia type 1B (PCH1B). EXOSC3 is one of three putative RNA-binding structural cap proteins that guide RNA into the RNA exosome, the cellular machinery that degrades RNA. Using RNAcompete, we identified a G-rich RNA motif binding to EXOSC3. Surface plasmon resonance (SPR) and microscale thermophoresis (MST) indicated an affinity in the low micromolar range of EXOSC3 for long and short G-rich RNA sequences. Although several PCH1B-causing mutations in EXOSC3 did not engage a specific RNA motif as shown by RNAcompete, they exhibited lower binding affinity to G-rich RNA as demonstrated by MST. To test the hypothesis that modification of the RNA-protein interface in EXOSC3 mutants may be phenocopied by small molecules, we performed an in-silico screen of 50 000 small molecules and used enzyme-linked immunosorbant assays (ELISAs) and MST to assess the ability of the molecules to inhibit RNA-binding by EXOSC3. We identified a small molecule, EXOSC3-RNA disrupting (ERD) compound 3 (ERD03), which ( i) bound specifically to EXOSC3 in saturation transfer difference nuclear magnetic resonance (STD-NMR), ( ii) disrupted the EXOSC3-RNA interaction in a concentration-dependent manner, and ( iii) produced a PCH1B-like phenotype with a 50% reduction in the cerebellum and an abnormally curved spine in zebrafish embryos. This compound also induced modification of zebrafish RNA expression levels similar to that observed with a morpholino against EXOSC3. To our knowledge, this is the first example of a small molecule obtained by rational design that models the abnormal developmental effects of a neurodegenerative disease in a whole organism.
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http://dx.doi.org/10.1021/acschembio.8b00745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6504997PMC
October 2018

Chemical shift perturbation mapping of the Ubc9-CRMP2 interface identifies a pocket in CRMP2 amenable for allosteric modulation of Nav1.7 channels.

Channels (Austin) 2018 6;12(1):219-227. Epub 2018 Aug 6.

a Departments of Pharmacology College of Medicine , University of Arizona , Tucson , Arizona USA.

Drug discovery campaigns directly targeting the voltage-gated sodium channel NaV1.7, a highly prized target in chronic pain, have not yet been clinically successful. In a differentiated approach, we demonstrated allosteric control of trafficking and activity of NaV1.7 by prevention of SUMOylation of collapsin response mediator protein 2 (CRMP2). Spinal administration of a SUMOylation incompetent CRMP2 (CRMP2 K374A) significantly attenuated pain behavior in the spared nerve injury (SNI) model of neuropathic pain, underscoring the importance of SUMOylation of CRMP2 as a pathologic event in chronic pain. Using a rational design strategy, we identified a heptamer peptide harboring CRMP2's SUMO motif that disrupted the CRMP2-Ubc9 interaction, inhibited CRMP2 SUMOylation, inhibited NaV1.7 membrane trafficking, and specifically inhibited NaV1.7 sodium influx in sensory neurons. Importantly, this peptide reversed nerve injury-induced thermal and mechanical hypersensitivity in the SNI model, supporting the practicality of discovering pain drugs by indirectly targeting NaV1.7 via prevention of CRMP2 SUMOylation. Here, our goal was to map the unique interface between CRMP2 and Ubc9, the E2 SUMO conjugating enzyme. Using computational and biophysical approaches, we demonstrate the enzyme/substrate nature of Ubc9/CRMP2 binding and identify hot spots on CRMP2 that may form the basis of future drug discovery campaigns disrupting the CRMP2-Ubc9 interaction to recapitulate allosteric regulation of NaV1.7 for pain relief.
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http://dx.doi.org/10.1080/19336950.2018.1491244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6104687PMC
August 2019

A novel variant in affects gene expression and is associated with X-linked intellectual disability syndrome.

Neuronal Signal 2018 Sep 16;2(3):NS20180141. Epub 2018 Jul 16.

