- Email: [email protected]
Structural basis for the inhibition of botulinum neurotoxin serotype A by potent peptidomimetics
Abstracts Toxins 2011 / Toxicon 68 (2013) 60–123
conventional methods such as PFGE. In the present study we planned to develop a Multiple-Locus, Variable-Number, Tandem-Repeat Analysis (MLVA) system, which would be useful for characterizing our Clostridium tetani stock that we could not analyze by the PFGE method. Methods used: 1) Strains: Thirty-six strains, consisted of eight human clinical isolates, seven yellow dust (Asian desert-generated and sand-transported by air) isolates and 21 soil isolates from Japan were subjected to the study. 2) MLVA analysis: Firstly, we identified many VNTR regions within the complete genomic sequence of C. tetani strain E88 by a web-based software Tandem Repeat Finder. Within many regions, we selected four regions as targets for MLVA analysis. Specific primers were designed for each region to amplify the entire target and flanking sequences. PCR reactions and data analyses were performed using an ABI automated fluorescent DNA sequencer. Summary of results: All of the four VNTR target sequences were successfully amplified in all of C. tetani strains used in this study. By MLVA analysis of the 4 regions, the 36 strains were classified into 3 genotypic groups. These groups consisted of 6 strains, 21 strains and 9 strains, respectively. PFGE analysis performed in parallel on the same strains could not distinguish those belonging to different VNTR subgroups, indicating that the MLVA system described here has a higher potential than conventional PFGE. Conclusions: Four VNTR markers identified in the genome sequence of C. tetani type strain E88 were used for MLVA analysis to subtype 36 C. tetani strains isolated in Japan. MLVA analysis may have higher potential for subtyping of C. tetani strains compared to PFGE typing. This method provides a useful tool for epidemiological studies, further characterization of archival strain collections, and for assisting in investigations of C. tetani infections. http://dx.doi.org/10.1016/j.toxicon.2012.07.120
Development of serotype-specific monoclonal antibodies against botulinum neurotoxins A, B and E, based on a trivalent immunization protocol and simultaneous differential robotic screen E. Diamant a, B. Lahmi b, A. Keren a, A. Barnea a, H. Marcus a, S. Cohen a, R. Zichel a a
Department of Biotechnology, Israel Institute for Biological Research, P.O.B. 19, Ness Ziona 76100, Israel Department of Infectious Diseases, Israel Institute for Biological Research, P.O.B. 19, Ness Ziona 76100, Israel E-mail address: [email protected] (R. Zichel). b
Botulinum neurotoxins (BoNT), produced by Clostridium botulinum strains, are considered the most lethal toxins known. Human botulism is mostly caused by serotypes A, B, and E, and might be fatal if not treated immediately. Differential in vitro diagnosis of botulinum toxins is challenging mainly due to cross-reactivity of polyclonal antibodies (pAbs) that stems from the homology between different botulinum toxin complexes. Purpose of study: In this study we describe the development of highly specific monoclonal antibodies (mAbs)
103
against A, B and E neurotoxins, using a tri-valent immunization protocol followed by a simultaneous differential robotic hybridoma screen. Methods used: Balb/C mice were immunized with a mixture of the recombinant carboxy-terminal fragment of each of the three neurotoxins (HcA, HcB and HcE), and serum antibody titers against the three holotoxins in hyper-immune mice were demonstrated by ELISA. Differential screening of secreting hybridoma cells against each of the three serotypes was simultaneously performed in three parallel ELISA assays using a high throughput robotic system. Summary of results: The specificity of the selected mAbs to botulinum toxin complexes was orders of magnitude higher than that of pAbs. Surprisingly, some of the mAbs had equally high titers as compared to their polyclonal counterparts. Moreover, few of the mAbs were capable of in vivo neutralization in a mouse bioassay. The results of competitive ELISA assays indicated that in serotypic groups A and B mAbs bound at least two distinct epitopes. Conclusions: Our approach for simultaneous production of mAbs to several antigens proved to be useful in generating highly specific mAbs to botulinum neurotoxins with a diagnostic and therapeutic potential. http://dx.doi.org/10.1016/j.toxicon.2012.07.121
Structural basis for the inhibition of botulinum neurotoxin serotype A by potent peptidomimetics J. Zuniga a, J. Hammill b, O. Drory a, J. Nuss c, J. Burnett d, R. Gussio e, P. Wipf b, S. Bavari c, A.T. Brunger a a
Howard Hughes Medical Institute and Stanford University, Stanford, CA, USA b University of Pittsburgh, Pittsburgh, PA, USA c U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD, USA d National Cancer Institute at Frederick, Frederick, MD, USA e Developmental Therapeutics Program, National Cancer Institute at Frederick, Frederick, MD, USA E-mail address: [email protected] (J. Zuniga).
