Small Molecules And Functionalized Peptides To Study Protein Prenylation

University of Minnesota Ph.D. dissertation September 2016. Major: Chemistry. Advisor: Mark Distefano. 1 computer file (PDF); xxii, 210 pages.Post-prenylation processing enzymes Rce1, Ste24, and Icmt function to increase the hydrophobicity of prenylated proteins and assist in targeting them to cellular membranes. Rce1 and Ste24 are CAAX endoproteases that cleave the –AAX residues off the C-termini of prenylated proteins that are then methylated by Icmt. Many prenylated proteins play key roles in the progression of cancer, such as the prenylated G-protein family Ras. Most therapeutic efforts in this area focused on preventing protein prenylation by inhibiting the prenyltransferase enzymes. However, other means of preventing oncogenic signal transduction can be accomplished by disrupting the function of Rce1, Ste24, and Icmt. This thesis focuses on the synthesis and application of small molecules and functionalized peptides to study isoprenoid recognizing enzymes. In order to evaluate Icmt’s topology, mechanism of methylation, and substrate recognition, isoprenoid-containing photoactivatable analogues were developed that crosslink to residues in or near the reactive sites. Initial studies focused on the synthesis and evaluation of benzophenone-containing peptide probes that resemble the natural, prenylated substrate. After the functionalized peptides were determined to be substrates, UV wavelength photolysis was used to cross-link the benzophenone moiety to adjacent Icmt isoprenoid binding site residues. To improve the efficiency of the substrate recognition and enhance labeling of Icmt, a new diazirine isoprenoid analogue was developed. Additionally, the peptide backbone was modified to contain both a biotin moiety and a fluorophore for facile in-gel fluoresces detection of the crosslinked material. These improvements should assist the ability for the cross-linked active site to be determined for Icmt after proteolysis and LC-MS-MS analysis, a task that has yet to be solved. Ste24p has two different proteolysis roles in the maturation of the yeast mating pheromone, a-factor. The first site is the CAAX motif, while the second site is upstream toward the N-terminus, each having a unique amino acid sequences. As a way to observe the reaction kinetics of both sites, peptide analogues that contain a fluorescent donor-quencher pair were developed. By monitoring the increase in fluorescence upon proteolysis, reaction kinetics of both cleavage positions were determined. To explore the pool of cellular proteins that recognize isoprenoid diphosphates, photoaffinity isoprenoid diphosphate analogues were synthesized. Two types of isoprenoid diphosphate derivatives were developed: diphosphate and phosphonophosphate. Each of the derivatives contain a diazirine motif as the photophore. The diphosphate analogue was shown to be an alternative substrate for both yeast and mammalian farnesyltransferase. The photoaffinity phosphonophosphate analogues were proven to be inhibitors of farnesyltransferase, demonstrated the ability to label isoprenoid binding proteins SmgGDS-607 and SmgGDS-558, and should be suitable for identifying unknown binding partners in future cellular lysate labeling experiments. Lastly, effort was put forth to synthesize a small molecule inhibitor for Icmt. The inhibitor incorporates both substrates recognized by Icmt: a prenylcysteine carboxylate, and the methyl donor S-adenosine methionine. In order to make the bisubstrate molecule an inhibitor, the appendage between the two substrates is through an amide bond that replaces the cysteine carboxylate moiety. It is hypothesized that by combining both substrates into one entity, the bisubstrate compound will bind more efficiently with Icmt and enhance inhibitor potency. The bisubstrate inhibitor was shown to inhibit mammalian Icmt, but had little effect on the yeast homologue

Effect of currently approved carriers and adjuvants on the pre-clinical efficacy of a conjugate vaccine against oxycodone in mice and rats.

Vaccination against the highly abused prescription opioid oxycodone has shown pre-clinical efficacy for blocking oxycodone effects. The current study further evaluated a candidate vaccine composed of oxycodone derivatized at the C6 position (6OXY) conjugated to the native keyhole limpet hemocyanin (nKLH) carrier protein. To provide an oxycodone vaccine formulation suitable for human studies, we studied the effect of alternative carriers and adjuvants on the generation of oxycodone-specific serum antibody and B cell responses, and the effect of immunization on oxycodone distribution and oxycodone-induced antinociception in mice and rats. 6OXY conjugated to tetanus toxoid (TT) or a GMP grade KLH dimer (dKLH) was as effective as 6OXY conjugated to the nKLH decamer in mice and rats, while the 6OXY hapten conjugated to a TT-derived peptide was not effective in preventing oxycodone-induced antinociception in mice. Immunization with 6OXY-TT s.c. absorbed on alum adjuvant provided similar protection to 6OXY-TT administered i.p. with Freund's adjuvant in rats. The toll-like receptor 4 (TLR4) agonist monophosphoryl lipid A (MPLA) adjuvant, alone or in combination with alum, offered no advantage over alum alone for generating oxycodone-specific serum antibodies or 6OXY-specific antibody secreting B cells in mice vaccinated with 6OXY-nKLH or 6OXY-TT. The immunogenicity of oxycodone vaccines may be modulated by TLR4 signaling since responses to 6OXY-nKLH in alum were decreased in TLR4-deficient mice. These data suggest that TT, nKLH and dKLH carriers provide consistent 6OXY conjugate vaccine immunogenicity across species, strains and via different routes of administration, while adjuvant formulations may need to be tailored to individual immunogens or patient populations

Evaluation of 6OXY-nKLH in different mouse strains.

