Showing papers by "Robert McKenna published in 2017"

Thermal Stability as a Determinant of AAV Serotype Identity.

Abstract: Currently, there are over 150 ongoing clinical trials utilizing adeno-associated viruses (AAVs) to target various genetic diseases, including hemophilia (AAV2 and AAV8), congenital heart failure (AAV1 and AAV6), cystic fibrosis (AAV2), rheumatoid arthritis (AAV2), and Batten disease (AAVrh.10). Prior to patient administration, AAV vectors must have their serotype, concentration, purity, and stability confirmed. Here, we report the application of differential scanning fluorimetry (DSF) as a good manufacturing practice (GMP) capable of determining the melting temperature (Tm) for AAV serotype identification. This is a simple, rapid, cost effective, and robust method utilizing small amounts of purified AAV capsids (∼25 μL of ∼1011 particles). AAV1-9 and AAVrh.10 exhibit specific Tms in buffer formulations commonly used in clinical trials. Notably, AAV2 and AAV3, which are the least stable, have varied Tms, whereas AAV5, the most stable, has a narrow Tm range in the different buffers, respectively. Vector stability was dictated by VP3 only, specifically, the ratio of basic/acidic amino acids, and was independent of VP1 and VP2 content or the genome packaged. Furthermore, stability of recombinant AAVs differing by a single basic or acidic amino acid residue are distinguishable. Hence, AAV DSF profiles can serve as a robust method for serotype identification of clinical vectors.

Discovery of New Sulfonamide Carbonic Anhydrase IX Inhibitors Incorporating Nitrogenous Bases

Abstract: Incorporation of the purine/pyrimidine moieties as tails to classical benzenesulfonamide scaffolds afforded two series of human (h) carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The compounds were designed according to the molecular hybridization approach, in order to modulate the interaction with different CA isozymes and exploit the antitumor effect of uracil and adenine derivatives in parallel and synergic mode to the inhibition of the tumor-associated hCA IX. The sulfonamides were investigated as inhibitors of four isoforms, cytosolic hCA I/II and transmembrane hCA IV/IX. The inhibitory profiles were dependent on the length and positioning of the spacer connecting the two pharmacophores. X-ray crystallography demonstrated the binding mode of an inhibitor to hCA II and hCA IX-mimic. Compounds endowed with the best hCA IX inhibitory efficacy were evaluated for antiproliferative activity against HT-29 colon cancer cell lines. The in vitro results suggest multiple mechanisms of action are responsible fo...

Structure-Activity Relationships of Benzenesulfonamide-Based Inhibitors towards Carbonic Anhydrase Isoform Specificity.

Abstract: Carbonic anhydrases (CAs) are implicated in a wide range of diseases, including the upregulation of isoforms CA IX and XII in many aggressive cancers. However, effective inhibition of disease-implicated CAs should minimally affect the ubiquitously expressed isoforms including CA I and II to improve directed distribution of the inhibitors to the cancer-associated isoforms and reduce side effects. Four benzenesulfonamide-based inhibitors were synthesized using the "tail approach" and display nM affinities for several CA isoforms. The crystal structures of the inhibitors bound to a CA IX-mimic and CA II are presented. Further in-silico modeling was performed with the inhibitors docked into CA I and XII to identify residues that contribute to or hinder their binding interactions. These structural studies demonstrate that active site residues lining the hydrophobic pocket, especially positions 92 and 131, dictate the positional binding and affinity of inhibitors and the tail groups modulate CA isoform specificity. Geometry optimizations were performed on each ligand in the crystal structures and showed the energetic penalties of the inhibitors' conformations were negligible compared to the gain of active site interactions. These studies further our understanding of obtaining isoform specificity when designing small molecule CA inhibitors.

Structural Insights into Human Bocaparvoviruses.

