The inhibition of EGF-dependent proliferation of keratinocytes by tyrphostin tyrosine kinase blockers.

Protein tyrosine kinase blockers of the tyrphostin family inhibited the EGF-dependent proliferation of human and guinea pig keratinocytes grown in culture and induced their growth arrest. These blockers also significantly inhibited the growth of epidermal keratinocytes, but not of dermal cells, in whole skin organ culture from both guinea pig and human origin. The antiproliferative activity of these tyrphostins correlated quantitatively with their potency as inhibitors of EGF receptor autophosphorylation and the EGF-dependent protein phosphorylation of intracellular target proteins in the keratinocyte. Furthermore, no significant cell cytotoxicity or reduction in serine and threonine phosphorylation of many intracellular polypeptides were observed upon incubation of the cells with tyrphostins like AG213. The complete growth arrest induced by the tyrphostins is fully reversible and upon their removal the keratinocytes resumed their growth with the original growth rate. Because of the nontoxic nature of these compounds and their growth-arresting properties, we suggest their use as agents to treat hyperproliferative conditions of human skin.

Blocking of EGF-dependent cell proliferation by EGF receptor kinase inhibitors.

A systematic series of low molecular weight protein tyrosine kinase inhibitors were synthesized; they had progressively increasing affinity over a 2500-fold range toward the substrate site of epidermal growth factor (EGF) receptor kinase domain. These compounds inhibited EGF receptor kinase activity up to three orders of magnitude more than they inhibited insulin receptor kinase, and they also effectively inhibited the EGF-dependent autophosphorylation of the receptor. The most potent compounds effectively inhibited the EGF-dependent proliferation of A431/clone 15 cells with little or no effect on the EGF-independent proliferation of these cells. The potential use of tyrosine protein kinase inhibitors as antiproliferative agents is demonstrated.

Substance P degrading systems of rat parotid and hypothalamus.

Inactivation of substance P and its C-terminal hexapeptide analog [p-Glu6]substance P6-11 was studied in rat parotid and hypothalamic slices. It was found that in the parotid slice system the decay of substance P induced K+ release occurs concurrently with a decrease in the biologically active concentration of the peptide in the medium. The inactivation was further studied using [p-Glu6]substance P6-11 as substrate in the parotid and in the hypothalamic slice systems. In both tissue preparations the hexapeptide is degraded to small peptide fragments by metalloendopeptidase. Separation of the peptide fragments by high performance liquid chromatography and determination of their amino acid composition showed that in the hypothalamic slice system the major cleavage of the hexapeptide analog occurs between Phe8-Gly9 with minor cleavage sites between Phe7-Phe8 and Gly9-Leu10. In the rat parotid slice system the major cleavage occurs between Gly9-Leu10 with a minor cleavage site between Phe7-Phe8. The degradation of the hexapeptide analog in the hypothalamic system was inhibited 77% and 67% by treatment with 1 mM p-chloromercuriphenylsulfonate and p-chloromercuribenzoate, respectively, whereas in the parotid system these reagents inhibited the degradation of the hexapeptide only by 15% and 8%. These results may indicate that different proteases in the parotid and hypothalamus are involved in degradation of substance P. Kinetic studies, including the use of various inhibitors as well as competition by the peptide hormones somatostatin, LHRH, TRH and Leu-enkephalin-NH2, revealed that in both tissues the hexapeptide analog is a preferred substrate for degradation by protease of considerable specificity towards the C-terminal sequence of substance P. It is suggested that this metalloendopeptidase may be important in the termination of the substance P response.

Miniaturized proteins: the backbone cyclic proteinomimetic approach.

The field of proteinomimetics utilizes peptide-based molecules to mimic native protein functions. We describe a novel general method for mimicking proteins by small cyclic peptides for the purpose of drug design, and demonstrate its applicability on bovine pancreatic trypsin inhibitor (BPTI). These unique cyclic peptides, which both embody discontinuous residues of proteins in their bio-active conformation and ensure an induced fit, may overcome some of the pharmacological drawbacks attributed to proteins and peptides. This method, which we call the backbone cyclic (BC) proteinomimetic approach, combines backbone cyclization of peptides with a suitable selection method, cycloscan. Following this procedure, we have prepared a bicyclic nonapeptide, which mimics the binding region of BPTI. The X-ray crystal structure of the complex trypsin:mimetic, as well as kinetic studies, show that the BPTI mimetic binds to the specificity pocket of trypsin in a similar manner to BPTI. Inhibition measurements of various constructs revealed that backbone cyclization imposed the conformation crucial to binding.