Department of Pathology, University of Arizona College of Medicine, Tucson, AZ 85724, U.S.A.

We investigated the genome of a 5-year-old male who presented with global developmental delay (motor, cognitive, and speech), hypotonia, possibly ataxia, and cerebellar hypoplasia of unknown origin. Whole genome sequencing (WGS) and mRNA sequencing (RNA-seq) were performed on a family having an affected proband, his unaffected parents, and maternal grandfather. To explore the molecular and functional consequences of the variant, we performed cell proliferation assays, quantitative real-time PCR (qRT-PCR) array, immunoblotting, calcium imaging, and neurite outgrowth experiments in SH-SY5Y neuroblastoma cells to compare the properties of the wild-type TATA-box-binding protein factor 1 (), deletion of , and variant p.Ser1600Gly samples. The whole genome data identified several gene variants. However, the genome sequence data failed to implicate a candidate gene as many of the variants were of unknown significance. By combining genome sequence data with transcriptomic data, a probable candidate variant, p.Ser1600Gly, emerged in . Moreover, the RNA-seq data revealed a 90:10 extremely skewed X-chromosome inactivation (XCI) in the mother. Our results showed that neuronal ion channel genes were differentially expressed between deletion and variant p.Ser1600Gly cells, when compared with their respective controls, and that the variant may impair neuronal differentiation and cell proliferation. Taken together, our data suggest that this novel variant in plays a key role in the development of a recently described X-linked syndrome, intellectual disability syndrome, and further extends our knowledge of a potential link between deficiency and defects in neuronal cell function.
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http://dx.doi.org/10.1042/NS20180141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7373232PMC
September 2018

Inhibition of the Ubc9 E2 SUMO-conjugating enzyme-CRMP2 interaction decreases NaV1.7 currents and reverses experimental neuropathic pain.

Pain 2018 Oct;159(10):2115-2127

Department of Pharmacology, Graduate Interdisciplinary Program, College of Medicine, Tucson, AZ, United States.

We previously reported that destruction of the small ubiquitin-like modifier (SUMO) modification site in the axonal collapsin response mediator protein 2 (CRMP2) was sufficient to selectively decrease trafficking of the voltage-gated sodium channel NaV1.7 and reverse neuropathic pain. Here, we further interrogate the biophysical nature of the interaction between CRMP2 and the SUMOylation machinery, and test the hypothesis that a rationally designed CRMP2 SUMOylation motif (CSM) peptide can interrupt E2 SUMO-conjugating enzyme Ubc9-dependent modification of CRMP2 leading to a similar suppression of NaV1.7 currents. Microscale thermophoresis and amplified luminescent proximity homogeneous alpha assay revealed a low micromolar binding affinity between CRMP2 and Ubc9. A heptamer peptide harboring CRMP2's SUMO motif, also bound with similar affinity to Ubc9, disrupted the CRMP2-Ubc9 interaction in a concentration-dependent manner. Importantly, incubation of a tat-conjugated cell-penetrating peptide (t-CSM) decreased sodium currents, predominantly NaV1.7, in a model neuronal cell line. Dialysis of t-CSM peptide reduced CRMP2 SUMOylation and blocked surface trafficking of NaV1.7 in rat sensory neurons. Fluorescence dye-based imaging in rat sensory neurons demonstrated inhibition of sodium influx in the presence of t-CSM peptide; by contrast, calcium influx was unaffected. Finally, t-CSM effectively reversed persistent mechanical and thermal hypersensitivity induced by a spinal nerve injury, a model of neuropathic pain. Structural modeling has now identified a pocket-harboring CRMP2's SUMOylation motif that, when targeted through computational screening of ligands/molecules, is expected to identify small molecules that will biochemically and functionally target CRMP2's SUMOylation to reduce NaV1.7 currents and reverse neuropathic pain.
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http://dx.doi.org/10.1097/j.pain.0000000000001294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6150792PMC
October 2018

A single structurally conserved SUMOylation site in CRMP2 controls NaV1.7 function.

