Circulating cell-free endometrial DNA level is unaltered during menstruation and in endometriosis.

STUDY QUESTION: Is circulating cell-free DNA (cirDNA) from the endometrium elevated during menstruation and in endometriosis? SUMMARY ANSWER: Endometrial cirDNA does not increase during menstruation and is not elevated in endometriosis. WHAT IS KNOWN ALREADY: Changes in cirDNA associated with common benign conditions are a potential source of false positives in cancer diagnostic applications, but also present an opportunity for biomarker development for diseases such as endometriosis. Elevated cirDNA has been reported in endometriosis patients compared to healthy community controls, but no difference in total or endometrial cirDNA has been found between patients with endometriosis and patients with other gynaecological conditions. Likewise, menstruation is a potential driver of changes in cirDNA levels and tissue profile, but total and endothelial cirDNA do not increase during menstruation. STUDY DESIGN, SIZE, DURATION: For endometriosis comparisons, 59 participants with surgically confirmed endometriosis and 27 laparoscopic patients without endometriosis (hospital controls) were prospectively recruited, while 25 healthy community participants (healthy controls) were recruited in a university setting. Total and endometrial cirDNA and cirDNA fragmentation were measured across the three groups. For menstrual comparisons, 36 matched non-menstruating and menstruating samples were collected from healthy women recruited within a university setting, and the endometrial cirDNA was compared between the two groups. PARTICIPANTS/MATERIALS, SETTING, METHODS: cirDNA was extracted from venous blood plasma then quantitated by quantitative PCR of ALU repetitive element (115 bp) and TP53 gene sequence (105 bp) for total concentration. cirDNA derived from the endometrium was quantitated by methylation-specific droplet digital PCR of a FAM101A region (69 bp) after bisulfite conversion of the DNA. A cirDNA size fragmentation ratio was obtained by quantifying a long segment of ALU repetitive element (247 bp) and expressing the amount relative to the 115 bp ALU target. MAIN RESULTS AND THE ROLE OF CHANCE: No differences in cirDNA level were found in any comparison populations in this study. Mean total cirDNA was unchanged between healthy controls (ALU-115-3.31 ng/ml; TP53-2.73 ng/ml), hospital controls (ALU-115-3.47 ng/ml; TP53-2.83 ng/ml) and endometriosis patients (ALU-115-3.35 ng/ml; TP53-2.66 ng/ml). Likewise, endometrial cirDNA was unchanged between healthy controls (18.3 copies/ml), hospital controls (20.6 copies/ml) and endometriosis patients (22 copies/ml). Endometrial cirDNA did not change during menstruation (non-menstruating: 38 copies/ml; menstruating: 33 copies/ml). Irrespective of endometriosis diagnosis, blood from patients undergoing laparoscopy (hospital controls: 0.77; endometriosis patients: 0.79), had a significantly higher cirDNA size ratio than community-recruited healthy controls (0.64), indicating increased abundance of long cirDNA fragments. LIMITATIONS, REASONS FOR CAUTION: It was not possible to completely match the age, BMI and parity between the three cohorts investigated, however of these, only age has been shown to influence circulating DNA levels and not within the age range of our cohort. Blood from community-recruited healthy women and women undergoing laparoscopy was collected via antecubital vein venepuncture (processed within 3 h) and with either peripheral cannula or venepuncture (processed within 6 h), respectively, which could potentially impact the size distribution of circulating DNA fragments. For the collection of non-menstruating phase blood samples, we did not differentiate between follicular phase, ovulation and luteal phase. Thus, only the mensturating samples were collected at a consistent phase, and any fluctuations in cirDNA that occur at the other phases may have obscured small changes during menstruation. WIDER IMPLICATIONS OF THE FINDINGS: There is no evidence that cirDNA has potential as a diagnostic biomarker for endometriosis. Endometriosis, representing a common benign gynaecological condition, and menstruation, representing a normal physiological occurrence in women, should not affect methylation-based diagnostics in other disease areas, including oncology. STUDY FUNDING/COMPETING INTEREST(S): N.L.Y.: Australian Government Research Training Program (RTP) Stipend through The University of New South Wales, Translational Cancer Research Network PhD Scholarship Top-Up Award via the Cancer Institute NSW, Beth Yarrow Memorial Award in Medical Science, UNSW Completion Scholarship; C.E.H.: Gynaecological Oncology Fund of the Royal Hospital for Women; K.W.: Ovarian Cancer Research Foundation and CAMILLA AND MARC. C.E.F.: UNSW Women's Wellbeing Academy and the Australian Human Rights Institute. We declare the following competing interest: K.W. holds stock in Guardant Health, Exact Sciences and Epigenomics AG. No other authors have competing interests. TRIAL REGISTRATION NUMBER: N/A.

