Structure-activity relationships of acyloxyamidine cytomegalovirus DNA polymerase inhibitors.

This paper describes the structure activity relationships of a new class of cytomegalovirus DNA polymerase inhibitors having two aryl groups joined by an acyloxyamidine linker. Examination of a series of analogues in which the terminal groups are varied revealed a very narrow SAR around the 2,4-dichlorophenyl group of the lead compound, but a variety of replacements for the benzothiazole ring are compatible with activity. The most notable of these is the isoxazole ring of compound 78, which provides a 30-fold enhancement in potency compared to the lead compound. We also describe the design, synthesis and evaluation of 10 analogues in which the acyloxyamidine linker is modified or replaced by an isosteric group. Structure-activity relationship studies identified the linker -NH2 group as a critical pharmacophoric element. Ab initio molecular orbital calculations combined with qualitative estimates of steric interaction energies suggest that the lowest energy conformations of the acyloxyamidine linker are characterized by an extended planar CAr-C=N-O-C arrangement and either a syn-periplanar or anti-periplanar N-O-C-C(Ar') arrangement. Only the anti-periplanar conformation was observed in the crystal structures of three acyloxyamidines. The most active of the linker-modified compounds designed on the basis of these studies is the amidine carbamate 20, which is approximately one-third as potent in the cytomegalovirus DNA polymerase inhibition assay as the comparator acyloxyamidine 53. The activity of 20 suggests that acyloxyamidines may bind to the cytomegalovirus DNA polymerase via an anti-periplanar conformation similar to that observed in the crystal structure of acyloxyamidine 36.

Binding of FGF-1 and FGF-2 to heparan sulphate proteoglycans of the mammalian lens capsule.

In the mammalian eye, FGF plays a key role in the induction of lens fibre differentiation and, in other systems, heparan sulphate proteoglycans (HSPGs) have been shown to modulate FGF activity. HSPGs were isolated from the anterior and posterior rat and bovine lens capsule and assessed in terms of their ability to bind FGF-1 and FGF-2. In the rat, at least four HSPGs were identified with molecular weights of 142, 166, 200 and approximately 250 kD, the latter species predominating. The capsule HSPGs bound both FGF-1 and FGF-2. There appeared to be little, if any, competition for binding between FGF-1 and FGF-2. The capsule contained substantial amounts of core protein, which did not bind FGF, with a higher core protein/HSPG ratio in the anterior than in the posterior capsule. This was the only major HSPG-related difference noted between anterior and posterior capsule.

Inhibition of transforming growth factor-beta-induced cataractous changes in lens explants by ocular media and alpha 2-macroglobulin.

PURPOSE: To investigate the ocular media for the presence of inhibitors of transforming growth factor-beta (TGF beta) using a lens epithelial explant system in which TGF beta induces cataractous changes. The effect of alpha 2-macroglobulin, an inhibitor of TGF beta in other systems, also was assessed. METHODS: Explants prepared from 21-day-old rats were cultured with TGF beta 2 with and without 50% bovine aqueous or vitreous or alpha 2-macroglobulin. alpha 2-macroglobulin was added to an aqueous concentrate, shown to contain endogenous TGF beta activity by blocking with anti-TGF beta. Explants were monitored by phase-contrast microscopy for 5 days and assessed in terms of capsule wrinkling, spindle-cell formation, blebbing, and cell loss. alpha 2-macroglobulin in the ocular media was assessed by enzyme-linked immunosorbent assay and Western blot analysis. RESULTS: At 50% strength, neither aqueous nor vitreous demonstrated TGF beta-like activity; however, aqueous partially and vitreous completely prevented cataractous changes induced by 25 and 100 pg/ml TGF beta 2, respectively. alpha 2-macroglobulin (50 to 200 micrograms/ml) also protected against these changes, with complete inhibition of TGF beta 2 or aqueous-derived TGF beta activity at the highest concentration. A threefold higher concentration of alpha 2-macroglobulin was detected in vitreous than aqueous. CONCLUSIONS: Both aqueous and vitreous contain molecule(s) that inhibit TGF beta 2 activity. alpha 2-macroglobulin has been identified in the ocular media and shown to block cataractous changes induced by TGF beta. Maintaining appropriate levels of alpha 2-macroglobulin or similar molecules in the ocular media may protect lens cells from the damaging effects of TGF beta, and reduced levels may predispose to cataract.

