Arezki Azzi

Molecular therapy of breast cancer: progress and future directions

Breast cancer is a major cause of death in Western women, with a 10% lifetime risk of the disease. Most breast cancers are estrogen-dependent. Molecular therapies for breast cancer have developed rapidly in the past few decades and future treatment strategies are being investigated. The selective estrogen receptor (ER) modulator tamoxifen, which until now has served as a standard therapy, functions not only as an estrogen antagonist but also as an estrogen agonist in terms of bone maintenance. Aromatase inhibitors have performed well in international trials and have become a new standard therapy for estrogen-dependent breast cancer. The systematic study of estrogen activation pathways suggests that the enzymes steroid sulfatase and 17β-hydroxysteroid dehydrogenase type 1, which both have pivotal roles in estrogen biosynthesis, are promising targets; the results of a phase I trial of steroid sulfatase inhibitors are encouraging. The activity of the human epidermal growth factor receptor (HER) pathway correlates negatively with that of the ER. HER2 is overexpressed in 22% of all breast cancers. In the decade since HER2 began being targeted, the monoclonal antibody trastuzumab has been used as well as pertuzumab and HER2 vaccines. Among the estrogen-independent breast cancers, the basal-like subtype has low survival, and therapeutic improvement is a priority. Crosstalk between ER and HER2 signaling pathways means that combinatory therapies may hold the key to enhancement of treatment responses. Other molecular therapies involving functional genomics and RNA interference studies also hold promise.

Structural basis of the multispecificity demonstrated by 17β-hydroxysteroid dehydrogenase types 1 and 5

17Beta-hydroxysteroid dehydrogenases/ketosteroid reductases (17beta-HSDs/KSRs) catalyze the last step of sex steroid synthesis or the first step of their degradation, and are thus critical for many physiological processes. The multispecificity demonstrated by 17beta-HSDs is important for steroid metabolism in gonadal and peripheral tissues, and is a consequence of the architecture of their binding and catalytic sites. Structurally, most of the family members are short chain dehydrogenase-reductases (SDRs) except the type 5 enzyme, which is an aldo-keto reductase (AKR). 17Beta-HSD type 1, a representative of the SDR family, has been studied extensively since the 1950s. However, its structure was not determined until the 1990s. It has always been considered as estrogen specific, in accord with the narrow binding tunnel that has been structurally determined and has been found to be complementary to estrogens. A recent study revealed that, in spite of the enzymes narrow binding tunnel, the pseudo-symmetry of C19 steroids leads to its alternative binding, resulting in the multispecificity of the enzyme. Expressed in ovary, breast and placenta, the enzyme catalyzes the formation of another estrogen A-diol from DHEA in addition to the biosynthesis of estradiol; it also inactivates the most active androgen DHT by both 17beta-hydroxysteroid oxidation and 3-ketosteroid reduction. Type 5 17beta-HSD (AKR1C3) differs significantly from the type 1 enzyme by possessing a spacious and flexible steroid-binding site. This is estimated to be about 960 or 470 A3 in ternary complex with testosterone or 4-dione, respectively, whereas the binding site volume of 17beta-HSD1 is only about 340 A3. This characteristic of the 17beta-HSD5 binding site permits the docking of various steroids in different orientations, which encompasses a wider range of activities from 20alpha-, 17beta- and 3alpha-HSD/KSR to prostaglandin 11-ketoreductase. The in vitro activities of the enzyme are significantly lower than the type 1 enzyme. In the ternary complex with testosterone, the steroid C3-C17 position is quasi-reversed as compared to the complex with 4-dione. The multi-specificity contributes significantly to steroid metabolism in peripheral tissues, due to the high levels of 17beta-HSD5 mRNA in both breast and prostate tissues.

Estrone and estradiol C-16 derivatives as inhibitors of type 1 17β-hydroxysteroid dehydrogenase

Three series of steroid derivatives, enones 1, enols 2 and saturated alcohols 3, were easily synthesized from estrone according to a sequence of three reactions: an aldol condensation with an aromatic aldehyde (R(a-g)CHO) to afford 1, the carbonyl reduction of 1 to obtain the enol 2, and the double bond reduction of 2 to give 3 with the R(a-g) group 16beta-oriented. All compounds were tested as inhibitors of type 1 17beta-HSD. The inhibitory potency increases in the following order 1<2<3, suggesting that the presence of a flexible 16beta-methylene group allows a better positioning of the aryl moiety. With an IC50 of 0.8 microM, the 16beta-benzyl-E2 (3a) is the best inhibitor in this series.

Three-dimensional modeling of cytomegalovirus DNA polymerase and preliminary analysis of drug resistance.

