Design and synthesis of Hsp90 inhibitors: Exploring the SAR of Sansalvamide A derivatives

Utilizing the structure–activity relationship we have developed during the synthesis of the first two generations and mechanism of action studies that point to the interaction of these molecules with the key oncogenic protein Hsp90, we report here the design of 32 new Sansalvamide A derivatives and their synthesis. Our new structures, designed from previously reported potent compounds, were tested for cytotoxicity on the HCT116 colon cancer cell line, and their binding to the biological target was analyzed using computational studies involving blind docking of derivatives using Autodock. Further, we show new evidence that our molecules bind directly to Hsp90 and modulate Hsp90’s binding with client proteins. Finally, we demonstrate that we have integrated good ADME properties into a new derivative.     

Serotype-Specific Structural Differences in the Protease-Cofactor Complexes of the Dengue Virus Family

With an estimated 40% of the world population at risk, dengue poses a significant threat to human health, especially in tropical and subtropical regions. Preventative and curative efforts, such as vaccine development and drug discovery, face additional challenges due to the occurrence of four antigenically distinct serotypes of the causative dengue virus (DEN1 to -4). Complex immune responses resulting from repeat assaults by the different serotypes necessitate simultaneous targeting of all forms of the virus. One of the promising targets for drug development is the highly conserved two-component viral protease NS2B-NS3, which plays an essential role in viral replication by processing the viral precursor polyprotein into functional proteins. In this paper, we report the 2.1-Å crystal structure of the DEN1 NS2B hydrophilic core (residues 49 to 95) in complex with the NS3 protease domain (residues 1 to 186) carrying an internal deletion in the N terminus (residues 11 to 20). While the overall folds within the protease core are similar to those of DEN2 and DEN4 proteases, the conformation of the cofactor NS2B is dramatically different from those of other flaviviral apoprotease structures. The differences are especially apparent within its C-terminal region, implicated in substrate binding. The structure reveals for the first time serotype-specific structural elements in the dengue virus family, with the reported alternate conformation resulting from a unique metal-binding site within the DEN1 sequence. We also report the identification of a 10-residue stretch within NS3pro that separates the substrate-binding function from the catalytic turnover rate of the enzyme. Implications for broad-spectrum drug discovery are discussed.Dengue fever (DF) affects tens of millions of people each year, with an average mortality rate of 5%, comprising mostly young children. The increasing spread and frequency of global epidemics of this disease have heightened the urgency for developing effective strategies for prevention, diagnosis, and treatment. Though several groups are involved in vaccine development (17, 23, 25), dengue presents a unique, complex challenge. Four antigenically distinct serotypes of the causative dengue virus (DENV) (DEN1 to -4) occur in nature, with differing pathogenicities in partially overlapping geographic locations. In individuals previously exposed to a certain serotype, repeat assault by a different serotype can lead to potentially fatal complications of the disease, dengue hemorrhagic fever and dengue shock syndrome. The presence of subneutralizing levels of antibodies against the first serotype, resulting in antibody-dependent enhancement (ADE), is believed to be a causative mechanism underlying this complication. Forty percent of the world population lives in dengue risk areas, mainly in tropical and subtropical regions, which necessitates the development of vaccines and therapeutics that can simultaneously protect against all four serotypes (24).DEN1 to -4 belong to the family Flaviviridae (genus Flavivirus) of positive-stranded RNA viruses transmitted by Aedes aegypti mosquitoes. Upon infection, the genomic RNA is translated by the host cell machinery into a 370-kDa polyprotein, which is subsequently cleaved and processed into 10 distinct structural (C, E, and prM) and nonstructural (NS1, 2A, 2B, 3, 4A, 4B, and 5) proteins. A majority of this processing (junctions of NS2A/2B, 2B/3, 3/4A, and 4B/5, as well as internal sites within C, 2A, 3, and 4A) is carried out by a virus-encoded two-component protease, NS2B-NS3. Additional processing, especially of sites toward the N terminus (junctions of C/prM, prM/E, E/NS1, NS1/NS2A, and NS4A/4B), employs cellular proteases, such as signalase. The NS2B-NS3-mediated cleavage forms an essential step in the viral replicative cycle, as evidenced by the lack of production of infectious virions in mutants carrying inactivating substitutions in the protease. Of the duo, the N terminus of NS3 encodes the enzymatic core, while a hydrophilic core within NS2B provides an essential cofactor function (9).Owing to its essential nature in viral replication and the promise and success of drugs targeting the proteases in the treatment of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections (4), several recent studies have concentrated on identifying inhibitors of the flaviviral protease (5, 10, 21, 26-28). An added advantage of targeting the protease is that it is highly conserved between the serotypes (63 to 74%). For such structure-based drug design efforts, the availability of three-dimensional (3D) structures is an essential prerequisite, and given the differing pathogenicities and immune complexity generated by the multiple serotypes, it is a prudent first step to determine the structures of all four DENV proteases. The crystal structures of the DEN2 and DEN4 proteases in complex with various lengths of NS2B are available (7, 15). In this paper, we present the structures of an optimal construct of DEN1pro in complex with the essential hydrophilic core of NS2B (NS2Bc), as well as its active-site mutant. These structures reveal a novel and unexpected serotype-specific structural element embedded in the DENV genome, with implications for the discovery of broad-spectrum drugs. We also report the identification of a 10-residue stretch within the NS3 sequence that allows the separation of the substrate-binding function of the enzyme from its catalytic efficiency.     

