Modulation of the binding affinity of myelopoietins for the interleukin-3 receptor by the granulocyte colony-stimulating factor receptor agonist.

Myelopoietins (MPOs) are a family of recombinant chimeric proteins that are both interleukin-3 (IL-3) receptor and granulocyte colony-stimulating factor (G-CSF) receptor agonists. In this study, MPO molecules containing one of three different IL-3 receptor agonists linked with a common G-CSF receptor agonist have been examined for their IL-3 receptor binding characteristics. Binding to the alpha-subunit of the IL-3 receptor revealed that the affinity of the MPO molecules was 1.7-3.4-fold less potent than those of their individual cognate IL-3 receptor agonists. The affinity decrease was reflected in the MPO chimeras having approximately 2-fold slower dissociation rates and 2.7-5.5-fold slower association rates than the corresponding specific IL-3 receptor agonists alone. The affinity of binding of the MPO molecules to the heteromultimeric alphabeta IL-3 receptor expressed on TF-1 cells was either 3-, 10-, or 42-fold less potent than that of the individual cognate IL-3 receptor agonist. Biophysical data from nuclear magnetic resonance, near-UV circular dichroism, dynamic light scattering, analytical ultracentrifugation, and size exclusion chromatography experiments determined that there were significant tertiary structural differences between the MPO molecules. These structural differences suggested that the IL-3 and G-CSF receptor agonist domains within the MPO chimera may perturb one another to varying degrees. Thus, the differential modulation of affinity observed in IL-3 receptor binding may be a direct result of the magnitude of these interdomain interactions.

Bivalent binding and signaling characteristics of Leridistim, a novel chimeric dual agonist of interleukin-3 and granulocyte colony-stimulating factor receptors.

Leridistim is a member of a novel family of engineered chimeric cytokines, myelopoietins, that contain agonists of both interleukin-3 (IL-3) receptors (IL-3R) and granulocyte colony-stimulating factor (G-CSF) receptors (G-CSFR). To more clearly understand Leridistim's function at the molecular level, binding to both IL-3R and G-CSFR and subsequent signaling characteristics have been delineated. The affinity of Leridistim for the human G-CSFR was found to be comparable to that of native G-CSF (IC(50) = 0.96 nM and 1.0 nM, respectively). Both Leridistim and G-CSF induced receptor tyrosine phosphorylation to a similar maximal level. Compared with native recombinant human IL-3 (rhIL-3), Leridistim was found to possess higher affinity for the IL-3R alpha chain (IL-3Ralpha) (IC(50) = 85 nM and 162 nM, respectively). However, the increase in Leridistim binding affinity to the functional, high-affinity heterodimeric IL-3Ralphabeta(c) receptor is lower than that observed with rhIL-3 (85 nM and 14 nM vs 162 nM and 3.5 nM, respectively). Leridistim induced tyrosine phosphorylation of beta(c) to a level comparable to native IL-3, and the level of JAK2 tyrosine phosphorylation in cells expressing both IL-3R and G-CSFR was comparable to that observed with IL-3 or G-CSF alone. The ability of Leridistim to interact with IL-3R and G-CSFR simultaneously was demonstrated using surface plasmon resonance analysis. These studies were extended to demonstrate that Leridistim exhibited a higher affinity for the IL-3R on cells that express both the IL-3Ralphabeta(c) and the G-CSFR (IC(50) = 2 nM) compared with cells that contain the IL-3Ralphabeta(c) alone (IC(50) = 14 nM). Leridistim binds to both IL-3R and G-CSFR simultaneously and has been shown to activate both receptors. The bivalent avidity may explain the unique biologic effects and unexpected potency of Leridistim in hematopoietic cells compared with rhIL-3 or G-CSF alone or in combination.

Circular permutation of the granulocyte colony-stimulating factor receptor agonist domain of myelopoietin.