Cytomegalovirus (CMV) is the leading cause of congenital infection and a frequent opportunistic agent in immunocompromised hosts such as transplant recipients and AIDS patients. CMV DNA polymerase, a member of the polymerase B family, is the primary target of all available antivirals (ganciclovir, cidofovir, and foscarnet) and certain variations of this enzyme could lead to drug resistance. However, understanding the drug resistance mechanisms at the atomic level is hampered by the lack of its three‐dimensional (3D) structure. In the present work, 3D models of two different conformations (closed and open) for CMV DNA polymerase have been built based on the crystal structures of bacteriophage RB69 DNA polymerase (a member of the polymerase B family) by using the 3D‐Jury Meta server and the program MODELLER. Most of the variations on CMV DNA polymerase pertinent to ganciclovir/cidofovir and foscarnet resistance can be explained well based on the open and closed conformation models, respectively. These results constitute a first step towards facilitating our understanding of drug resistance mechanisms for CMV and the interpretation of novel viral mutations. Proteins 2006.

Recombinant Phenotyping of Cytomegalovirus UL54 Mutations That Emerged during Cell Passages in the Presence of either Ganciclovir or Foscarnet

ABSTRACT Selection of human cytomegalovirus variants in the presence of ganciclovir or foscarnet led to 18 DNA polymerase mutations, 14 of which had not been previously studied. Using bacterial artificial chromosome technology, each of these mutations was individually transferred into the genome of a reference strain. Following reconstitution of infectious viral stocks, each mutant was assessed for its drug susceptibility and growth kinetics in cell culture. Computer-assisted three-dimensional (3D) modeling of the polymerase was also used to position each of the mutations in one of four proposed structural domains and to predict their influence on structural stability of the protein. Among the 10 DNA polymerase mutations selected with ganciclovir, 7 (P488R, C539R, L545S, V787L, V812L, P829S, and L862F) were associated with ganciclovir resistance, whereas 2 (F595I and V946L) conferred only foscarnet resistance. Among the eight mutations selected with foscarnet, only two (T552N and S585A) conferred foscarnet resistance, whereas four (N408D, K500N, L802V, and L957F) had an impact on ganciclovir susceptibility. Surprisingly, the combination of mutations, some of which were not associated with resistance for a specific antiviral, resulted in increasing resistance effects. 3D modeling suggested that none of the mutated residues were directly involved in the polymerase catalytic site but rather had an influence on drug susceptibility by modifying the structural flexibility of the protein. Our study significantly adds to the number of DNA polymerase mutations conferring in vitro drug resistance and emphasizes the point that evaluation of individual mutations may not accurately reflect the phenotype conferred by multiple mutations.

Opposite effect of two cytomegalovirus DNA polymerase mutations on replicative capacity and polymerase activity.

BACKGROUND Human cytomegalovirus (HCMV) infections cause significant morbidity in immunocompromised hosts. The aim of this study was to characterize the role of two HCMV DNA polymerase mutations (K805Q and T821I) found in a ganciclovir- and foscarnet-resistant clinical isolate from an AIDS patient. METHODS The effects of single and dual DNA polymerase mutations on virus susceptibility and replicative capacity, as well as on enzymatic activity, were studied using recombinant viruses generated from overlapping cosmids and DNA polymerase enzymes expressed in rabbit reticulocyte lysates. RESULTS Recombinant viruses containing mutations K805Q, T821I and K805Q+T821I had 0.8-fold, 5.3-fold and 4.8-fold increases in ganciclovir 50% inhibitory concentration (IC(50)) values and 0.3-fold, 23.3-fold and 15.6-fold increases in foscarnet IC(50) values, respectively, compared with those of the wild-type virus. The recombinant virus T821I had impaired replication in fibroblastic cells on day 2 post-infection with a decrease in viral titres of 3.5-fold, 4.3-fold and 2.6-fold compared to the recombinant wild-type, K805Q and K805Q+T821I viruses, respectively. Enzymatic studies of wild-type and mutant DNA polymerase enzymes in presence of foscarnet resulted in IC(50) values that were similar to those of the recombinant viruses. Steady-state kinetic constants K(m) and V(max) derived from Michaelis-Menten equations showed that the activity of the mutant T821I enzyme was diminished compared with those of wild-type, K805Q and K805Q+T821I mutant enzymes. Thermodynamic stability of the two single mutant enzymes was opposed as shown by computer-assisted three-dimensional modelling studies. CONCLUSIONS The HCMV DNA polymerase mutation K805Q improved the fitness of the T821I mutation associated with high levels of resistance to foscarnet.