Incorporation and replication of 8-oxo-deoxyguanosine by the human mitochondrial DNA polymerase

To assess the role of oxidative stress on the replication of mitochondrial DNA, we examined the kinetics of incorporation of 8-oxo-7,8-dihydroguanosine (8-oxodG) triphosphate catalyzed by the human mitochondrial DNA polymerase. Using transient state kinetic methods, we quantified the kinetics of incorporation, excision, and extension beyond a base pair containing 8-oxodG. The 8-oxodGTP was incorporated opposite dC in the template with a specificity constant of 0.005 microM(-1) s(-1), a value approximately 10,000-fold lower than that for dGTP. Once incorporated, 96% of the time 8-oxodGMP was extended by continued polymerization rather than being excised by the proofreading exonuclease. The specificity constant for incorporation of 8-oxodGTP opposite a template dA was 0.2 microM(-1) s(-1), a value 13-fold higher than incorporation opposite a template dC. The 8-oxodG:dA mispair was extended rather than excised at least 70% of the time. Examination of the kinetics of polymerization with 8-oxodG in the template strand also revealed relatively low fidelity in that dCTP would be incorporated only 90% of the time. In nearly 10% of events, dATP would be incorporated, and once incorporated dA (opposite 8-oxodG) was extended rather than excised. The greatest fidelity was against a dTTP:8-oxodG mismatch affording a discrimination value of only 1800. These data reveal that 8-oxodGTP is a potent mutagen. Once it is incorporated into DNA, 8-oxodGMP codes for error prone DNA synthesis. These reactions are likely to play important roles in oxidative stress in mitochondria related to aging and as compounded by nucleoside analogs used to treat human immunodeficiency virus infections.     

A novel gammaherpesvirus associated with genital lesions in a Blainville's beaked whale (Mesoplodon densirostris)

An adult male Blainville's beaked whale (Mesoplodon densirostris) was found stranded on the Atlantic coast of the USA on 28 January 2004. Necropsy revealed a focal papilloma-like penile lesion, the cells from which revealed single 4-6 microm basophilic intranuclear inclusions. Total DNA extracted from lesion material was tested using a pan-herpes-virus PCR assay that targets the DNA polymerase gene and found to be positive. When the amplified DNA fragment was cloned, sequenced, and compared to GenBank-deposited herpesvirus DNA polymerase sequences, the detected virus was determined to be a distinct member of the Gammaherpesvirinae subfamily of herpesviruses. This new virus, tentatively named Ziphiid herpesvirus type 1, was associated with but not determined to be the cause of genital disease in the Blainville's beaked whale.     