Purpose of study: We aimed to unveil the inhibitory interactions of peptides on the botulinum neurotoxin type A catalytic domain (BoNT/A LC). This structural information would then be used for the rational design of more potent inhibitors. Methods used: We generated crystals of BoNT/A LC, with subsequent soaking in solutions containing the I1 peptidomimetic. The structure of this first complex was solved, and a SAR analysis was performed with related derivatives and their corresponding inhibition potencies (Ki). Next, the key interactions were used to design a new series of heptapeptides, whose complex with BoNT/A LC was also characterized by X-ray crystallography, further unveiling features of BoNT/A LC that will enable the structure-based development and screening of potent inhibitors. Summary of results: These structural data revealed novel and unexpected interactions for effective binding to, and inhibition of, BoNT/A LC. The inhibition by non-Znchelating peptidic inhibitors relies on the terminal amino
104
Abstracts Toxins 2011 / Toxicon 68 (2013) 60–123
group, which displaces a water molecule from the active site, crucial for efficient proteolysis of SNAP-25. In these two structures P1’Arg engages into a salt bridge with Asp370 in the toxin. In addition, aromatic side-chains in the P2’ position dock into an induced hydrophobic pocket in the protease. Conclusions: Effective inhibition of BoNT/A LC activity can be achieved by placing a chemical group with the same orientation as the terminal amino groups of the peptides used in the present study and similar capability to coordinate the Glu224 in the enzyme. This interaction, together with the non-polar contacts by the P2’ residue, and some others, can be used to define a pharmacophore to guide in silico screening for drugable organic compounds. http://dx.doi.org/10.1016/j.toxicon.2012.07.122
Clinical science poster abstracts Communication calendar for botulinum toxin therapy of spasticity in an outpatient setting F. Adib Saberi a, H. Pickenbrock b, D. Dressler b a Department of Neurology, Asklepios Klinik Nord Heidberg, Hamburg, Germany b Movement Disorders Section, Department of Neurology, Hannover Medical School, Hannover, Germany E-mail address: [email protected] (F. Adib Saberi).
Purpose of study: Botulinum toxin (BT) therapy of spasticity is best applied together in combination with additional therapies including physiotherapy and occupational therapy. To improve this collaboration, we developed a communication calendar (CC) and used it in our outpatient clinics. Methods used: The CC includes the following items: 1) target muscle description, 2) list of therapy goals, 3) goal attainment scale (GAS), 4) evaluation of the injection scheme, 5) assessment of BT dosing in target muscles, 6) additional information. It is accompanied by an instruction manual. All items are evaluated 4-6 weeks after the last injection and directly before the next BT application throughout all injections cycles. The patient, together with the therapist, determines the therapy goal as to function, pain, nursing or cosmetic aspects. The BT injector records the target muscles. The therapist evaluates the therapeutic effect on the goal and the target muscles, suggests modifications of the injection scheme or complementary measures and documents interfering incidents. Summary of results: Time to evaluate items was less than 5 min for each party. None of the CC was rejected. It monitors the results of the ongoing treatment. Conclusions: The CC is easy to use and well accepted. It improves flow of information between the BT injector and the other therapists involved and facilitates adjustments of treatment strategies. http://dx.doi.org/10.1016/j.toxicon.2012.07.123
RimabotulinumtoxinB in the treatment of refractory pain in fibromyalgia: A case series M.V. Alvarez a, P.M. Grogan b a b
Center for Neurological Care and Research, San Antonio, TX, USA Wilford Hall Medical Center, Lackland Air Force Base, TX, USA E-mail address: [email protected] (M.V. Alvarez).