Identification of a nuclear transport inhibitory signal (NTIS) in the basic domain of HIV-1 Vif protein.

The HIV-1 auxiliary protein Vif contains a basic domain within its sequence. This basic region,90RKKR93, is similar to the prototypic nuclear localization signal (NLS). However, Vif is not a nuclear protein and does not function in the nucleus. Here we have studied the karyophilic properties of this basic region. We have synthesized peptides corresponding to this positively charged NLS-like region and observed that these peptides inhibited nuclear transport via the importin pathway in vitro with IC50values in the micromolar range. Inhibition was observed only with peptides derived from the positively charged region, but not from other regions of the Vif protein, showing sequence specificity. On the other hand, the Vif inhibitory peptide Vif88-98 did not confer karyophilic properties when conjugated to BSA. The inactive Vif conjugate and the active SV40-NLS-BSA conjugate both contained a similar number of peptides conjugated to each BSA molecule, as was determined by amino acid analysis of the peptide-BSA conjugates. Thus, the lack of nuclear import of the Vif peptide-BSA conjugate cannot be attributed to insufficient number of conjugated peptide molecules per BSA molecule. Our results suggest that the HIV-1 Vif protein carries an NLS-like sequence that inhibits, but does not mediate, nuclear import via the importin pathway. We have termed such signals as nuclear transport inhibitory signals (NTIS). The possible role of NTIS in controlling nuclear uptake, and specifically during virus infection, is discussed herein. Our results raise the possibility that NLS-like sequences of certain low molecular weight viral proteins may serve as regulators of nucleocytoplasmic trafficking and not neccessarily as mediators of nuclear import.

Peptides derived from HIV-1 Vif: a non-substrate based novel type of HIV-1 protease inhibitors.

The retroviral protease (PR) is absolutely essential for completion of human immunodeficiency virus multiplication cycle, and cannot be replaced by any cellular function. Thus PR, like reverse transcriptase, is an ideal target for the development of anti-AIDS therapy. A large number of human immunodeficiency virus type-1 (HIV-1) PR inhibitors have been developed, and several are currently used as anti-AIDS drugs. These inhibitors are mainly based on the natural PR cleavage sites within the viral Gag and Gag-Pol precursors. The major difficulty encountered while using anti-HIV therapeutic agents in patients has been the rapid emergence of drug-resistant viral strains. Most of the mutations which convert the PR into inhibitor-resistant are located within the substrate binding subsites of the enzyme. Recently, it has been shown that the HIV-1 auxiliary protein Vif, and especially the N-terminal half of Vif (N'-Vif) specifically interacts with the viral PR and inhibits its activity. We now show that efficient inhibition of HIV-1 PR activity can be achieved using Vif-derived peptides. Based on the above model we have performed peptide mapping of N'-Vif in order to find a small peptidic lead compound which inhibits PR activity. The screening revealed that peptides derived from two regions in Vif spanning from residues 30-65 and 78-98 inhibit PR activity in vitro, specifically bind HIV-PR and inhibit HIV-1 production in vivo. Further mapping of these regions revealed the lead compounds Vif81-88 and Vif88-98. These peptides specifically inhibit and bind HIV-1 PR, but do not affect pepsin and rous sarcoma virus protease. In contrast to other known PR inhibitors, these peptides are not substrate-based and their sequences do not resemble the sequences of the natural PR substrates (cleavage sites). Moreover, the Vif-derived peptides themselves are not cleaved by HIV-1 PR. Conversion of the lead peptides into small backbone cyclic peptidomimetics is taking place nowadays in order to turn these lead compounds into metabolically stable selective novel type of HIV-PR non-substrate-based inhibitors.