Abstract: Bocaparvoviruses are emerging pathogens of the Parvoviridae family. Human bocavirus 1 (HBoV1) causes severe respiratory infections and HBoV2 to HBoV4 cause gastrointestinal infections in young children. Recent reports of life-threatening cases, lack of direct treatment or vaccination, and a limited understanding of their disease mechanisms highlight the need to study these pathogens on a molecular and structural level for the development of therapeutics. Toward this end, the capsid structures of HBoV1, HBoV3, and HBoV4 were determined to a resolution of 2.8 to 3.0 A by cryo-electron microscopy and three-dimensional image reconstruction. The bocaparvovirus capsids, which display different tissue tropisms, have features in common with other parvoviruses, such as depressions at the icosahedral 2-fold symmetry axis and surrounding the 5-fold symmetry axis, protrusions surrounding the 3-fold symmetry axis, and a channel at the 5-fold symmetry axis. However, unlike other parvoviruses, densities extending the 5-fold channel into the capsid interior are conserved among the bocaparvoviruses and are suggestive of a genus-specific function. Additionally, their major viral protein 3 contains loops with variable regions at their apexes conferring capsid surface topologies different from those of other parvoviruses. Structural comparisons at the strain (HBoV) and genus (bovine parvovirus and HBoV) levels identified differences in surface loops that are functionally important in host/tissue tropism, pathogenicity, and antigenicity in other parvoviruses and likely play similar roles in these viruses. This study thus provides a structural framework to characterize determinants of host/tissue tropism, pathogenicity, and antigenicity for the development of antiviral strategies to control human bocavirus infections.IMPORTANCE Human bocaviruses are one of only a few members of the Parvoviridae family pathogenic to humans, especially young children and immunocompromised adults. There are currently no treatments or vaccines for these viruses or the related enteric bocaviruses. This study obtained the first high-resolution structures of three human bocaparvoviruses determined by cryo-reconstruction. HBoV1 infects the respiratory tract, and HBoV3 and HBoV4 infect the gastrointestinal tract, tissues that are likely targeted by the capsid. Comparison of these viruses provides information on conserved bocaparvovirus-specific features and variable regions resulting in unique surface topologies that can serve as guides to characterize HBoV determinants of tissue tropism and antigenicity in future experiments. Based on the comparison to other existing parvovirus capsid structures, this study suggests capsid regions that likely control successful infection, including determinants of receptor attachment, host cell trafficking, and antigenic reactivity. Overall, these observations could impact efforts to design antiviral strategies and vaccines for HBoVs.

Parvovirus capsid structures required for infection: Mutations controlling receptor recognition and protease cleavages

Abstract: Parvovirus capsids are small but complex molecular machines responsible for undertaking many of the steps of cell infection, genome packing, and cell-to-cell as well as host-to-host transfer. The details of parvovirus infection of cells are still not fully understood, but the processes must involve small changes in the capsid structure that allow the endocytosed virus to escape from the endosome, pass through the cell cytoplasm, and deliver the single-stranded DNA (ssDNA) genome to the nucleus, where viral replication occurs. Here, we examine capsid substitutions that eliminate canine parvovirus (CPV) infectivity and identify how those mutations changed the capsid structure or altered interactions with the infectious pathway. Amino acid substitutions on the exterior surface of the capsid (Gly299Lys/Ala300Lys) altered the binding of the capsid to transferrin receptor type 1 (TfR), particularly during virus dissociation from the receptor, but still allowed efficient entry into both feline and canine cells without successful infection. These substitutions likely control specific capsid structural changes resulting from TfR binding required for infection. A second set of changes on the interior surface of the capsid reduced viral infectivity by >100-fold and included two cysteine residues and neighboring residues. One of these substitutions, Cys270Ser, modulates a VP2 cleavage event found in ∼10% of the capsid proteins that also was shown to alter capsid stability. A neighboring substitution, Pro272Lys, significantly reduced capsid assembly, while a Cys273Ser change appeared to alter capsid transport from the nucleus. These mutants reveal additional structural details that explain cell infection processes of parvovirus capsids. IMPORTANCE Parvoviruses are commonly found in both vertebrate and invertebrate animals and cause widespread disease. They are also being developed as oncolytic therapeutics and as gene therapy vectors. Most functions involved in infection or transduction are mediated by the viral capsid, but the structure-function correlates of the capsids and their constituent proteins are still incompletely understood, especially in relation to identifying capsid processes responsible for infection and release from the cell. Here, we characterize the functional effects of capsid protein mutations that result in the loss of virus infectivity, giving a better understanding of the portions of the capsid that mediate essential steps in successful infection pathways and how they contribute to viral infectivity.

Exploring Heteroaryl-pyrazole Carboxylic Acids as Human Carbonic Anhydrase XII Inhibitors

Abstract: We report the synthesis, biological evaluation, and structural study of a series of substituted heteroaryl-pyrazole carboxylic acid derivatives. These compounds have been developed as inhibitors of specific isoforms of carbonic anhydrase (CA), with potential as prototypes of a new class of chemotherapeutics. Both X-ray crystallography and computational modeling provide insights into the CA inhibition mechanism. Results indicate that this chemotype produces an indirect interference with the zinc ion, thus behaving differently from other related nonclassical inhibitors. Among the tested compounds, 2c with Ki = 0.21 μM toward hCA XII demonstrated significant antiproliferative activity against hypoxic tumor cell lines. Taken together, the results thus provide the basis of structural determinants for the development of novel anticancer agents.