Diet of chimpanzees (Pan troglodytes schweinfurthii) at Ngogo, Kibale National Park, Uganda, 2. Temporal variation and fallback foods

Highly frugivorous primates like chimpanzees (Pan trogolodytes) must contend with temporal variation in food abundance and quality by tracking fruit crops and relying more on alternative foods, some of them fallbacks, when fruit is scarce. We used behavioral data from 122 months between 1995 and 2009 plus 12 years of phenology records to investigate temporal dietary variation and use of fallback foods by chimpanzees at Ngogo, Kibale National Park, Uganda. Fruit, including figs, comprised most of the diet. Fruit and fig availability varied seasonally, but the exact timing of fruit production and the amount of fruit produced varied extensively from year to year, both overall and within and among species. Feeding time devoted to all major fruit and fig species was positively associated with availability, reinforcing the argument that chimpanzees are ripe fruit specialists. Feeding time devoted to figs-particularly Ficus mucuso (the top food)--varied inversely with the abundance of nonfig fruits and with foraging effort devoted to such fruit. However, figs contributed much of the diet for most of the year and are best seen as staples available most of the time and eaten in proportion to availability. Leaves also contributed much of the diet and served as fallbacks when nonfig fruits were scarce. In contrast to the nearby Kanywara study site in Kibale, pith and stems contributed little of the diet and were not fallbacks. Fruit seasons (periods of at least 2 months when nonfig fruits account for at least 40% of feeding time; Gilby & Wrangham., Behavioral Ecology and Sociobiology 61:1771-1779, 2007) were more common at Ngogo than Kanyawara, consistent with an earlier report that fruit availability varies less at Ngogo [Chapman et al., African Journal of Ecology 35:287-302, 1997]. F. mucuso is absent at Kanyawara; its high density at Ngogo, combined with lower variation in fruit availability, probably helps to explain why chimpanzee population density is much higher at Ngogo.     

How the Electronic Health Record Will Change the Future of Health Care

Genetic testing is expected to play a critical role in patient care in the near future. Advances in genomic research have the potential to impact medicine in very tangible and direct ways, from carrier screening to disease diagnosis and prognosis to targeted treatments and personalized medicine. However, numerous barriers to widespread adoption of genetic testing continue to exist, and health information technology will be a critical means of addressing these challenges. Electronic health records (EHRs) are a digital replacement for the traditional paper-based patient chart designed to improve the quality of patient care. EHRs have become increasingly essential to managing the wealth of existing clinical information that now includes genetic information extracted from the patient genome. The EHR is capable of changing health care in the future by transforming the way physicians use genomic information in the practice of medicine.     

Comparison of the cytotoxic response against ovarian cancer by immune effector cells isolated and expanded from normal donors and ovarian cancer patients

Background/AimsThe aim of this study was to compare the cytotoxic response against ovarian cancer (OC) cells elicited by different immune effector cells in combination with the cytokines interleukin (IL)-2 and interferon (IFN) α-2b.MethodsOC cells were co-cultured with peripheral blood mononuclear cells (PBMC) from normal donors or OC patients and IL-2 or IFN α-2b alone or in combination, in order to determine the cytotoxicity. T cells were isolated from healthy donors to determine T cell cytotoxic activity. PBMC from healthy donors and OC patients were expanded in an IL-2/IL-7/IL-12 cocktail with and without anti-CD3 antibody, and the cytotoxic activity measured. Flow cytometry was performed on primary, selected and expanded cells to determine T, B, and natural killer- (NK) cell percentages.ResultsHealthy donor PBMC elicited a significant cytotoxic response (59%) compared with OC patient PBMC (7%). T cells enriched from normal donors elicited a significant cytotoxic response (18%) compared with controls lacking effector cells (1.4%); however, the cytotoxicity observed was significantly less compared with unselected PBMC. Expanded effector cells consisted primarily of T cells (98%) and the fold-expansion was significantly higher in the presence of anti-CD3 (19- versus 132-fold). No significant difference in the expansion (either fold-expansion or cell type) was observed between OC patients and healthy donors. Expanded cells from both healthy donors and OC patients elicited a significant cytotoxic response in the presence of IL-2 (19% and 22%) compared with controls.ConclusionsPBMC from OC patients do not elicit a significant cytotoxic response; however, ex vivo-expanded cells from OC patients are capable of cytotoxic killing similar to unexpanded T cells isolated from normal donors. These data provide the groundwork for further development of cellular therapy against OC.     