Novel long-acting somatostatin analog with endocrine selectivity: potent suppression of growth hormone but not of insulin.

Somatostatin, also known as somatotropin release-inhibiting factor (SRIF), is a natural cyclic peptide inhibitor of pituitary, pancreatic, and gastrointestinal secretion. Its long-acting analogs are in clinical use for treatment of various endocrine syndromes and gastrointestinal anomalies. These analogs are more potent inhibitors of the endocrine release of GH, glucagon, and insulin than the native SRIF; hence, they do not display considerable physiological selectivity. Our goal was to design effective and physiologically selective SRIF analogs with potential therapeutic value. We employed an integrated approach consisting of screening of backbone cyclic peptide libraries constructed on the basis of molecular modeling of known SRIF agonists and of high throughput receptor binding assays with each of the five cloned human SRIF receptors (hsst1-5). By using this approach, we identified a novel, high affinity, enzymatically stable, and long-acting SRIF analog, PTR-3173, which binds with nanomolar affinity to human SRIF receptors hsst2, hsst4, and hsst5. The hsst5 and the rat sst5 (rsst5) forms have the same nanomolar affinity for this analog. In the human carcinoid-derived cell line BON-1, PTR-3173 inhibits forskolin-stimulated cAMP accumulation as efficiently as the drug octreotide, indicating its agonistic effect in this human cell system. In hormone secretion studies with rats, we found that PTR-3173 is 1000-fold and more than 10,000-fold more potent in inhibiting GH release than glucagon and insulin release, respectively. These results suggest that PTR-3173 is the first highly selective somatostatinergic analog for the in vivo inhibition of GH secretion, with minimal or no effect on glucagon and insulin release, respectively.

Tyrphostins inhibit the epidermal growth factor receptor-mediated breakdown of phosphoinositides.

In response to epidermal growth factor (EGF) and the Ca2+ ionophore A23187, the total phosphatidylinositides (IPT) increased in A431 human epidermoid carcinoma cells 1.8- and 2.0-fold and in the EGF-dependent A431/Clone 15-2 cells 3.0- and 8.0-fold, respectively, over basal levels. Both responses were inhibited by the antiproliferative agents tyrphostins, but the EGF-induced increase in IPT was inhibited to a much greater extent than that induced by the ionophore. Tyrphostins which are potent EGF-receptor kinase inhibitors were also potent in blocking the EGF-induced production of phosphoinositides. The less potent tyrphostins were found to inhibit the EGF-dependent IPT formation more weakly. These results support the notion that phospholipase C is activated through its phosphorylation by the EGF receptor.

Tyrphostin-induced differentiation of mouse erythroleukemia cells.

Inhibitors of protein-tyrosine kinases (TPKs) from the tyrphostins family induce terminal erythroid differentiation of mouse erythroleukemia (MEL) cells. The most potent tyrphostin was found to be AG-568 which was therefore investigated in more detail. Just prior to differentiation the inhibition of tyrosine phosphorylation of a pp97 protein band was noted. We also found that AG-568 treatment induces the appearance of a putative differentiation factor which could induce tyrphostin-independent differentiation in untreated cells. Our study suggests that the inhibition of tyrosine phosphorylation by AG-568 leads to the production of differentiating factor(s) which induce the MEL cells to differentiate.

A peptide derived from the N-terminal region of HIV-1 Vpr promotes nuclear import in permeabilized cells: elucidation of the NLS region of the Vpr.

Viral protein r (Vpr), a HIV-1 auxiliary protein which mediates nuclear import of the viral preintegration complex (PIC), contains two regions, N- and C-terminal, which have been proposed to function as a nuclear localization signal (NLS). We have synthesized peptides corresponding to both regions (designated as VprN and VprC), conjugated them to bovine serum albumin (BSA), and tested their ability to mediate nuclear import in permeabilized cells. Only VprN, and not VprC, functioned as an active NLS and promoted translocation of the conjugate into nuclei. Nuclear import of the conjugate was found to be energy and temperature dependent and was inhibited by wheat germ agglutinin (WGA). However, it did not require the addition of cytosolic factors and was not inhibited by the prototypic SV40 large T-antigen NLS peptide. Our results show that Vpr harbours a non-conventional negatively charged NLS and therefore suggest that Vpr may use a distinct nuclear import pathway.