Myelopoietins (MPOs) are a family of engineered dual interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF) receptor agonists that are superior in comparison to the single agonists in their ability to promote the growth and maturation of hematopoietic cells of the myeloid lineage. A series of MPO molecules were created which incorporated circularly permuted G-CSF (cpG-CSF) sequences with an IL-3 receptor (IL-3R) agonist moiety attached at locations that correspond to the loops that connect the helices of the G-CSF four-helix bundle structure. The cpG-CSF linkage sites (using the original sequence numbering) were residue 39, which is at the beginning of the first loop connecting helices 1 and 2; residue 97, which is in the turn connecting helices 2 and 3; and residues 126, 133, and 142, which are at the beginning, middle, and end, respectively, of the loop connecting helices 3 and 4. The N- and C-terminal helices of each cpG-CSF domain were constrained, either by direct linkage of the termini (L0) or by replacement of the amino-terminal 10-residue segment with a seven-residue linker composed of SGGSGGS (L1). All of the MPO molecules stimulated the proliferation of both IL-3-dependent (EC50 = 13-95 pM) and G-CSF-dependent (EC50 = 35-710 pM) cell lines. MPOs with the IL-3R agonist domain linked to cpG-CSFs in the first (residue 39) or second (residue 133) long overhand loops were found by CD spectroscopy to have helical contents similar to that expected for a protein comprised of two linked four-helix bundles. The MPOs retained the ability to bind to the IL-3R with affinities similar to that of the parental MPO. Using both a cell surface competitive binding assay and surface plasmon resonance detection of binding kinetics, the MPOs were found to bind to the G-CSF receptor with low nanomolar affinities, similar to that of G-CSF(S17). In a study of isolated cpG-CSF domains [Feng, Y., et al. (1999) Biochemistry 38, 4553-4563], domains with the L1 linker had lower G-CSF receptor-mediated proliferative activities and conformational stabilities than those which had the L0 linker. A similar trend was found for the MPOs in which the G-CSFR agonist activity is mostly a property of the cpG-CSF domain. Important exceptions were found in which the linkage to the IL-3R agonist domain either restored (e.g., attachment at residue 142) or further decreased (linkage at residue 39) the G-CSFR-mediated proliferative activity. MPO in which the IL-3R agonist domain is attached to the cpG-CSF(L1)[133/132] domain was shown to be more potent than the coaddition of the IL-3R agonist and G-CSF in stimulating the production of CFU-GM colonies in a human bone marrow-derived CD34+ colony-forming unit assay. Several MPOs also had decreased proinflammatory activity in a leukotriene C4 release assay using N-formyl-Met-Leu-Phe-primed human monocytes. It was found that circular permutation of the G-CSF domain can alter the ratio of G-CSFR:IL-3R agonist activities, demonstrating that it is a useful tool in engineering chimeric proteins with therapeutic potential.

Use of combinatorial mutagenesis to select for multiply substituted human interleukin-3 variants with improved pharmacologic properties.

A combinatorial mutagenesis strategy was used to create a collection of nearly 500 variants of human interleukin 3 (IL-3), each with four to nine amino acid substitutions clustered within four linear, nonoverlapping regions of the polypeptide. The variants were secreted into the periplasm of Escherichia coli and supernatants were assayed for IL-3 receptor-dependent cell proliferation activity. Sixteen percent of the variants, containing "region-restricted" substitutions, retained substantial proliferative activity through two rounds of screening. A subset of these was combined to yield variants with substitutions distributed through approximately half of the polypeptide. With one exception, "half-substituted" variants exhibited proliferative activity within 3.5-fold of native IL-3. A subset of the "half-substituted" variants was combined to yield "fully substituted" IL-3 variants having 27 or more substitutions. The combination of the substitutions resulted in a set of polypeptides, some of which exhibit increased proliferative activity relative to native IL-3. The elevated hematopoietic potency was confirmed in a methylcellulose colony-forming unit assay using freshly isolated human bone marrow cells. A subset of the multiply substituted proteins exhibited only a modest increase in inflammatory mediator (leukotriene C4) release. The molecules also exhibited 40- to 100-fold greater affinity for the alpha subunit of the IL-3 receptor and demonstrated a 10-fold faster association rate with the alpha-receptor subunit. The multiply substituted IL-3 variants described in this study provide a unique collection of molecules from which candidates for clinical evaluation may be defined and selected.

Saturation mutagenesis of human interleukin-3.