Channels (Austin) 2017 Jul 28;11(4):316-328. Epub 2017 Feb 28.

a Department of Pharmacology, College of Medicine , University of Arizona , Tucson , AZ , USA.

The neuronal collapsin response mediator protein 2 (CRMP2) undergoes several posttranslational modifications that codify its functions. Most recently, CRMP2 SUMOylation (addition of small ubiquitin like modifier (SUMO)) was identified as a key regulatory step within a modification program that codes for CRMP2 interaction with, and trafficking of, voltage-gated sodium channel NaV1.7. In this paper, we illustrate the utility of combining sequence alignment within protein families with structural analysis to identify, from several putative SUMOylation sites, those that are most likely to be biologically relevant. Co-opting this principle to CRMP2, we demonstrate that, of 3 sites predicted to be SUMOylated in CRMP2, only the lysine 374 site is a SUMOylation client. A reduction in NaV1.7 currents was the corollary of the loss of CRMP2 SUMOylation at this site. A 1.78-Å-resolution crystal structure of mouse CRMP2 was solved using X-ray crystallography, revealing lysine 374 as buried within the CRMP2 tetramer interface but exposed in the monomer. Since CRMP2 SUMOylation is dependent on phosphorylation, we postulate that this state forces CRMP2 toward a monomer, exposing the SUMO site and consequently, resulting in constitutive regulation of NaV1.7.
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http://dx.doi.org/10.1080/19336950.2017.1299838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555265PMC
July 2017

Homology-guided mutational analysis reveals the functional requirements for antinociceptive specificity of collapsin response mediator protein 2-derived peptides.

Br J Pharmacol 2018 06 17;175(12):2244-2260. Epub 2017 Mar 17.

Department of Pharmacology, University of Arizona, Tucson, AZ, USA.

Background And Purpose: N-type voltage-gated calcium (Ca 2.2) channels are critical determinants of increased neuronal excitability and neurotransmission accompanying persistent neuropathic pain. Although Ca 2.2 channel antagonists are recommended as first-line treatment for neuropathic pain, calcium-current blocking gabapentinoids inadequately alleviate chronic pain symptoms and often exhibit numerous side effects. Collapsin response mediator protein 2 (CRMP2) targets Ca 2.2 channels to the sensory neuron membrane and allosterically modulates their function. A 15-amino-acid peptide (CBD3), derived from CRMP2, disrupts the functional protein-protein interaction between CRMP2 and Ca 2.2 channels to inhibit calcium influx, transmitter release and acute, inflammatory and neuropathic pain. Here, we have mapped the minimal domain of CBD3 necessary for its antinociceptive potential.

Experimental Approach: Truncated as well as homology-guided mutant versions of CBD3 were generated and assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons, binding between CRMP2 and Ca 2.2 channels, whole-cell voltage clamp electrophysiology and behavioural effects in two models of experimental pain: post-surgical pain and HIV-induced sensory neuropathy induced by the viral glycoprotein 120.

Key Results: The first six amino acids within CBD3 accounted for all in vitro activity and antinociception. Spinal administration of a prototypical peptide (TAT-CBD3-L5M) reversed pain behaviours. Homology-guided mutational analyses of these six amino acids identified at least two residues, Ala1 and Arg4, as being critical for antinociception in two pain models.

Conclusions And Implications: These results identify an antinociceptive scaffold core in CBD3 that can be used for development of low MW mimetics of CBD3.

Linked Articles: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.
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http://dx.doi.org/10.1111/bph.13737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5980424PMC
June 2018

(S)-Lacosamide Binding to Collapsin Response Mediator Protein 2 (CRMP2) Regulates CaV2.2 Activity by Subverting Its Phosphorylation by Cdk5.

Mol Neurobiol 2016 Apr 7;53(3):1959-1976. Epub 2015 Apr 7.

Department of Pharmacology, College of Medicine, University of Arizona, 1501 North Campbell Drive, P.O. Box 245050, Tucson, AZ, 85742, USA.