Translocation of NLS-BSA conjugates into nuclei of permeabilized mammalian cells can be supported by protoplast extract. An experimental system for studying plant cytosolic factors involved in nuclear import.

An heterologous experimental system, which allows the study of the yet unknown cytosolic factors involved in nuclear import of nuclear localization signal (NLS)-containing proteins in plants, has been established. The ability of plant cell extract to substitute mammalian cytosol and to promote translocation of NLS-containing proteins into nuclei of permeabilized HeLa cells was demonstrated. The results described in the present work show that nuclear import of fluorescently labeled BSA conjugates bearing the NLS sequence of SV40 large T antigen could be supported by petunia cell cytoplasmic extract. This heterologous system shows the characteristic features of the homologous mammalian system, namely, is ATP dependent and is inhibited by WGA, GTPgammaS as well as by non-fluorescent NLS-BSA conjugates. The system described here offers an experimental method to study and characterise cytosolic factors which are required for nuclear import in plants.

Human immunodeficiency virus type 1 Vif-derived peptides inhibit the viral protease and arrest virus production.

Human immunodeficiency virus type 1 (HIV-1) Vif protein is required for productive HIV-1 infection of peripheral blood lymphocytes and macrophages in cell culture and for pathogenesis in the SCID-hu mouse model of HIV-1 infection. Vif inhibits the viral protease (PR)-dependent autoprocessing of truncated HIV-1 Gag-Pol precursors expressed in bacterial cells and efficiently inhibits the PR-mediated hydrolysis of peptides in cell-free systems. The obstructive activity of Vif has been assigned to the 92 amino acids residing at its N'-terminus (N-Vif). To determine the minimal Vif sequence required to inhibit PR, we synthesized overlapping peptides derived from N-Vif. These peptides were then assessed, using two in vitro and two in vivo systems: (i) inhibition of purified PR, (ii) binding of PR, (iii) inhibition of the autoprocessing of the Gag-Pol polyprotein expressed by a vaccinia virus vector, and (iv) inhibition of mature virus production in human cells. The peptides derived from two regions of N-Vif encompassing residues Tyr-30-Val-65 and Asp-78-Val-98, inhibited PR activity in both the in vitro and the in vivo assays. Thus, these peptides can be used as lead compounds to design new PR inhibitors.

Neurochemical mediators of the behavioural effects of receptor-selective substance P agonists administered intrathecally in the rat.

Recently, two compounds have been developed, designated septide and senktide, which are highly selective agonists for the substance P receptor, types NK-1 and NK-3, respectively. Each of these, when injected intrathecally in awake rats, produced a distinct and non-overlapping constellation of sensory and behavioural effects which were subsets of the symptoms evoked by intrathecal administration of substance P. Prior systemic administration of 5-hydroxytryptamine (5-HT), alpha-adrenergic and opiate receptor antagonists, at doses sufficient to block the behavioural effects of the corresponding receptor agonists, did not alter responses to intrathecally injected septide or senktide. This was so, even for symptoms which suggested inhibitory mediation, hypoalgesia and (transient) motor flaccidity. Septide and senktide, administered by lumbar puncture and by indwelling catheter, produced identical results. Finally, in contrast to some other peptides, flaccid paralysis induced by senktide was not accompanied by spinal necrosis.

Tyrphostins I: synthesis and biological activity of protein tyrosine kinase inhibitors.

A novel class of low molecular weight protein tyrosine kinase inhibitors is described. These compounds constitute a systematic series of molecules with a progressive increase in affinity toward the substrate site of the EGF receptor kinase domain. These competitive inhibitors also effectively block the EGF-dependent autophosphorylation of the receptor. The potent EGF receptor kinase blockers examined were found to competitively inhibit the homologous insulin receptor kinase at 10(2)-10(3) higher inhibitor concentrations in spite of the significant homology between these protein tyrosine kinases. These results demonstrate the ability to synthesize selective tyrosine kinase inhibitors. The most potent EGF receptor kinase inhibitors also inhibit the EGF-dependent proliferation of A431/clone 15 cells with little or no effect on EGF independent cell growth. These results demonstrate the potential use of protein tyrosine kinase inhibitors as selective antiproliferative agents for proliferative diseases caused by the hyperactivity of protein tyrosine kinases. We have suggested the name "tyrphostins" for this class of antiproliferative compounds which act as protein tyrosine kinase blockers.