Purpose of study: To report a series of patients with fibromyalgia (FM) who achieved significant, reproducible improvement in pain following injections of rimabotulinumtoxinB (rbtx-B). Methods used: Case Series. Nine patients diagnosed with FM based on American College of Rheumatology criteria were followed (8 females, 1 male; ages 38-63 years old). All had been previously treated with oral medications for FM pain, including PregabalinÒ, GabapentinÒ, and/or DuloxetineÒ, but all noted suboptimal response in pain relief. Pain scores were graded using Visual Analog Score (VAS). Patients were given intramuscular injections of rbtxB, 5000 U/1 ml volume without dilution into muscles of the torso (N¼10) and/or extremities (N¼4) overlying painful pressure points, each injection with 500 U per site. Total dosages utilized per cycle: 5000-10,000 U, depending on the number of painful tender points. All patients received between 3-12 repeat injection cycles (average 6.8) at a frequency of every 12 weeks. Summary of results: All patients reported reductions in FM-related pain. VAS for pain prior to injections ranged from 5–8 (average 6.2); post treatment VAS ranged from 2–4 (average 2.7; 43% reduction from baseline). VAS score reductions were sustained over repeat injection cycles. Two patients were able to discontinue oral medications for FM and achieve adequate pain reduction with rbtx-B injections alone. No significant adverse effects to rbtx-B were reported, and there were no drug-drug interactions. Conclusions: In our series of nine patients with FMrelated pain refractory to available approved oral medications, we found that rbtx-B can be a safe and efficacious adjunctive therapy for pain management. http://dx.doi.org/10.1016/j.toxicon.2012.07.124
Is there a role for rimabotulinumtoxinB In the treatment of medication-resistant low back pain? M.V. Alvarez a, P.M. Grogan b a
Center for Neurological Care and Research, San Antonio, TX, USA Department of Neurology, Brooke Army Medical Center, Fort Sam Houston, TX, USA E-mail address: [email protected] (M.V. Alvarez). b
Purpose of study: In one statistical analysis low back pain can be seen in as many as 31 million Americans in any given time, and it runs second, after the common cold, as the top reason for visiting a healthcare provider in the United States. Several studies have shown the potential role of botulinum toxin type A in the management of chronic low back pain. We report the efficacy and safety of
conventional methods such as PFGE. In the present study we planned to develop a Multiple-Locus, Variable-Number, Tandem-Repeat Analysis (MLVA) system, which would be useful for characterizing our Clostridium tetani stock that we could not analyze by the PFGE method. Methods used: 1) Strains: Thirty-six strains, consisted of eight human clinical isolates, seven yellow dust (Asian desert-generated and sand-transported by air) isolates and 21 soil isolates from Japan were subjected to the study. 2) MLVA analysis: Firstly, we identified many VNTR regions within the complete genomic sequence of C. tetani strain E88 by a web-based software Tandem Repeat Finder. Within many regions, we selected four regions as targets for MLVA analysis. Specific primers were designed for each region to amplify the entire target and flanking sequences. PCR reactions and data analyses were performed using an ABI automated fluorescent DNA sequencer. Summary of results: All of the four VNTR target sequences were successfully amplified in all of C. tetani strains used in this study. By MLVA analysis of the 4 regions, the 36 strains were classified into 3 genotypic groups. These groups consisted of 6 strains, 21 strains and 9 strains, respectively. PFGE analysis performed in parallel on the same strains could not distinguish those belonging to different VNTR subgroups, indicating that the MLVA system described here has a higher potential than conventional PFGE. Conclusions: Four VNTR markers identified in the genome sequence of C. tetani type strain E88 were used for MLVA analysis to subtype 36 C. tetani strains isolated in Japan. MLVA analysis may have higher potential for subtyping of C. tetani strains compared to PFGE typing. This method provides a useful tool for epidemiological studies, further characterization of archival strain collections, and for assisting in investigations of C. tetani infections. http://dx.doi.org/10.1016/j.toxicon.2012.07.120
Development of serotype-specific monoclonal antibodies against botulinum neurotoxins A, B and E, based on a trivalent immunization protocol and simultaneous differential robotic screen E. Diamant a, B. Lahmi b, A. Keren a, A. Barnea a, H. Marcus a, S. Cohen a, R. Zichel a a