Characteristics of candidate genes associated with embryonic development in the cow: Evidence for a role for WBP1 in development to the blastocyst stage.

Abstract: The goal was to gain understanding of how 12 genes containing SNP previously related to embryo competence to become a blastocyst (BRINP3, C1QB, HSPA1L, IRF9, MON1B, PARM1, PCCB, PMM2, SLC18A2, TBC1D24, TTLL3 and WBP1) participate in embryonic development Gene expression was evaluated in matured oocytes and embryos BRINP3 and C1QB were not detected at any stage For most other genes, transcript abundance declined as the embryo developed to the blastocyst stage Exceptions were for PARM1 and WBP1, where steady-state mRNA increased at the 9-16 cell stage The SNP in WBP1 caused large differences in the predicted three-dimensional structure of the protein while the SNP in PARM1 caused smaller changes The mutation in WBP1 causes an amino acid substitution located close to a P-P-X-Y motif involved in protein-protein interactions Moreover, the observation that the reference allele varies between mammalian species indicates that the locus has not been conserved during mammalian evolution Knockdown of mRNA for WBP1 decreased the percent of putative zygotes becoming blastocysts and reduced the number of trophectoderm cells and immunoreactive CDX2 in the resulting blastocysts WBP1 is an important gene for embryonic development in the cow Further research to identify how the SNP in WBP1 affects processes leading to differentiation of the embryo into TE and ICM lineages is warranted

The Combination of IV and Perineural Dexamethasone Prolongs the Analgesic Duration of Intercostal Nerve Blocks Compared with IV Dexamethasone Alone.

Abstract: Objective. The use of multiple-level, single-injection intercostal nerve blocks for pain control following video-assisted thorascopic surgery (VATS) is limited by the analgesic duration of local anesthetics. This study examines whether the combination of perineural and intravenous (IV) dexamethasone will prolong the duration of intraoperatively placed intercostal nerve blocks following VATS compared with IV dexamethasone and a perineural saline placebo. Design. Prospective, double-blind, randomized placebo-controlled trial. Setting. Single level-1 academic trauma center. Subjects. Forty patients undergoing a unilateral VATS under the care of a single surgeon. Methods. Patients were randomly assigned to two groups and received an intercostal nerve block containing 1) 0.5% bupivacaine with epinephrine and 1 ml of 0.9% saline or 2) 0.5% bupivacaine with epinephrine and 1 ml of a 4 mg/ml dexamethasone solution. All patients received 8 mg of IV dexamethasone. Results. Group 2 had lower NRS-11 scores at post-operative hours 8 (5.05, SD = 2.13 vs 3.50, SD = 2.50; p = 0.04), 20 (4.30, SD = 2.96 vs 2.26, SD = 2.31; p = 0.02), and 24 (4.53, SD = 1.95 vs 2.26, SD = 2.31; p = 0.02). Equianalgesic opioid requirement was decreased in group 2 at 32 hours (5.78 mg, SD = 5.77 vs 1.67 mg, SD = 3.49; p = 0.02). Group 2 also had greater FEV1 measured at 8, 12, 24, and 44 hours; greater FVC at 24 hours; greater PEF at 28 through 48 hours; and greater FEV1/FVC at 8 and 36 hours. Conclusions. The combination of IV and perineural dexamethasone prolonged the duration of a single-injection bupivacaine intercostal nerve block as measured by NRS-11 compared with IV dexamethasone alone at 24 hours. Reduced NRS-11 at other times, reduced opioid requirements, and increased PFTs were observed in group 2.

Atomic Resolution Structure of the Oncolytic Parvovirus LuIII by Electron Microscopy and 3D Image Reconstruction.

