Vlad K. Kumirov

Novel fentanyl-based dual μ/δ-opioid agonists for the treatment of acute and chronic pain.

UNLABELLED Approximately one third of the adult U.S. population suffers from some type of on-going, chronic pain annually, and many more will have some type of acute pain associated with trauma or surgery. First-line therapies for moderate to severe pain include prescriptions for common mu opioid receptor agonists such as morphine and its various derivatives. The epidemic use, misuse and diversion of prescription opioids have highlighted just one of the adverse effects of mu opioid analgesics. Alternative approaches include novel opioids that target delta or kappa opioid receptors, or compounds that interact with two or more of the opioid receptors. AIMS Here we report the pharmacology of a newly synthesized bifunctional opioid agonist (RV-Jim-C3) derived from combined structures of fentanyl and enkephalin in rodents. RV-Jim-C3 has high affinity binding to both mu and delta opioid receptors. MAIN METHODS Mice and rats were used to test RV-Jim-C3 in a tailflick test with and without opioid selective antagonist for antinociception. RV-Jim-C3 was tested for anti-inflammatory and antihypersensitivity effects in a model of formalin-induced flinching and spinal nerve ligation. To rule out motor impairment, rotarod was tested in rats. KEY FINDINGS RV-Jim-C3 demonstrates potent-efficacious activity in several in vivo pain models including inflammatory pain, antihyperalgesia and antiallodynic with no significant motor impairment. SIGNIFICANCE This is the first report of a fentanyl-based structure with delta and mu opioid receptor activity that exhibits outstanding antinociceptive efficacy in neuropathic pain, reducing the propensity of unwanted side effects driven by current therapies that are unifunctional mu opioid agonists.

Phospholipase D Toxins of Brown Spider Venom Convert Lysophosphatidylcholine and Sphingomyelin to Cyclic Phosphates

Venoms of brown spiders in the genus Loxosceles contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These toxins cleave the substrates sphingomyelin and lysophosphatidylcholine in mammalian tissues, releasing the choline head group. The other products of substrate cleavage have previously been reported to be monoester phospholipids, which would result from substrate hydrolysis. Using 31P NMR and mass spectrometry we demonstrate that recombinant toxins, as well as whole venoms from diverse Loxosceles species, exclusively catalyze transphosphatidylation rather than hydrolysis, forming cyclic phosphate products from both major substrates. Cyclic phosphates have vastly different biological properties from their monoester counterparts, and they may be relevant to the pathology of brown spider envenomation.

Studying protein fold evolution with hybrids of differently folded homologs

To study the sequence determinants governing protein fold evolution, we generated hybrid sequences from two homologous proteins with 40% identity but different folds: Pfl 6 Cro, which has a mixed α + β structure, and Xfaso 1 Cro, which has an all α-helical structure. First, we first examined eight chimeric hybrids in which the more structurally conserved N-terminal half of one protein was fused to the more structurally divergent C-terminal half of the other. None of these chimeras folded, as judged by circular dichroism spectra and thermal melts, suggesting that both halves have strong intrinsic preferences for the native global fold pattern, and/or that the interfaces between the halves are not readily interchangeable. Second, we examined 10 hybrids in which blocks of the structurally divergent C-terminal region were exchanged. These hybrids showed varying levels of thermal stability and suggested that the key residues in the Xfaso 1 C terminus specifying the all-α fold were concentrated near the end of helix 4 in Xfaso 1, which aligns to the end of strand 2 in Pfl 6. Finally, we generated hybrid substitutions for each individual residue in this critical region and measured thermal stabilities. The results suggested that R47 and V48 were the strongest factors that excluded formation of the α + β fold in the C-terminal region of Xfaso 1. In support of this idea, we found that the folding stability of one of the original eight chimeras could be rescued by back-substituting these two residues. Overall, the results show not only that the key factors for Cro fold specificity and evolution are global and multifarious, but also that some all-α Cro proteins have a C-terminal subdomain sequence within a few substitutions of switching to the α + β fold.

Synthesis and Investigation of Mixed μ-Opioid and δ-Opioid Agonists as Possible Bivalent Ligands for Treatment of Pain

Several studies have suggested functional association between μ-opioid and δ-opioid receptors and showed that μ-activity could be modulated by δ-ligands. The general conclusion is that agonists for the δ-receptor can enhance the analgesic potency and efficacy of μ-agonists. Our preliminary investigations demonstrate that new bivalent ligands constructed from the μ-agonist fentanyl and the δ-agonist enkephalin-like peptides are promising entities for creation of new analgesics with reduced side effects for treatment of neuropathic pain. A new superposition of the mentioned pharmacophores led to novel μ-bivalent/δ-bivalent compounds that demonstrate both μ-opioid and δ-opioid receptor agonist activity and high efficacy in anti-inflammatory and neuropathic pain models with the potential of reduced unwanted side effects.

Multistep mutational transformation of a protein fold through structural intermediates: Intermediate structures in protein fold evolution

New protein folds may evolve from existing folds through metamorphic evolution involving a dramatic switch in structure. To mimic pathways by which amino acid sequence changes could induce a change in fold, we designed two folded hybrids of Xfaso 1 and Pfl 6, a pair of homologous Cro protein sequences with ~40% identity but different folds (all‐α vs. α + β, respectively). Each hybrid, XPH1 or XPH2, is 85% identical in sequence to its parent, Xfaso 1 or Pfl 6, respectively; 55% identical to its noncognate parent; and ~70% identical to the other hybrid. XPH1 and XPH2 also feature a designed hybrid chameleon sequence corresponding to the C‐terminal region, which switched from α‐helical to β‐sheet structure during Cro evolution. We report solution nuclear magnetic resonance (NMR) structures of XPH1 and XPH2 at 0.3 Å and 0.5 Å backbone root mean square deviation (RMSD), respectively. XPH1 retains a global fold generally similar to Xfaso 1, and XPH2 retains a fold similar to Pfl 6, as measured by TM‐align scores (~0.7), DALI Z‐scores (7‐9), and backbone RMSD (2–3 Å RMSD for the most ordered regions). However, these scores also indicate significant deviations in structure. Most notably, XPH1 and XPH2 have different, and intermediate, secondary structure content relative to Xfaso 1 and Pfl 6. The multistep progression in sequence, from Xfaso 1 to XPH1 to XPH2 to Pfl 6, thus involves both abrupt and gradual changes in folding pattern. The plasticity of some protein folds may allow for “polymetamorphic” evolution through intermediate structures.