The neuronal circuit remodels during development as well as in human neuropathologies such as epilepsy. Neurite outgrowth is an obligatory step in these events. We recently reported that alterations in the phosphorylation state of an axon specification/guidance protein, the collapsin response mediator protein 2 (CRMP2), play a major role in the activity-dependent regulation of neurite outgrowth. We also identified (S)-LCM, an inactive stereoisomer of the clinically used antiepileptic drug (R)-LCM (Vimpat®), as a novel tool for preferentially targeting CRMP2-mediated neurite outgrowth. Here, we investigated the mechanism by which (S)-LCM affects CRMP2 phosphorylation by two key kinases, cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3β (GSK-3β). (S)-LCM application to embryonic cortical neurons resulted in reduced levels of Cdk5- and GSK-3β-phosphorylated CRMP2. Mechanistically, (S)-LCM increased CRMP2 binding to both Cdk5- and GSK-3β without affecting binding of CRMP2 to its canonical partner tubulin. Saturation transfer difference nuclear magnetic resonance (STD NMR) and differential scanning fluorimetry (DSF) experiments demonstrated direct binding of (S)-LCM to CRMP2. Using an in vitro luminescent kinase assay, we observed that (S)-LCM specifically inhibited Cdk5-mediated phosphorylation of CRMP2. Cross-linking experiments and analytical ultracentrifugation showed no effect of (S)-LCM on the oligomerization state of CRMP2. The increased association between Cdk5-phosphorylated CRMP2 and CaV2.2 was reduced by (S)-LCM in vitro and in vivo. This reduction translated into a decrease of calcium influx via CaV2.2 in (S)-LCM-treated neurons compared to controls. (S)-LCM, to our knowledge, is the first molecule described to directly inhibit CRMP2 phosphorylation and may be useful for delineating CRMP2-facilitated functions.
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http://dx.doi.org/10.1007/s12035-015-9141-2DOI Listing
April 2016

Expression and purification of functional human glycogen synthase-1 (hGYS1) in insect cells.

Protein Expr Purif 2013 Aug 24;90(2):78-83. Epub 2013 May 24.

Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202-5126, USA.

We have successfully expressed and purified active human glycogen synthase-1 (hGYS1). Successful production of the recombinant hGYS1 protein was achieved by co-expression of hGYS1 and rabbit glycogenin (rGYG1) using the MultiBac baculovirus expression system (BEVS). Functional measurements of activity ratios of hGYS1 in the absence and presence of glucose-6-phosphate and treatment with phosphatase indicate that the expressed protein is heavily phosphorylated. We used mass spectrometry to further characterize the sites of phosphorylation, which include most of the known regulatory phosphorylation sites, as well as several sites unique to the insect cell over-expression. Obtaining large quantities of functional hGYS1 will be invaluable for future structural studies as well as detailed studies on the effects on specific sites of phosphorylation.
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http://dx.doi.org/10.1016/j.pep.2013.05.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3720772PMC
August 2013

Catalytic contribution of threonine 244 in human ALDH2.

Chem Biol Interact 2013 Feb 4;202(1-3):32-40. Epub 2013 Jan 4.

Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202-5122, United States.