A deletion variant of human interleukin-3, hIL-3(15-125), was produced in the periplasmic space of Escherichia coli and had full activity in an AML193.1.3 cell proliferation assay. Libraries of random single-amino acid substitutions were constructed at each of 105 positions in the gene for hIL-3(15-125). Approximately eight single-site substitutions at each position were produced in osmotic shock fractions and screened for activity. 15 mutants were found with bioactivity of 5-26-fold greater than that of native hIL-3. The majority of amino acids in hIL-3(15-125) could be substituted without substantial loss of activity. Substitution of residues predicted to be in the hydrophobic core of the protein often resulted in reduced activity and/or low accumulation levels. Only five residues predicted to be on the surface of the protein were intolerant of substitution and hence are candidates for sites of interaction with the receptor. We therefore propose that the majority of residues in hIL-3 serve a structural role and permit the display of a few key residues in the correct configuration for recognition by the receptor.

Two novel heat shock genes encoding proteins produced in response to heterologous protein expression in Escherichia coli.

In Escherichia coli high-level production of some heterologous proteins (specifically, human prorenin, renin, and bovine insulin-like growth factor 2) resulted in the induction of two new E. coli heat shock proteins, both of which have molecular masses of 16 kDa and are tightly associated with inclusion bodies formed during heterologous protein production. We named these inclusion body-associated proteins IbpA and IbpB. The coding sequences for IbpA and IbpB were identified and isolated from the Kohara E. coli gene bank. The genes for these proteins (ibpA and ibpB) are located at 82.5 min on the chromosome. Nucleotide sequencing of the two genes revealed that they are transcribed in the same direction and are separated by 110 bp. Putative Shine-Dalgarno sequences are located upstream from the initiation codons of both genes. A putative heat shock promoter is located upstream from ibpA, and a putative transcription terminator is located downstream from ibpB. A temperature upshift experiment in which we used a wild-type E. coli strain and an isogenic rpoH mutant strain indicated that a sigma 32-containing RNA polymerase is involved in the regulation of expression of these genes. There is 57.5% identity between the genes at the nucleotide level and 52.2% identity at the amino acid level. A search of the protein data bases showed that both of these 16-kDa proteins exhibit low levels of homology to low-molecular-weight heat shock proteins from eukaryotic species.

Effects of signal peptide changes on the secretion of bovine somatotropin (bST) from Escherichia coli.

Bovine somatotropin (bST) was secreted from Escherichia coli at moderate levels of 1-2 micrograms/ml/OD using expression vectors in which the bST gene was fused to the lamB secretion signal. To study the secretion properties of bST in E.coli further, two approaches for modifying the secretion signal were employed. In the first case, fusion proteins were constructed with six alternative bacterial secretion signals: three from E.coli proteins (HisJ, MalE and OmpA), two from bacteriophage proteins (M13 coat protein and PA-2 Lc) and one from the chitinase A protein of Serratia marcescens. The results, as monitored by Western blot analysis of both total cell protein and the periplasmic fraction, showed that these changes in the secretion signal did not significantly affect the secretion properties of bST. In the second approach, a library of random mutations was created in the lamB secretion signal and 200 independent clones were screened. The level of secreted bST was determined by growing individual clones in duplicate in microtiter wells, inducing protein expression and measuring the bST released by osmotic shock using a particle concentration fluorescent immunoassay. The secretion properties of several novel variants in the LamB signal peptide are presented.

Complete nucleotide sequence of a gene conferring polymyxin B resistance on yeast: similarity of the predicted polypeptide to protein kinases.

Polymyxin B is an antibiotic that kills sensitive cells by disrupting their membranes. We have cloned a wild-type yeast gene that, when present on a high-copy-number plasmid, renders the cells resistant to the drug. The nucleotide sequence of this gene is presented. A single open reading frame within the sequence has the potential to encode a polypeptide (molecular mass of 77.5 kDa) that shows strong homologies to polypeptides of the protein kinase family. The gene, PBS2, located on chromosome X, is not allelic to the previously described PBS1 gene (where PBS signifies polymyxin B sensitivity). Although pbs1 mutations confer resistance to high levels of polymyxin B, double mutants, pbs1 pbs2, are not resistant to the drug, indicating that PBS2 is essential for pbs1 activity. Models based on the proposed protein kinase activity of the PBS2 gene product are presented to explain the interaction between PBS1 and PBS2 gene products involved in conferring polymyxin B resistance on yeast cells.