[pGlu6,Pro9]SP6-11, a selective agonist for the substance P P-receptor subtype.

Substitution of a single amino acid residue, proline for glycine-9 in [pGlu6]SP6-11, a hexapeptide analogue of substance P, confers on the peptide selective agonist activity toward the SP-P receptor subtype. [pGlu6,Pro9]SP6-11 had 20% and 75% of the activity of [pGlu6]SP6-11 in stimulating, respectively, K+ release from rat parotid slices and contraction of the isolated guinea pig ileum, via the SP-P receptor subtype. In contrast, [pGlu6,Pro9]SP6-11 had substantially reduced activity on SP-E systems such as the hamster urinary bladder and rat duodenum, being about 20-fold less potent than [pGlu6]SP6-11 and 200-670-fold less potent than neurokinin B. In the guinea pig ileum [pGlu6,Pro9]SP6-11 had very low activity on the neuronal tachykinin receptor, being 325 times less potent than [pGlu6]SP6-11 and 1000 times less potent than neurokinin B. Because of its discrimination between the muscular and neuronal receptors in the guinea pig ileum (muscular/neuronal potency ratio = 600), [pGlu6,Pro9]SP6-11 can be used to specifically desensitize the muscular receptor of this tissue. This procedure enables a selective and sensitive bioassay of the neuronal receptor.

Tyrphostins. 6. Dimeric benzylidenemalononitrile tyrophostins: potent inhibitors of EGF receptor tyrosine kinase in vitro.

Benzylidenemalononitrile (BMN) tyrphostins were previously found to be potent inhibitors of EGF receptor (EGFR) tyrosine kinase activity. Since these compounds were found to compete for the substrate and sometimes with the ATP site and since EGFR acts as a dimer, we prepared a series of dimeric tyrphostins. These dimeric tyrphostins were built from two BMN units linked by various spacers and designed to fit the dimeric cross-autophosphorylation signal transduction intermediate of the EGFR tyrosine kinases. Structure-activity relationship of these potent dimeric EGF receptor tyrosine kinase inhibitors is reported.

Ketomethylene pseudopeptide analogues of substance P: synthesis and biological activity.

Two pseudopeptide analogues [Bz-(RS)Phe8 psi (COCH2)Gly9]SP8-11 (I) and [pGlu6,(RS)Phe8 psi (COCH2)Gly9]SP6-11 (II) of the substance P related C-terminal hexapeptide [pGlu6]SP6-11 were prepared as follows. The pseudodipeptidic unit H(RS)Phe psi (COCH2)GlyOH was synthesized by using a modified Dakin-West reaction between Bz-Phe-OH and monomethyl succinoyl chloride. The N alpha-protected pseudopeptidic unit was then incorporated into the appropriate peptide by using various coupling methods. The two pseudopeptide analogues were purified, characterized, and tested for their biological activity and inhibitory effect on SP degrading enzymes. Analogue II was a full agonist contracting the isolated guinea pig ileum with a potency of 70% compared to the parent hexapeptide [pGlu6]SP6-11. It was also a potent inhibitor of SP degrading activity in rat diencephalon membranes with a Ki of 20 microM whereas analogue I was a weak inhibitor.

Dehydro keto methylene and keto methylene analogues of substance P. Synthesis and biological activity.