Amongst the numerous conserved residues in the aldehyde dehydrogenase superfamily, the precise role of Thr-244 remains enigmatic. Crystal structures show that this residue lies at the interface between the coenzyme-binding and substrate-binding sites with the side chain methyl substituent oriented toward the B-face of the nicotinamide ring of the NAD(P)(+) coenzyme, when in position for hydride transfer. Site-directed mutagenesis in ALDH1A1 and GAPN has suggested a role for Thr-244 in stabilizing the nicotinamide ring for efficient hydride transfer. Additionally, these studies also revealed a negative effect on cofactor binding which is not fully explained by the interaction with the nicotinamide ring. However, it is suggestive that Thr-244 immediately precedes helix αG, which forms one-half of the primary binding interface for the coenzyme. Hence, in order to more fully investigate the role of this highly conserved residue, we generated valine, alanine, glycine and serine substitutions for Thr-244 in human ALDH2. All four substituted enzymes exhibited reduced catalytic efficiency toward substrate and coenzyme. We also determined the crystal structure of the T244A enzyme in the absence and presence of coenzyme. In the apo-enzyme, the alpha G helix, which is key to NAD binding, exhibits increased temperature factors accompanied by a small displacement toward the active site cysteine. This structural perturbation was reversed in the coenzyme-bound complex. Our studies confirm a role for the Thr-244 beta methyl in the accurate positioning of the nicotinamide ring for efficient catalysis. We also identify a new role for Thr-244 in the stabilization of the N-terminal end of helix αG. This suggests that Thr-244, although less critical than Glu-487, is also an important contributor toward coenzyme binding.
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http://dx.doi.org/10.1016/j.cbi.2012.12.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3602351PMC
February 2013

Discovery of a novel class of covalent inhibitor for aldehyde dehydrogenases.

J Biol Chem 2011 Dec 21;286(50):43486-94. Epub 2011 Oct 21.

Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.

Human aldehyde dehydrogenases (ALDHs) comprise a family of 17 homologous enzymes that metabolize different biogenic and exogenic aldehydes. To date, there are relatively few general ALDH inhibitors that can be used to probe the contribution of this class of enzymes to particular metabolic pathways. Here, we report the discovery of a general class of ALDH inhibitors with a common mechanism of action. The combined data from kinetic studies, mass spectrometric measurements, and crystallographic analyses demonstrate that these inhibitors undergo an enzyme-mediated β-elimination reaction generating a vinyl ketone intermediate that covalently modifies the active site cysteine residue present in these enzymes. The studies described here can provide the basis for rational approach to design ALDH isoenzyme-specific inhibitors as research tools and perhaps as drugs, to address diseases such as cancer where increased ALDH activity is associated with a cellular phenotype.
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http://dx.doi.org/10.1074/jbc.M111.293597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3234859PMC
December 2011

High resolution X-ray structures of mouse major urinary protein nasal isoform in complex with pheromones.

Protein Sci 2010 Aug;19(8):1469-79

Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.

In mice, the major urinary proteins (MUP) play a key role in pheromonal communication by binding and transporting semiochemicals. MUP-IV is the only isoform known to be expressed in the vomeronasal mucosa. In comparison with the MUP isoforms that are abundantly excreted in the urine, MUP-IV is highly specific for the male mouse pheromone 2-sec-butyl-4,5-dihydrothiazole (SBT). To examine the structural basis of this ligand preference, we determined the X-ray crystal structure of MUP-IV bound to three mouse pheromones: SBT, 2,5-dimethylpyrazine, and 2-heptanone. We also obtained the structure of MUP-IV with 2-ethylhexanol bound in the cavity. These four structures show that relative to the major excreted MUP isoforms, three amino acid substitutions within the binding calyx impact ligand coordination. The F103 for A along with F54 for L result in a smaller cavity, potentially creating a more closely packed environment for the ligand. The E118 for G substitution introduces a charged group into a hydrophobic environment. The sidechain of E118 is observed to hydrogen bond to polar groups on all four ligands with nearly the same geometry as seen for the water-mediated hydrogen bond network in the MUP-I and MUP-II crystal structures. These differences in cavity size and interactions between the protein and ligand are likely to contribute to the observed specificity of MUP-IV.
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http://dx.doi.org/10.1002/pro.426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2923500PMC
August 2010

Alda-1 is an agonist and chemical chaperone for the common human aldehyde dehydrogenase 2 variant.

Nat Struct Mol Biol 2010 Feb 10;17(2):159-64. Epub 2010 Jan 10.

Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

In approximately one billion people, a point mutation inactivates a key detoxifying enzyme, aldehyde dehydrogenase (ALDH2). This mitochondrial enzyme metabolizes toxic biogenic and environmental aldehydes, including the endogenously produced 4-hydroxynonenal (4HNE) and the environmental pollutant acrolein, and also bioactivates nitroglycerin. ALDH2 is best known, however, for its role in ethanol metabolism. The accumulation of acetaldehyde following the consumption of even a single alcoholic beverage leads to the Asian alcohol-induced flushing syndrome in ALDH2*2 homozygotes. The ALDH2*2 allele is semidominant, and heterozygotic individuals show a similar but less severe phenotype. We recently identified a small molecule, Alda-1, that activates wild-type ALDH2 and restores near-wild-type activity to ALDH2*2. The structures of Alda-1 bound to ALDH2 and ALDH2*2 reveal how Alda-1 activates the wild-type enzyme and how it restores the activity of ALDH2*2 by acting as a structural chaperone.
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http://dx.doi.org/10.1038/nsmb.1737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2857674PMC
February 2010

Light chain C-terminal region reinforces the stability of clathrin heavy chain trimers.

Traffic 2007 Aug 6;8(8):1101-10. Epub 2007 Jun 6.

Department of Biology, Indiana University, Bloomington, IN 47405, USA.

The self-assembly of clathrin into lattices relies on the ability of heavy chain legs to form a three-legged pinwheel structure. We investigated the role of light chains in clathrin trimerization by challenging recombinant hub (plus and minus light chain) with an anionic detergent. The binding of light chain increases the amount of detergent needed to induce detrimerization, suggesting light chains reinforced hub trimers. We also show that light chain C-terminal residues are important for enhancing the in vitro assembly of hub at low pH. We assessed how much the C-terminus of light chain contributed to the stability of the trimerization domain by adding full-length and truncated light chains to trimer-defective hub mutants, C1573S and C1573A. Adding full-length LCb to C1573S caused some retrimerization, but little activity was restored, suggesting the majority of oligomeric C1573S was nonnative. A larger percentage of monomeric C1573A could be retrimerized into an assembly-competent form by adding intact LCb. We also discovered that C-terminally deleted light chains produced a heterogeneous population of hubs that were smaller than native hubs, but were assembly active. We propose a model showing how light chains reinforce the puckered clathrin triskelion. Finally, the ability of light chains to retrimerize C1573A hub suggests that the structural role of light chain may be conserved in yeast and mammals.
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http://dx.doi.org/10.1111/j.1600-0854.2007.00597.xDOI Listing
August 2007

Coenzyme isomerization is integral to catalysis in aldehyde dehydrogenase.

Biochemistry 2003 Jun;42(23):7100-9

Program in Medical Biophysics and Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.

Crystal structures of many enzymes in the aldehyde dehydrogenase superfamily determined in the presence of bound NAD(P)(+) have exhibited conformational flexibility for the nicotinamide half of the cofactor. This has been hypothesized to be important in catalysis because one conformation would block the second half of the reaction, but no firm evidence has been put forth which shows whether the oxidized and reduced cofactors preferentially occupy the two observed conformations. We present here two structures of the wild type and two structures of a Cys302Ser mutant of human mitochondrial aldehyde dehydrogenase in binary complexes with NAD(+) and NADH. These structures, including the Cys302Ser mutant in complex with NAD(+) at 1.4 A resolution and the wild-type enzyme in complex with NADH at 1.9 A resolution, provide strong evidence that bound NAD(+) prefers an extended conformation ideal for hydride transfer and bound NADH prefers a contracted conformation ideal for acyl-enzyme hydrolysis. Unique interactions between the cofactor and the Rossmann fold make isomerization possible while allowing the remainder of the active site complex to remain intact. In addition, these structures clarify the role of magnesium in activating the human class 2 enzyme. Our data suggest that the presence of magnesium may lead to selection of particular conformations and speed isomerization of the reduced cofactor following hydride transfer.
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http://dx.doi.org/10.1021/bi034182wDOI Listing
June 2003