Human SARS‐coronavirus RNA‐dependent RNA polymerase: Activity determinants and nucleoside analogue inhibitors

A coronavirus, an RNA virus, has been identified in humans as the pathogen responsible for the recent worldwide Severe Acute Respiratory Syndrome (SARS) outbreak. Rapid study of the critical SARS enzymes will provide useful inhibitors for this disease as well as a methodology that may be applicable to fight emerging pathogens in the future. The necessity of this rapid approach is further justified by the fact that the SARS virus reservoir has not been identified and future viral outbreaks are still possible. Indeed, new cases have already been reported in 2004. The SARS coronavirus (SARS CoV) genome is a positive strand of RNA with 11 open reading frames and a genomic organization similar to other coronaviruses. However, phylogenic analysis and sequence alignments have shown that the SARS CoV has developed distinctly from other coronaviruses. The viral replication proceeds via synthesis of a complementary minus strand RNA using the genome as a template and the subsequent synthesis of genomic plus strand RNA from the minus strand RNA template. The key enzyme responsible for both steps is the RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 12 (nsP12). Since RdRp activity is not essential for the host, its inhibition will not cause undesirable side effects during therapy. With the enzyme characterization in its early stages, a homology model based on the amino acid sequence can provide some clues concerning the residues critical for its function. We have built a homology model of the SARS polymerase using the 3D jury system. Recently, during preparation and revision of this study, Xu et al. reported an RdRp model using the program Modeller. Although their and our models have been built using a different approach, the r.m.s between the two models is about 1.6 Å (for C atoms). The similarity between the two models can be partially explained by the fact that the 3D-jury system uses a set of prediction methods that includes the Pcons method on which the Modeller program is based on. The capability of the 3D-jury in using consensus between Meta prediction methods makes it a very reliable tool for model building as proven by its highest ranking in the CASP5 (Critical Assessment of Techniques for Protein Structure Prediction) competition. This predictor has been used recently to successfully assign a methyl-transferase function to nsP13 and to build a 3D model that identified residues critical for the enzyme function. Substrate and ligand docking in the active site were carried out with the Genetic Optimization Ligand Docking Program (Gold) giving us an estimate of ligand binding in the active site. This docking method, with a success rate of around 70% through its validation process, proved to be very reliable when used with caution.

Two non-reactive ternary complexes of estrogenic 17β-hydroxysteroid dehydrogenase: crystallization and preliminary structural analysis

Human estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD1, EC1.1.1.62) is an important enzyme that catalyses the last step of active estrogen formation. 17Beta-HSD1 plays a key role in the proliferation of breast cancer cells. The three-dimensional structures of this enzyme and of the enzyme-estradiol complex have been solved (Zhu et al., 1993, J. Mol. Biol. 234:242; Ghosh et al., 1995, Structure 3:503; Azzi et al., 1996, Nature Struct. Biol. 3:665). The determination of the non-reactive ternary complex structure, which could mimic the transition state, constitutes a further critical step toward the rational design of inhibitors for this enzyme (Ghosh et al. 1995, Structure 3:503; Penning, 1996, Endocrine-Related Cancer, 3:41). To further study the transition state, two non-reactive ternary complexes, 17beta-HSD1-EM519-NADP+ and 17beta-HSD1-EM553-NADP+ were crystallized using combined methods of soaking and co-crystallization. Although they belong to the same C2 space group, they have different unit cells, with a = 155.59 A, b = 42.82 A, c = 121.15 A, beta = 128.5 degrees for 17beta-HSD1-EM519-NADP+, and a = 124.01 A, b = 45.16 A, c = 61.40 A, beta = 99.2 degrees for 17beta-HSD1-EM553-NADP+, respectively. Our preliminary results revealed that the inhibitors interact differently with the enzyme than do the natural substrates.

Crystallization and preliminary crystallographic analysis of the snake muscle fructose 1,6-bisphosphatase

The snake muscle fructose 1,6-bisphosphatase, a typical allosteric enzyme which plays important roles in gluconeogenesis, was crystallized in the presence of polyethylene glycol 3350 and magnesium chloride at pH 8.5. The crystals diffract to 2.3 A on a rotating-anode X-ray source. The space group was determined to be either P3121 or its enantiomorph P3221, with unit-cell parameters a = b = 83.7, c = 202.41 A, alpha = beta = 90 and gamma = 120 degrees. There are two subunits in the asymmetric unit. Preliminary molecular-replacement studies indicate that the first enantiomorph is the correct one.

Three-dimensional modeling of DCIR and identification of new drugs blocking HIV-1 attachment and propagation

The HIV-1 pandemic continues to expand while no effective vaccine is yet available. Finding new therapeutic targets and drugs is therefore crucial. We have previously shown that the dendritic cell immunoreceptor (DCIR), a C-type lectin receptor expressed in dendritic cells (DCs), acts as an attachment factor for HIV-1 to DCs and contributes to HIV-1 transmission to CD4+ T lymphocytes (CD4TL). Directly involved in HIV-1 infection, DCIR is expressed in apoptotic or infected CD4TL and promotes trans-infection to bystander cells. The aim of the present study is to characterize the extracellular domain of DCIR and to test chemical inhibitors of HIV-1 attachment thereto.