Nitric oxide-mediated death of cultured neonatal retinal ganglion cells: neuroprotective properties of glutamate and chondroitin sulfate proteoglycan.

The release of nitric oxide and stimulation of glutamate receptors by excitatory amino acids has been linked to neuronal degeneration and toxicity. In the rat retina approximately 60% of retinal ganglion cells (RGCs) die during the first postnatal week. In this study we examined the effects of nitric oxide synthase blockers and glutamate on the survival of neonatal RGCs in vitro over a 16 h assay period. Less than 10% of P1 RGCs survived in serum free defined media alone (control), however survival was increased, in a dose-dependent manner, when L-glutamate (10 microM-10 mM) was added to the media; a maximum of 70% of RGCs could be maintained with the addition of 5 mM glutamate. This effect was blocked by the NMDA and non-NMDA receptor blockers APV and DNQX and was age dependent; the survival of RGCs from P5 but not P7 rats was enhanced by the addition of glutamate even in high calcium concentrations (10 mM). When the nitric oxide synthase blockers L-NAME (5 mM) or haemoglobin (25 microM) were added to the culture media, up to 61% of P1 RGCs survived. The addition of the 480 kDa chondroitin sulfate proteoglycan (SCCP) previously shown to enhance RGC survival in vivo and in vitro, potentiated the action of glutamate and L-NAME and increased RGC survival to over 90% with almost all RGCs expressing a profusion of processes. These results suggest that the release of nitric oxide and glutamate by cells within the retina may contribute to the regulation of RGC numbers in vivo during development.

TGF-beta 1 induces lens cells to accumulate alpha-smooth muscle actin, a marker for subcapsular cataracts.

Spindle-shaped myofibroblast-like cells, which contain alpha-smooth muscle actin, have been described in anterior subcapsular cataract and after-cataract. In a previous study in this laboratory, it was shown that transforming growth factor-beta (TGF beta) induces the formation of spindle-shaped cells in lens epithelial explants. The aim of this investigation was to determine whether these TGF beta-induced spindle-shaped cells contain alpha-smooth muscle actin. Lens epithelial explants were prepared from 21-day-old rats and cultured with either TGF beta 1 or basic FGF alone, a combination of both growth factors, or without added growth factors. After three days, cellular changes were monitored by phase contrast microscopy, localisation of filamentous actin with rhodamine-phalloidin, and immunolocalisation and immunoblotting of alpha-smooth muscle actin. TGF beta induced rapid cell elongation and formation of characteristic spindle-shaped cells in lens epithelial explants in the presence or absence of FGF. These cells contained alpha-smooth muscle actin, a marker for myofibroblastic cells and a protein not normally found in the lens. The present study thus provides molecular evidence that TGF beta induces cataractous changes in lens epithelial cells. As TGF beta is potentially available to lens cells in situ throughout life, these findings are consistent with a key role for TGF beta in the aetiology of major forms of subcapsular cataract.

Acidic and basic FGF in ocular media and lens: implications for lens polarity and growth patterns.

We have shown previously that FGF induces lens epithelial cells in explant culture to proliferate, migrate and differentiate into fibre cells in a progressive concentration-dependent manner. In situ, these processes occur in a distinct anterior-posterior pattern in clearly defined regions of the lens. Thus anterior-posterior differences in the bio-availability of FGF in the lens environment may play a role in determining lens polarity and growth patterns. In this study, using heparin chromatography and western blotting (or ELISA), we established that both acidic and basic FGF are present in the aqueous and vitreous (the ocular media that bathe the anterior and posterior compartments of the lens, respectively). In addition, substantially more FGF was recovered from vitreous than from aqueous. Both forms of FGF were also detected in lens fibre cells and capsule. A truncated form of basic FGF (less than 20 x 10(3) M(r)) predominated in every case with traces of higher M(r) forms in lens cells. For acidic FGF, the classical full-length form (about 20 x 10(3) M(r)) predominated in lens cells and a truncated form was found in vitreous. The capsule contained a higher M(r) form. Using our explant system, we also tested the biological activity of ocular media and FGF fractions obtained from vitreous and lens cells. Vitreous but not aqueous contained fibre-differentiating activity. Furthermore, virtually all the fibre-differentiating activity of vitreous was shown to be FGF-associated, as follows: (a) this activity remained associated with FGF during fractionation of vitreous by heparin and Mono-S chromatography and (b) the activity of the major FGF-containing fraction was blocked by antibodies to acidic and basic FGF. Posterior, but not anterior, capsule was shown to have mitogenic activity, which was neutralised by FGF antibodies and associated only with the cellular surface. These results support our hypothesis that FGF is involved in determining the behaviour of lens cells in situ. In particular, a key role for FGF in determining lens polarity and growth patterns is suggested by the anterior-posterior differences in the bio-availability of FGF in the ocular media and capsule.