Abstract: LuIII, a protoparvovirus pathogenic to rodents, replicates in human mitotic cells, making it applicable for use to kill cancer cells. This virus group includes H-1 parvovirus (H-1PV) and minute virus of mice (MVM). However, LuIII displays enhanced oncolysis compared to H-1PV and MVM, a phenotype mapped to the major capsid viral protein 2 (VP2). This suggests that within LuIII VP2 are determinants for improved tumor lysis. To investigate this, the structure of the LuIII virus-like-particle was determined using single particle cryo-electron microscopy and image reconstruction to 3.17 A resolution, and compared to the H-1PV and MVM structures. The LuIII VP2 structure, ordered from residue 37 to 587 (C-terminal), had the conserved VP topology and capsid morphology previously reported for other protoparvoviruses. This includes a core β-barrel and α-helix A, a depression at the icosahedral 2-fold and surrounding the 5-fold axes, and a single protrusion at the 3-fold axes. Comparative analysis identified surface loop differences among LuIII, H-1PV, and MVM at or close to the capsid 2- and 5-fold symmetry axes, and the shoulder of the 3-fold protrusions. The 2-fold differences cluster near the previously identified MVM sialic acid receptor binding pocket, and revealed potential determinants of protoparvovirus tumor tropism.

The Serpintine Solution

Abstract: Alexandra Lucas, MD, FRCP(C)1,2,*, Sriram Ambadapadi, PhD1, Brian Mahon, PhD3, Kasinath Viswanathan, PhD4, Hao Chen, MD, PhD5, Liying Liu, MD6, Erbin Dai, MD6, Ganesh Munuswami-Ramanujam, PhD7, Jacek M. Kwiecien, DVM, MSc, PhD8, Jordan R Yaron, PhD1, Purushottam Shivaji Narute, BVSc & AH, MVSc, PhD1,9, Robert McKenna, PhD10, Shahar Keinan, PhD11, Westley Reeves, MD, PhD12, Mark Brantly, MD, PhD13, Carl Pepine, MD, FACC14 and Grant McFadden, PhD1 1Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe AZ, USA 2Saint Josephs Hospital, Dignity Health Phoenix, Phoenix, AZ, USA 3NIH/ NIDDK, Bethesda MD, USA 4Zydus Research Centre, Ahmedabad, India 5Department of tumor surgery, The Second Hospital of Lanzhou University, Lanzhou, Gansu, P.R.China 6Beth Israel Deaconess Medical Center, Harvard, Boston, MA, USA 7Interdisciplinary Institute of Indian system of Medicine (IIISM), SRM University, Chennai, Tamil Nadu, India 8MacMaster University, Hamilton, ON, Canada 9Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda MD, USA 10Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL, USA 11Cloud Pharmaceuticals, Durham, North Carolina, USA 12Division of Rheumatology, University of Florida, Gainesville, FL, USA 13Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, University of Florida, Gainesville, FL, USA 14Division of Cardiovascular Medicine, University of Florida, Gainesville, FL, USA *Corresponding author: Alexandra Lucas, MD FRCP(C), Director Cardiovascular Fellowship Research, Saint Josephs Hospital, Dignity Health Phoenix, Professor , Biodesign Institute / Arizona State University, Center for Personalized Diagnostics, Room A220D, Tempe, AZ, 727 E Tyler St, 85287, Tel: 352-672-2301; E-mail: alexluc1@asu.edu

Entropic Anomaly Observed in Lipid Polymorphisms Induced by Surfactant Peptide SP-B(1-25).

Abstract: The N-terminal 25 amino-acid residues of pulmonary surfactant protein B (SP-B1–25) induces unusual lipid polymorphisms in a model lipid system, 4:1 DPPC/POPG, mirroring the lipid composition of native pulmonary surfactant. It is widely suggested that SP-B1–25-induced lipid polymorphisms within the alveolar aqueous subphase provide a structural platform for rapid lipid adsorption to the air–water interface. Here, we characterize in detail the phase behavior of DPPC and POPG in hydrated lipid assemblies containing therapeutic levels of SP-B1–25 using 2H and 31P solid state NMR spectroscopy. The appearance of a previously observed isotropic lipid phase is found to be highly dependent on the thermal cycling of the samples. Slow heating of frozen samples leads to phase separation of DPPC into a lamellar phase whereas POPG lipids interact with the peptide to form an isotropic phase at physiologic temperature. Rapid heating of frozen samples to room temperature leads to strongly isotropic phase behavior for both...

Stable poliovirus variants and uses thereof

Abstract: Provided herein are compositions of virus like particles (VLPs) of poliovirus (PV) that have one or more stabilizing mutations that confer a higher degree of thermostability to the N-antigenic form of the VLPs. These VLPs are non-infectious, and thus safer for use in vaccine development and administration to clinical subjects.