Mark D. Prairie

Mechanism of action of didemnin B, a depsipeptide from the sea

With the brush border membrane vesicles prepared from the rat kidney cortex, didemnin B and its parent compound, didemnin A function neither as a K+-specific ionophore nor as an ionophore for Na+ ions while other depsipeptide antibiotics such as valinomycin and gramicidin promote transmembrane movement of K+ and Na+ ions, respectively. Didemnin B inhibits protein synthesis and DNA synthesis much more than RNA synthesis and is in general more potent than didemnin A. Time course studies reveal that the action of didemnin B is rapid and cannot be reversed after 2 h in contact with the cells. The inhibition of protein synthesis is almost superimposable to that of L1210 cells growth. DNA synthesis is also markedly inhibited. These results collectively suggest that didemnin B acts differently, at least in part, from other depsipeptide antibiotics and its biological effect is primarily mediated through its inhibition of protein synthesis and to a lesser extent its inhibition of DNA synthesis.

Structure and activity relationship of several novel CC-1065 analogs

SummaryCC-1065 was found to cause delayed toxicity at therapeutic doses, therefore, a large number of analogs have since been synthesized. A series of analogs with simplified but closely related structures were chosen for this investigation because some were found to be superior to CC-1065 in the treatment of several experimental tumors. The inhibition of L1210 cell growth by U-68,415 was comparable to that by CC-1065. A similar situation was true in terms of their in vivo potency; however, U-68,415 was superior to CC-1065 in terms of anti-P 388 leukemia activity. At the optimal dosage, U-68,415 produced 4 out of 6 long-term (> 30 day) survivors; whereas CC-1065 produced a mere 62% increase of life span (ILS) and no long-term survivors. The order of antitumor potency and effectiveness of the CC-1065 analogs was U-68,415 > U-66,694 > U-68,819 > U-66,664, which was parallel to the inhibition of L1210 cell growth. CC-1065 and all the analogs tested here inhibited DNA synthesis approximately 10 times more than RNA synthesis. Protein synthesis was the least inhibited. On a molar basis, U-68,415 was about 6–9 times more inhibitory toward cellular DNA synthesis than CC-1065, yet the interaction and/or binding of CC-1065 to DNA determined by circular dichroism, DNA melting or differential cytotoxicity assay was much stronger than that of U-68,415. The order of binding of these analogs to calf thymus DNA was U-68,415 > U-66,694 > U-68,819 > U-66,664, and was parallel to that of DNA synthesis inhibition which was in turn parallel to cell growth inhibition and antitumor potential. These results collectively suggest that the cellular DNA is a major site of the action of CC-1065 analogs; however, time course studies reveal that the inhibition of cellular DNA synthesis could not wholly account for their cytotoxicity. Hence, the precise mechanism of action of these agents is not yet fully understood. U-68,415, which exhibited superior activity against a number of tumors and did not cause delayed death in mice, warrants further investigation. U-68,415 is a racemate and two chiral isomers were recently isolated. Therefore, further investigation of both U-68,415 and its chiral isomers is necessary.

The binding of CC-1065 to thymidine and deoxyadenosine oligonucleotides and to poly(dA) · poly(dT)

In this work, we report on the binding of the novel antitumor agent CC-1065 to poly(dA).poly(dT) and to mixtures of dA and dT oligomers as determined by electronic absorption and circular dichroism (CD) methods. In addition, the DNA binding properties of CC-1065 and its binding mechanism are compared to those of netropsin. CC-1065 binds to the polymer by at least three mechanisms to produce one irreversibly and two reversibly bound species. One reversibly bound species is moderately stable, but in time (days), it converts to the irreversibly bound species. Both of these species bind within the minor groove of the polymer and exhibit intense CC-1065 induced CD spectra. The other reversibly bound species does not acquire an induced CD. CC-1065 forces B-form duplex formation between mixtures of single strand dA and dT oligomers and binds irreversibly to the duplexes without showing the presence of an intermediate, reversibly bound species. The induced CD increases with increasing length of the oligomer, from the 5-mer (barely detectable CD) to the 14-mer (intense CD). The 7-, 10- and 14-mer mixtures bind about 1, between 1 and 2, and between 2 and 3 CC-1065 molecules, respectively. Computer graphic models of the CC-1065-DNA complex show that the covalent adduct of CC-1065 and unreacted CC-1065 can attain the same close van der Waals contacts between adenine C2 hydrogens and antibiotic CH groups that were observed in the crystal structure of the netropsin-DNA complex. These contacts may account for the dA-dT base pair binding specificity of CC-1065 and for the stability of the reversibly bound CC-1065 species.