An energy-based approach to packing the 7-helix bundle of bacteriorhodopsin.

Based on the heavy-atom coordinates determined by the electron microscopy for the seven main helical regions of bacteriorhodopsin with the all-trans retinal isomer, energy optimizations were carried out for helix bundles containing the all-trans retinal and 13-cis retinal chromophores, respectively. A combination of simulated annealing and energy minimization was utilized during the process of energy optimization. It was found that the 7-helix bundle containing the all-trans isomer is about 10 kcal/mol lower in conformational energy than that containing the 13-cis isomer. An energetic analysis indicates that such a difference in energy is consistent with the observation that absorption of a 570-nm proton is required for the conversion of a bacteriorhodopsin from its all-trans to 13-cis form. It was also found that the above conversion process is accompanied by a significant conformational perturbation around the chromophore, as reflected by the fact that the beta-ionone ring of retinal moves about 5.6 A along the direction perpendicular to the membrane plane. This is consistent with the observation by Fodor et al. (Fodor, S.P.A., Ames, J.B., Gebhard, R., van der Berg, E.M.M., Stoeckenius, W., Lugtenburg, J., & Mathies, R.A., 1988, Biochemistry 27, 7097-7101). Furthermore, it is interesting to observe that although the retinal chromophore undergoes a significant change in its spatial position, the orientation of its transition dipole changes only slightly, in accord with experimental observations. In other words, even though orientation of the retinal transition dipole is very restricted, there is sufficient room, and degrees of freedom, for the retinal chromophore to readjust its position considerably. This finding provides new insight into the subtle change of the retinal microenvironment, which may be important for revealing the proton-pumping mechanism of bacteriorhodopsin. The importance of electrostatic and nonbonded interactions in stabilizing the 7-helix bundle structure has also been analyzed. Electrostatic interactions favor an antiparallel arrangement among adjacent helices. Nonbonded interactions, however, drive most of the closely packed helices into an arrangement in which the packing angles lie around -160 degrees, a value very near the -154 degrees value computed earlier as the most favorable packing arrangement of two poly(Ala) alpha-helices (Chou, K.-C., Némethy, G., & Scheraga, H.A., 1983, J. Phys. Chem. 87, 2869-2881). The structural features of the 7-helix bundle and their relationship to those found in typical 4-helix bundle proteins are also discussed.(ABSTRACT TRUNCATED AT 400 WORDS)

Geometric and energy parameters in lysine-retinal chromophores.

The parameters used in the computer program ECEPP (Empirical Conformational Energy Program for Peptides) have been expanded to cover some key elements in retinal-containing proteins. These elements are 'all-trans retinal lysine with unprotonated imine', 'all-trans retinal lysine with protonated imine', '13-cis retinal lysine with unprotonated imine' and '13-cis retinal lysine with protonated imine' respectively. The geometric parameters of these four new 'amino acid residues' were derived by optimizing their molecular structures with the AM1 Hamiltonian included in MOPAC (Molecular Orbital PACkage), and their partial atomic charges were determined with a CNDO/2 (Complete Neglect of Differential Overlap) calculation. The parameters for nonbonded interactions and torsional potentials were obtained from the existing ECEPP parameters through a logical extension. The augmented ECEPP system thus obtained can be employed to investigate the conformation of bacteriorhodopsin and its proton-pumping mechanism from an energetic point of view. The computer modeling study on bacteriorhodopsin and other seven-helix membrane proteins, e.g. serotonin receptor and dopamine receptor, is under way in the Upjohn Laboratories.