The synthesis of dehydro keto methylene and keto methylene analogues of substance P using classical peptide synthesis is described. The following analogues were prepared: [pGlu6,Gly9psi(COCH2)(RS)Leu10]SP6-11 (4) and [pGlu6,-(RS)Phe7 psi(COCH2)(RS)Phe8]SP6-11 (8). The use of an improved deprotection scheme employing Meerwein's reagent (Et3OBF4) made possible the syntheses of the novel dehydro keto methylene analogue [pGlu6,(RS)Phe7 psi-(COCH2)delta(E)Phe8]SP6-11 (26) adn the tetrapeptide analogue [pGlu6,(RS)Phe8 psi(COCH2)Gly9]SP6-9(-OMe) (23). Compound 4 was a weak agonist in provoking contractions of the guinea pig ileum. Compound 26 was a potent inhibitor of SP degradation in rat hypothalamus preparations, with an IC50 value of 1.8 microM.

Synthesis of partially modified retro-inverso substance P analogues and their biological activity.

Partial retro-inverso modification of a single peptide bond was applied to pGlu-Phe-Phe-Gly-Leu-Met-NH2 (I), a C-terminal hexapeptide analogue of the neuropeptide substance P. Two analogues with reversed peptide bonds, between the pGlu-Phe and Phe-Gly residues, were prepared, purified and characterized. The analogue gpGlu-(RS)-mPhe-Phe-Gly-Leu-Met-NH2 (II) was devoid of either agonistic or antagonistic activity. The second pseudopeptide analogue, i.e., pGlu-Phe-gPhe-mGly-Leu-Met-NH2 (III), was found to be a full agonist with 22% of the potency of I in the guinea pig ileum assay.

Pseudopeptide analogues of substance P and leucine enkephalinamide containing the psi (CH2O) modification: synthesis and biological activity.

The isosteric methyleneoxy psi (CH2O) function was employed as a novel peptide-bond surrogate and incorporated into sequences of two neuropeptides, substance P (SP) and enkephalin. A pseudopeptide analogue [pGlu6,Phe8 psi(CH2O)Gly9]SP6-11 (7) of SP related C-terminal hexapeptide [pGlu6]SP6-11 and two pseudopeptide analogues of [Leu5]enkephalinamide, [Tyr1 psi (CH2O)Gly2, Leu5] enkephalinamide (11) and [Gly2 psi (CH2O)-Gly3, Leu5]enkephalinamide (17), were synthesized. The N alpha-protected pseudodipeptidic units were incorporated in the appropriate peptide sequences by using conventional coupling methods in solution. Compound 7 was a potent agonist (EC50 = 4.8 nM) of substance P as compared to the parent peptide [pGlu6]SP6-11 (EC50 = 1.2 nM), in stimulating contraction of the isolated guinea pig ileum (GPI). Analogue 7 was more potent on the neuronal (NK-3) than on the muscular (NK-1) tachykinin receptors in the GPI as shown by the ratio of activities, EC50 (NK-1)/EC50 (NK-3) = 3.16, thus displaying an improved selectivity for the NK-3 tachykinin receptor subtype as compared to that of [pGlu6]SP6-11, EC50 (NK-1)/EC50 (NK-3) = 0.44. In the rat vas deferens (RVD) assay, a typical NK-2 system, the pseudopeptide analogue 7 was (EC50 = 2 microM) 10-fold more potent than the parent peptide and 20-fold less potent than eledoisin, an NK-2 selective tachykinin. The pseudopeptide enkephalin analogue 17 had low biological activity when tested in the electrically induced GPI (EC50 = 2.3 microM) and was inactive in the mouse vas deferens (MVD) assay. In the rat brain membrane (RBM) binding assay analogue 17 had low affinity (in the micromolar range) for both the mu and delta binding sites. In contrast, analogue 11 was a potent enkephalin agonist (EC50 = 30 nM), being equipotent to [D-Ala2, Leu5]enkephalinamide (DALE) in the GPI assay. In the MVD, analogue 11 showed a substantially reduced activity (EC50 = 92 nM), being about 10-fold less potent than DALE. In the RBM binding assay analogue 11 showed high affinity (in the nanomolar range) for both mu and delta binding sites with increased selectivity for the delta sites as shown by the ratio of the apparent affinities for both receptors, Ki (delta)/Ki (mu) = 2.1. The contribution of the modified peptide bonds in the mode of interaction of SP and enkephalin at their corresponding receptors is discussed.