<p>The immunogenicity of 6OXY-nKLH in C57Bl/10ScNJ TRL4^-/- mice was reduced versus C57Bl/10ScSnJ wt controls. A) Oxycodone-specific serum IgG antibody titers in mice vaccinated s.c. with 6OXY-nKLH in alum adjuvant; and B) Number of 6OXY-specific antibody secreting B cells in C57Bl/10ScNJ TRL4^-/- and C57Bl/10ScSnJ wt mice immunized s.c. with 6OXY-nKLH in alum. Statistical symbols: * p< 0.05 and **p< 0.01 compared to C57Bl/10ScSnJ wt control mice.</p

Evaluation of 6OXY-TT in rats.

<p>A) oxycodone-specific serum antibody titers in rats immunized with 6OXY-TT using alum s.c. or Freund’s i.p.; and B) their ability in preventing oxycodone-induced antinociception, shown as % decrease compared to TT control group. Statistical symbols: * p< 0.05 compared to TT control.</p

MPLA decreases immunogenicity of 6OXY-nKLH vaccine.

<p>Oxycodone-specific serum antibody titers in BALB/c mice immunized with alum adjuvant alone, MPLA alone or in combination with alum. Statistical symbols: *** p< 0.001 compared to 6OXY-nKLH in alum s.c. as control.</p

The ability of vaccination with different carrier-containing immunogens in preventing oxycodone nociception and its distribution to the brain in BALB/c mice.

<p>A) Oxycodone antinociception in mice immunized with either 6OXY-TT, 6OXY-TT peptide or unconjugated TT. The % maximal possible effect (MPE%) is calculated as (post-drug – baseline)/ (maximal cutoff-baseline) * 100; and B) Oxycodone distribution to the brain in the same mice as in previous panel. In both panels, percent (%) decrease compared to TT control is shown on top of each group. Statistical symbols: * p< 0.05 compared to TT control.</p

Evaluation of GMP grade dKLH in rats.

<p>A) oxycodone-specific serum antibody titers in rats immunized with 6OXY-nKLH or 6OXY-dKLH using alum s.c. or Freund’s i.p.; B) their ability in preventing oxycodone-induced antinociception; C) their effect on oxycodone serum concentration; and D) vaccination effect in preventing brain distribution. Percent (%) decrease compared to nKLH control is shown on top of each group. Statistical symbols: * p< 0.05, **p< 0.01 and *** p< 0.001 compared to KLH control. Brackets to indicate between groups differences.</p

MPLA decreases immunogenicity of 6OXY-TT vaccine.

<p>A) Oxycodone-specific and TT-specific serum IgG antibody titers in mice vaccinated s.c. with 6OXY-TT in alum or MPLA adjuvants; B) Number of 6OXY-specific antibody secreting B cells (ASC) in mice immunized s.c. with 6OXY-TT using alum or MPLA; and C) Relationship between oxycodone-specific IgG titers and 6OXY-specific antibody secreting B cells including all subjects immunized with 6OXY-TT. Statistical symbols: * p< 0.05 and **p< 0.01 between groups.</p

Diazirine-Containing Photoactivatable Isoprenoid: Synthesis and Application in Studies with Isoprenylcysteine Carboxyl Methyltransferase

Photoaffinity labeling is a useful technique employed to identify protein–ligand and protein–protein noncovalent interactions. Photolabeling experiments have been particularly informative for probing membrane-bound proteins where structural information is difficult to obtain. The most widely used classes of photoactive functionalities include aryl azides, diazocarbonyls, diazirines, and benzophenones. Diazirines are intrinsically smaller than benzophenones and generate carbenes upon photolysis that react with a broader range of amino acid side chains compared with the benzophenone-derived diradical; this makes diazirines potentially more general photoaffinity-labeling agents. In this article, we describe the development and application of a new isoprenoid analogue containing a diazirine moiety that was prepared in six steps and incorporated into an <b>a</b>-factor-derived peptide produced via solid-phase synthesis. In addition to the diazirine moiety, fluorescein and biotin groups were also incorporated into the peptide to aid in the detection and enrichment of photo-cross-linked products. This multifuctional diazirine-containing peptide was a substrate for Ste14p, the yeast homologue of the potential anticancer target Icmt, with <i>K</i>_m (6.6 μM) and <i>V</i>_max (947 pmol min^–1 mg^–1) values comparable or better than <b>a</b>-factor peptides functionalized with benzophenone-based isoprenoids. Photo-cross-linking experiments demonstrated that the diazirine probe photo-cross-linked to Ste14p with observably higher efficiency than benzophenone-containing <b>a</b>-factor peptides