The mRNA for a proteinase inhibitor related to the HI-30 domain of inter-alpha-trypsin inhibitor also encodes alpha-1-microglobulin (protein HC).

Inter-alpha-trypsin inhibitor (ITI) is a 180 kd serine proteinase inhibitor found in human serum. Treatment of 180 kd ITI with trypsin releases a 30 kd fragment (HI-30) which contains the anti-proteolytic activity of the high molecular weight form. We have isolated a cDNA clone from a human liver library which codes for HI-30, and have determined its DNA sequence. The mRNA not only codes for HI-30 but also another serum protein, alpha-1-microglobulin, which has not been previously associated with ITI or HI-30. The alpha-1-microglobulin sequence is found in the amino-terminus of the protein and is preceded by a signal sequence. HI-30 is found at the carboxy-terminus. The two protein sequences are separated by two arginine residues.

Analgesic narcotic antagonists. 15. Potent narcotic agonist 7 beta-(arylalkyl)-4,5 alpha-epoxymorphinans.

Structure-activity correlations in 7 beta-(arylalkyl)-3-methoxy- or hydroxy-4,5 alpha-epoxymorphinans have been investigated. 6 beta-Hydroxy-7 alpha-hydroxymethyl compounds 7 with 7 beta-substituents CH2CH2R [a, R = H; b, R = CH2CH3; c, R = C6H5; d, R = CH2C6H5; f, R = CH2CH2C6H5; g, R = (CH2)3C6H5; h, R = (CH2)4C6H5] were prepared. Wittig condensations with previously reported 4,5 alpha-epoxy-7 beta-formyl-7 alpha-(hydroxymethyl)-6 beta, 7 alpha-O-isopropylidene-3-methoxy-17-methylmorphinan-6 beta-ol (3), followed by dilute acid removal of the blocking group and then hydrogenation, gave saturated compounds 7. Compounds with a 6 alpha, 7 alpha-oxymethylene ring. 18c,d,f,g, were prepared from 7 beta-formyl derivative 16 and the appropriate Wittig reagent, followed by hydrogenation. Both the 6 beta-hydroxy-7 alpha-hydroxymethyl and 6 alpha, 7 alpha-oxymethylene series containing 7 beta-arylalkyl groups with an alkyl chain length of 2 to 4 ar potent narcotic agonists. The most potent 17-methyl compound, 4,5 alpha-epoxy-7 alpha-(hydroxymethyl)-17-methyl-17 beta-(4-phenylbutyl)morphinan-3,6 beta-diol (8f) was 700 times more potent than morphine in the acetic acid induced mouse writhing assay. 17-Methyl compounds in the c, d, f, g series were converted to 17-cyclopropylmethyl (P series) or 17-cyclobutylmethyl (B series) derivatives. Narcotic antagonistic activity could not be demonstrated for these potent agonist 17-cycloalkylmethyl derivatives. These pharmacological results parallel those previously reported for tertiary alcohol derivatives of the endo-ethenotetrahydrooripavines. Structureal considerations confirm the existence of a lipophilic site extending upward and outward from where the C ring of morphine and congeners bind to opiate receptors.

Analgesic narcotic antagonists. 8. 7 alpha-Alkyl-4,5 alpha-epoxymorphinan-6-ones.

The preparation of a series of 7 alpha-alkylated dihydrocodeinones is described. N-(Cyclopropylmethyl) (P series) or N-(cyclobutylmethyl) (B series) 7 alpha-methyl (a series) or 7 alpha, 8 beta-dimethyl (b series) substituted dihydronorcodeinones (7) were prepared from the appropriately substituted N-(cycloalkylmethyl)-4-hydroxymorphinan-6-ones (5) by dibromination, 4,5-epoxy ring closure, and catalytic debromination. Treatment of 7 with BBr3 gave low yields of the corresponding 3-phenols 8. Alternatively, reaction of dihydrocodeinone (10) with dimethylformamide dimethyl acetal gave the 7-[(dimethylamino)methylene] adduct 11, which was hydrogenated to 7 alpha-methyl- (12) or 7 alpha-(hydroxymethyl)dihydrocodeinone (13). Treatment of 11 with lithium reagents, followed by hydrogenation, gave a mixture of 7 alpha-alkyl (15c-f) compounds and the corresponding 4,5-epoxy-cleaved products 16. Reaction of 11 with alpha-ethoxyvinyllithium gave intermediate 17, which on hydrolysis and hydrogenation yielded the 6,7-furyl (18) or pyrroyl (19) derivative. N-(Cycloalkylmethyl)-14-hydroxydihydronorcodeinones 23P,B reacted with dimethylformamide dimethyl acetal to give 25P,B, which were hydrogenated to the 7 alpha-methyl compounds 26P,B and O-demethylated to give 27P,B. The 7 alpha-methyl-N-methyl compounds were about equipotent with dihydrocodeinone. Derivatives with larger alkyl groups were less potent. Corresponding N-(cycloalkylmethyl) compounds did not show strong mixed agonist-narcotic antagonist activity.