Department of Biotechnology, Israel Institute for Biological Research, P.O.B. 19, Ness Ziona 76100, Israel Department of Infectious Diseases, Israel Institute for Biological Research, P.O.B. 19, Ness Ziona 76100, Israel E-mail address: [email protected] (R. Zichel). b
Botulinum neurotoxins (BoNT), produced by Clostridium botulinum strains, are considered the most lethal toxins known. Human botulism is mostly caused by serotypes A, B, and E, and might be fatal if not treated immediately. Differential in vitro diagnosis of botulinum toxins is challenging mainly due to cross-reactivity of polyclonal antibodies (pAbs) that stems from the homology between different botulinum toxin complexes. Purpose of study: In this study we describe the development of highly specific monoclonal antibodies (mAbs)
103
against A, B and E neurotoxins, using a tri-valent immunization protocol followed by a simultaneous differential robotic hybridoma screen. Methods used: Balb/C mice were immunized with a mixture of the recombinant carboxy-terminal fragment of each of the three neurotoxins (HcA, HcB and HcE), and serum antibody titers against the three holotoxins in hyper-immune mice were demonstrated by ELISA. Differential screening of secreting hybridoma cells against each of the three serotypes was simultaneously performed in three parallel ELISA assays using a high throughput robotic system. Summary of results: The specificity of the selected mAbs to botulinum toxin complexes was orders of magnitude higher than that of pAbs. Surprisingly, some of the mAbs had equally high titers as compared to their polyclonal counterparts. Moreover, few of the mAbs were capable of in vivo neutralization in a mouse bioassay. The results of competitive ELISA assays indicated that in serotypic groups A and B mAbs bound at least two distinct epitopes. Conclusions: Our approach for simultaneous production of mAbs to several antigens proved to be useful in generating highly specific mAbs to botulinum neurotoxins with a diagnostic and therapeutic potential. http://dx.doi.org/10.1016/j.toxicon.2012.07.121
Structural basis for the inhibition of botulinum neurotoxin serotype A by potent peptidomimetics J. Zuniga a, J. Hammill b, O. Drory a, J. Nuss c, J. Burnett d, R. Gussio e, P. Wipf b, S. Bavari c, A.T. Brunger a a
Howard Hughes Medical Institute and Stanford University, Stanford, CA, USA b University of Pittsburgh, Pittsburgh, PA, USA c U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD, USA d National Cancer Institute at Frederick, Frederick, MD, USA e Developmental Therapeutics Program, National Cancer Institute at Frederick, Frederick, MD, USA E-mail address: [email protected] (J. Zuniga).
Purpose of study: We aimed to unveil the inhibitory interactions of peptides on the botulinum neurotoxin type A catalytic domain (BoNT/A LC). This structural information would then be used for the rational design of more potent inhibitors. Methods used: We generated crystals of BoNT/A LC, with subsequent soaking in solutions containing the I1 peptidomimetic. The structure of this first complex was solved, and a SAR analysis was performed with related derivatives and their corresponding inhibition potencies (Ki). Next, the key interactions were used to design a new series of heptapeptides, whose complex with BoNT/A LC was also characterized by X-ray crystallography, further unveiling features of BoNT/A LC that will enable the structure-based development and screening of potent inhibitors. Summary of results: These structural data revealed novel and unexpected interactions for effective binding to, and inhibition of, BoNT/A LC. The inhibition by non-Znchelating peptidic inhibitors relies on the terminal amino
104
Abstracts Toxins 2011 / Toxicon 68 (2013) 60–123
group, which displaces a water molecule from the active site, crucial for efficient proteolysis of SNAP-25. In these two structures P1’Arg engages into a salt bridge with Asp370 in the toxin. In addition, aromatic side-chains in the P2’ position dock into an induced hydrophobic pocket in the protease. Conclusions: Effective inhibition of BoNT/A LC activity can be achieved by placing a chemical group with the same orientation as the terminal amino groups of the peptides used in the present study and similar capability to coordinate the Glu224 in the enzyme. This interaction, together with the non-polar contacts by the P2’ residue, and some others, can be used to define a pharmacophore to guide in silico screening for drugable organic compounds. http://dx.doi.org/10.1016/j.toxicon.2012.07.122
Clinical science poster abstracts Communication calendar for botulinum toxin therapy of spasticity in an outpatient setting F. Adib Saberi a, H. Pickenbrock b, D. Dressler b a Department of Neurology, Asklepios Klinik Nord Heidberg, Hamburg, Germany b Movement Disorders Section, Department of Neurology, Hannover Medical School, Hannover, Germany E-mail address: [email protected] (F. Adib Saberi).