Association of monosodium glutamate intake with overweight in Chinese adults: the INTERMAP Study

Animal studies indicate that monosodium glutamate (MSG) can induce hypothalamic lesions and leptin resistance, possibly influencing energy balance, leading to overweight. This study examines the association between MSG intake and overweight in humans. We conducted a cross-sectional study involving 752 healthy Chinese (48.7% women), aged 40-59 years, randomly sampled from three rural villages in north and south China. The great majority of participants prepared their foods at home, without use of commercially processed foods. Diet was assessed with four in-depth multipass 24-h recalls. Participants were asked to demonstrate MSG amounts added in food preparation. Amounts shaken out were weighed by trained interviewers. Overweight was defined as BMI > or =25.0 or > or =23.0 kg/m(2)(based on World Health Organization recommendations for Asian populations). Eighty-two percent of participants were MSG users. Average intake was 0.33 g/day (s.d. = 0.40). With adjustment for potential confounders including physical activity and total energy intake, MSG intake was positively related to BMI. Prevalence of overweight was significantly higher in MSG users than nonusers. For users in the highest tertile of MSG intake compared to nonusers, the multivariable-adjusted odds ratios of overweight (BMI > or =23.0 and > or =25.0) were 2.10 (95% confidence interval, 1.13-3.90, P for trend across four MSG categories = 0.03) and 2.75 (95% confidence interval, 1.28-5.95, P = 0.04). This research provides data that MSG intake may be associated with increased risk of overweight independent of physical activity and total energy intake in humans.     

Human Thromboxane A2 Receptor Genetic Variants: In Silico,In Vitro and “In Platelet” Analysis

Thromboxane and its receptor have emerged as key players in modulating vascular thrombotic events. Thus, a dysfunctional hTP genetic variant may protect against (hypoactivity) or promote (hyperactivity) vascular events, based upon its activity on platelets. After extensive in silico analysis, six hTP-α variants were selected (C⁶⁸S, V⁸⁰E, E⁹⁴V, A¹⁶⁰T, V¹⁷⁶E, and V²¹⁷I) for detailed biochemical studies based on structural proximity to key regions involved in receptor function and in silico predictions. Variant biochemical profiles ranged from severe instability (C⁶⁸S) to normal (V²¹⁷I), with most variants demonstrating functional alteration in binding, expression or activation (V⁸⁰E, E⁹⁴V, A¹⁶⁰T, and V¹⁷⁶E). In the absence of patient platelet samples, we developed and validated a novel megakaryocyte based system to evaluate human platelet function in the presence of detected dysfunctional genetic variants. Interestingly, variant V80E exhibited reduced platelet activation whereas A160T demonstrated platelet hyperactivity. This report provides the most comprehensive in silico, in vitro and “in platelet” evaluation of hTP variants to date and highlightscurrent inherent problems in evaluating genetic variants, with possible solutions. The study additionally provides clinical relevance to characterized dysfunctional hTP variants.     

Forest succession in Kibale National Park, Uganda: implications for forest restoration and management

Forest succession was studied in four plots in former grasslands at the Ngogo study area in Kibale National Park, Uganda. The plots were located in areas that had been protected from fire for 0.58, 25, 9 and ≈30 years for plots 1, 2, 3 and 4, respectively. Species richness reflected the length of time that the plot had been protected from fire; it was highest in plot 4 and lowest in plot 1. Species density, stem density and basal area were all highest in plot 4 and lowest in plot 1. The species densities of plots 2 and 3 were not different. Similarly, plots 2 and 4 did not differ with regard to stem density or basal area. Animal seed dispersers played a vital role in the colonization of grasslands by forest tree species.