Analgesic narcotic antagonists. 9. 6-Methylene-8 beta-alkyl-N-(cycloalkylmethyl)-3-hydroxy- or -methoxymorphinans.

Series of N-(cyclopropylmethyl) (P series) or N-(cyclobutylmethyl) (B series) 3-methoxy (1) or 3-hydroxy (2) morphinan-6-ones with hydrogen (a), methyl (b), or ethyl (c) groups in the 8 beta position were converted to the 6-methylene compounds 3 or 4 by reaction with Ph3P = CH2. One member of this new series, N-(cyclobutylmethyl)-8 beta-methyl-6-methylenemorphinan-3-ol (4Bb), had potent mixed agonist-narcotic antagonist properties and, in contrast to the previously studied 6-oxo compound 2Bb, did not substitute for morphine in dependent rats or monkeys.

Analgesic narcotic antagonists. 2. 8-Alkymorphinan-6-ones.

A series of 8-alkyl-3-methoxy-17-methylmorphinan-6-ones (3C) and -isomorphinan-6-ones (3T) were prepared by conjugate addition of lithium dialkylcuprates to the corresponding 7,8-didehydro-6-ones 2C and 2T. These 17-methyl compounds were potent analgesics and were converted to mixed narcotic agonists-antagonists 7-10, by replacement of the 17-methyl groups with cycloalkylmethyl moieties. The 8 substituent modified the type of activity observed. One of these compounds, 17-(cyclobutylmethyl)-3-hydroxy-8 beta-methylmorphinan-6-one (10Ca), had an agonist-antagonist ratio of 0.1. Compound 10Ca did not support or cause dependence in rats. This compound, however, appeared to be a typical narcotic agent in morphine-dependent monkeys.

Analgesic narcotic antagonists. 1. 8 beta-Alkyl-, 8 beta-acyl-, and 8 beta-(tertiary alcohol)dihydrocodeinones and -dihydromorphinones.

Conjugate addition of lithium dialkyl cuprates to codeinone (3) gave as the major product a series of 8 beta-alkyldihydrocodeinones 4a-m. A low yield of the 8 alpha-isomer 6 was isolated in several cases. 8 beta-Acyldihydrocodeinones 10 were prepared by the addition of acyl carbanion equivalents (protected cyanohydrin method or lithium bis(alpha-ethoxyvinyl)cuprate) to 3 followed by hydrolysis. 8 beta-Acetyldihydrocodeine (12) was reacted with MeLi or n-BuLi to give tertiary alcohols 13, which were oxidized to target dihydrocodeinones 14. The 8 beta-substituted compounds with unsaturated (4c,f,m), branched (4d,g,i-k), or large straight-chain (4h,l) alkyl groups, as well as the acyl (10a-d) and tertiary alcohol (14a,b) derivatives, were less active than dihydrocodeinone (4n) in the mouse writhing and rat tail-flick analgesic assays. The analgesically active 8 beta-methyl (4a) and 8 beta-ethyl (4b) compounds were converted to N-(cyclopropylmethyl)- and N-(cyclobutylmethyl)dihydronorcodeinones (17 and 18) and -dihydronormorphinones (19 and 20). Some of these compounds had mixed agonist-antagonist profiles of action. One of these compounds, N-(cyclopropylmethyl)-8 beta-ethyldihydronorcodeinone (17b), has been selected for further study in man.