Evaluation of DNA binding characteristics of some CC-1065 analogs

The factors influencing the binding of CC-1065 to DNA were examined using racemic analogs with varying chain lengths. The ability of these agents to bind DNA appeared to be related to cytotoxic potency, however this did not appear to be a direct quantitative correlation. Two enantiomers of a bis-indole analog of CC-1065 were studied for DNA binding and cytotoxic activity. The agent with the same stereochemical configuration as CC-1065 was a potent cytotoxin, but its enantiomer was essentially inactive. Both enantiomers showed significant binding to DNA, but the biologically less active isomer showed less overall binding. In all cases, the agents preferred AT-rich DNA, and all bound to similar regions in DNA as evidenced by positions of drug-initiated thermal breaks in single end-labelled fragments of phi X 174RF DNA. The overall similarity in site specificity for binding of the structurally diverse agents suggests that much of the specificity observed in binding of the agent to DNA lies in the DNA itself. Thus, it may be difficult to change minor groove specificity for agents of this type simply by designing structures that can encompass guanine or cytosine residues. Other modifications, such as changing the specificity of the alkylating moiety, may be required to achieve this goal.

The origin of the DNA induced circular dichroism of CC-1065 and analogs.

The calf thymus DNA (CT-DNA) and poly(dI-dC).poly(dI-dC) binding properties of the natural antitumor antibiotic CC-1065 and selected analogs of CC-1065 were studied by circular dichroism (CD) and absorbance methods. The results indicate that the intense long wavelength DNA-induced CD band of these molecules originates from a chiral electronic transition which is delocalized over the whole molecule. Both the covalently bound species (N-3 adenine adduct) and the reversibly bound species exhibit the characteristic spectral behavior of an inherently dissymmetric chromophore when these agents bind within the minor groove of B-form DNA. This mechanism of optical activity accounts for why CC-1065 shows a weak CD in buffer but a very intense induced CD at long wavelength when bound to DNA, why the intensity of the induced CD of CC-1065 analogs depends upon how many fused ring systems the analog contains, and why covalently bound analogs having the mirror image configuration of the natural configuration also exhibit an intense positive induced CD band at long wavelength.

Purification and structural characterization of the CD11b/CD18 integrin α subunit I domain reveals a folded conformation in solution

The α subunits of the leukocyte CD11/CD18 integrins contain a ∼200 amino acid ‘inserted’ or I domain. The I domain of the cell‐surface Mac‐1(CD11b/CD18) integrin has been shown to be the major recognition site for several adhesion ligands, including iC3b, fibrinogen, factor X, and ICAM‐1. The I domain from the Mac‐1 α subunit has been expressed in Escherichia coli as a soluble GST‐fusion protein containing a factor Xa sensitive cleavage site. Analytical characterization of the purified I domain reveals that it is obtained in very high quality at high yields. CD and NMR spectra indicate that I domain adopts a predominantly folded structure in solution, independent of the remainder of the α subunit. Addition of Ca2+ and Mg2+ did not significantly perturb the structural conformation.

Calf thymus DNA binding/bonding properties of CC-1065 and analogs as related to their biological activities and toxicities

CC-1065 is a potent natural antitumor antibiotic that binds non-covalently and covalently (N-3 adenine adduct) in the minor groove of B-form DNA. Synthetic analogs of CC-1065 do not exhibit the delayed death toxicity of CC-1065 and are efficacious anticancer agents, some of them curative in murine tumor models. In an attempt to understand the different biological properties of CC-1065 and analogs, we have determined the following quantities for CC-1065, enantiomeric CC-1065, and three biologically active analogs and their enantiomers: the calf thymus DNA (CT-DNA) induced molar ellipticity of the adduct (or how rigidly the adduct is held in the right-hand conformation of the minor groove); the stability of the adduct with respect to long incubation times and to digestion by snake venom phosphodiesterase I (SVPD); the stabilizing effect on the CT-DNA helix of the covalently and non-covalently bound species with respect to thermal melting; and the CT-DNA binding/bonding (non-covalent/covalent) profiles at a low molar ratio of nucleotide to drug. The major observations from these studies are as follows: (i) molecules which show large DNA interaction parameters, stable adducts, and significant non-covalent binding exhibit delayed death toxicity; (ii) molecules which show intermediate DNA interaction parameters and stable adducts, but do not show significant non-covalent binding, do not exhibit delayed death toxicity and are biologically active; (iii) molecules which show small DNA interaction parameters and unstable DNA adducts are biologically inactive. The results suggest that a window exists in the affinity for the minor groove of DNA wherein an analog may possess the correct balance of toxicity and activity to make a useful anticancer agent. Outside of this window, the analog causes delayed deaths or has no significant biological activity.