Purpose of study: Botulinum toxin (BT) therapy of spasticity is best applied together in combination with additional therapies including physiotherapy and occupational therapy. To improve this collaboration, we developed a communication calendar (CC) and used it in our outpatient clinics. Methods used: The CC includes the following items: 1) target muscle description, 2) list of therapy goals, 3) goal attainment scale (GAS), 4) evaluation of the injection scheme, 5) assessment of BT dosing in target muscles, 6) additional information. It is accompanied by an instruction manual. All items are evaluated 4-6 weeks after the last injection and directly before the next BT application throughout all injections cycles. The patient, together with the therapist, determines the therapy goal as to function, pain, nursing or cosmetic aspects. The BT injector records the target muscles. The therapist evaluates the therapeutic effect on the goal and the target muscles, suggests modifications of the injection scheme or complementary measures and documents interfering incidents. Summary of results: Time to evaluate items was less than 5 min for each party. None of the CC was rejected. It monitors the results of the ongoing treatment. Conclusions: The CC is easy to use and well accepted. It improves flow of information between the BT injector and the other therapists involved and facilitates adjustments of treatment strategies. http://dx.doi.org/10.1016/j.toxicon.2012.07.123
RimabotulinumtoxinB in the treatment of refractory pain in fibromyalgia: A case series M.V. Alvarez a, P.M. Grogan b a b
Center for Neurological Care and Research, San Antonio, TX, USA Wilford Hall Medical Center, Lackland Air Force Base, TX, USA E-mail address: [email protected] (M.V. Alvarez).
Purpose of study: To report a series of patients with fibromyalgia (FM) who achieved significant, reproducible improvement in pain following injections of rimabotulinumtoxinB (rbtx-B). Methods used: Case Series. Nine patients diagnosed with FM based on American College of Rheumatology criteria were followed (8 females, 1 male; ages 38-63 years old). All had been previously treated with oral medications for FM pain, including PregabalinÒ, GabapentinÒ, and/or DuloxetineÒ, but all noted suboptimal response in pain relief. Pain scores were graded using Visual Analog Score (VAS). Patients were given intramuscular injections of rbtxB, 5000 U/1 ml volume without dilution into muscles of the torso (N¼10) and/or extremities (N¼4) overlying painful pressure points, each injection with 500 U per site. Total dosages utilized per cycle: 5000-10,000 U, depending on the number of painful tender points. All patients received between 3-12 repeat injection cycles (average 6.8) at a frequency of every 12 weeks. Summary of results: All patients reported reductions in FM-related pain. VAS for pain prior to injections ranged from 5–8 (average 6.2); post treatment VAS ranged from 2–4 (average 2.7; 43% reduction from baseline). VAS score reductions were sustained over repeat injection cycles. Two patients were able to discontinue oral medications for FM and achieve adequate pain reduction with rbtx-B injections alone. No significant adverse effects to rbtx-B were reported, and there were no drug-drug interactions. Conclusions: In our series of nine patients with FMrelated pain refractory to available approved oral medications, we found that rbtx-B can be a safe and efficacious adjunctive therapy for pain management. http://dx.doi.org/10.1016/j.toxicon.2012.07.124
Is there a role for rimabotulinumtoxinB In the treatment of medication-resistant low back pain? M.V. Alvarez a, P.M. Grogan b a
Center for Neurological Care and Research, San Antonio, TX, USA Department of Neurology, Brooke Army Medical Center, Fort Sam Houston, TX, USA E-mail address: [email protected] (M.V. Alvarez). b
Purpose of study: In one statistical analysis low back pain can be seen in as many as 31 million Americans in any given time, and it runs second, after the common cold, as the top reason for visiting a healthcare provider in the United States. Several studies have shown the potential role of botulinum toxin type A in the management of chronic low back pain. We report the efficacy and safety of