Chemical modification of Interleukin-1β: Biochemical characterization of a carbodiimide-catalyzed intramolecular cross-linked protein

We have modified recombinant human Interleukin-1β using 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide atpH 6.5, resulting in the formation of an internally cross-linked protein. The major product (30% yield) of the reaction (17 kD; pI=6.2) was purified and fully characterized by peptide mapping using Endoproteinase Lys C. When digests were conducted under nondenaturing conditions, we found that the modified protein is different from the native protein. The native protein yielded 14 peptides after digestion, whereas only two large peptides and a tetrapeptide, Asn-Tyr-Pro-Lys, were released from the cross-linked protein (i.e., cleavage occurs only at residues Lys88 and Lys92). Using gel filtration, the two peptides were found to co-elute as a single species (15 kD), which represent a noncovalent complex of the amino terminal and C-terminal portions of the molecule. Further analysis of the modified protein by peptide mapping under denaturing conditions and by FAB MS analysis showed that Glu111 and Lys138 were internally cross-linked. The cross-linked protein had bioactivity (T-cell proliferation), fluorescence, and circular dichroism spectra similar to native IL-1β. In contrast, while having similar secondary structure, the digested cross-linked protein had less than 1% of T-cell proliferative activity of the undigested protein. These data show that the structural integrity surrounding and perhaps including the Asn-Tyr-Pro-Lys region may be crucial for the biological activity of rIL-1β and may be important for the binding of IL-1 to its receptor.

Design and Structure/Conformation-Activity Studies of a Prototypic Corticotropin-Releasing Factor (CRF) Antagonist: Multiple Alanine Substitutions of CRF12-41

The known physiological role(s) and proposed pathophysiological properties of the neuroendocrine peptide CRF have been previously described (for review, see 1), and CRF has been shown to exert a variety of CNS-mediated effects on behavior2,3, cardiovascular system4,5, reproduction6,7, gastrointestinal secretion8,9, motility10, and transit11. Of particular significance is that CRF may, therefore, be involved in stress stimuli-induced activation of neural/humoral pathways leading towards anxiety and depressive disorders (e.g.,depiession, panic and anorexia nervosa). Nevertheless, the molecular pharmacology and mechanisms which are involved in stress-induced behavioral, endocrine and metabolic activities are not well defined. The discovery and development of potent CRF antagonists may provide key molecular probes to investigate the biological activities of endogenous CRF in animal models as well as for studying the molecular pharmacology of CRF-receptor interactions. Such studies have been reported12,13 and have been primarily based upon synthetic modification of CRF; yet the emergence of a high affinity analog of low molecular mass (i.e., small peptide or peptidomimetic) has remained elusive to date. Nevertheless, studies14-18 on the blockade of endogenous CRF using CRF antiserum or prototypic CRF antagonists have probed the possible role that endogenous CRF may have on the effects of stress in different animal models. Of noteworthy contribution to such CRF research has been both structure-activity and structure-conformation studies12,13,19,20 to investigate CRFreceptor binding and functional properties (agonism/antagonism). These studies have culminated in the identification of prototypic CRF antagonists (or partial agonists) which were modified fragment analogs of the native peptide. Specifically, compound I (Fig. 1) has been advanced13 as a significant lead towards the development of high affinity CRF receptor antagonists. In this report we describe analogs of I to further explore the role of side-chain functionlization in CRF receptor binding using a strategy of multiple (iterative) Ala substitution with a particular focus on the central domain of this CRF analog corresponding to CRF22-31 In addition, the structure-conformation properties of these analogs were investigated by circular dichroism spectroscopy.

DNA Base Pair Binding Specificity of CC-1065

Abstract Oligomer duplexes were prepared by a solid-phase phosphoramidite triester coupling approach in order to study the DNA base pair binding specificity of the antitumor antibiotic CC-1065 by CD spectroscopy.