ROR1 and ROR2 play distinct and opposing roles in endometrial cancer.

OBJECTIVE: In recent years, the Wnt signalling pathway and the ROR1 and ROR2 receptors have been implicated in a range of gynecological cancers. These receptors have been described as prospective therapeutic targets, and this study investigated such potential in an endometrial cancer context. METHOD: Immunohistochemistry for ROR1 and ROR2 was performed in a patient cohort, and expression was correlated with clinicopathological parameters including type, stage, grade, myometrial invasion, lymphovascular involvement, patient age and survival. The functional role of these receptors in endometrial cancer was investigated via siRNA knockdown of ROR1 and ROR2 in three cell line models (KLE, RL95-2 and MFE-319). Effects on proliferation, adhesion, migration and invasion were measured. RESULTS: High ROR1 expression in patient samples correlated with worse overall survival (p = 0.0169) while high ROR2 expression correlated with better overall survival (p = 0.06). ROR1 knockdown in KLE cells significantly decreased proliferation (p = 0.047) and reduced migration and invasion. ROR2 knockdown in RL95-2 cells increased cell migration and invasion (p = 0.011). Double ROR1 and ROR2 knockdown in MFE-319 cells decreased adhesion and significantly increased cell migration (P = 0.008) and invasion (p < 0.001). CONCLUSION: ROR1 and ROR2 play distinct roles in endometrial cancer. ROR1 may promote tumor progression, similar to its role in ovarian cancer, while ROR2 may act as a tumor suppressor in endometrioid endometrial cancer, similar to its role in colorectal cancer. With several ROR-targeting therapies currently in development and phase I clinical trials for other tumor types, this study supports the potential of these receptors as therapeutic targets for women with endometrial cancer.

Wnt signalling in gynaecological cancers: A future target for personalised medicine?

The three major gynaecological cancers, ovarian, uterine and cervical, contribute a significant burden to global cancer mortality, and affect women in both developed and developing countries. However, unlike other cancer types that have seen rapid advances and incorporation of targeted treatments in recent years, personalised medicine is not yet a reality in the treatment of gynaecological cancers. Advances in sequencing technology and international collaborations and initiatives such as The Cancer Genome Atlas are now revealing the molecular basis of these cancers, and highlighting key signalling pathways involved. One pathway which plays a role in all three cancer types, is the Wnt signalling pathway. This complex developmental pathway is altered in most human malignancies, and members of this pathway, particularly the recently linked ROR receptor tyrosine kinases may be attractive future therapeutic targets. This review provides an up-to-date summary of research into Wnt signalling and ovarian, uterine and cervical cancers, and discusses the potential of the Wnt pathway as a future target for personalised medicine in gynaecological cancers.

The non-canonical Wnt ligand, Wnt5a, is upregulated and associated with epithelial to mesenchymal transition in epithelial ovarian cancer.

OBJECTIVE: Aberrant Wnt signalling has previously been associated with gynaecological cancers, and the aim of this study was to investigate the expression of Wnt5a in epithelial ovarian cancer, and clarify its role in activating or inhibiting ß-catenin dependent and independent Wnt signalling pathways. METHOD: Wnt5a expression was investigated in a large cohort of epithelial ovarian cancer patient samples using immunohistochemistry and correlated with clinicopathological variables. Wnt5a function was investigated in vitro in ovarian cell lines. RESULTS: Wnt5a expression was found to be upregulated in all major subtypes (serous, endometrioid, clear cell and mucinous) of epithelial ovarian cancer compared to borderline tumours and benign controls. Treatment of ovarian surface epithelial cells with recombinant Wnt5a decreased cell adhesion and was associated with increased epithelial to mesenchymal transition (EMT). In addition, downstream targets of ß-catenin dependent Wnt signalling were inhibited, and ß-catenin independent targets increased following Wnt5a upregulation. Knockdown of Wnt5a in ovarian cancer cells was associated with a mesenchymal to epithelial transition (MET), but had no significant effect on cell migration or proliferation. CONCLUSION: This study adds to the increasing evidence that Wnt signalling may play an important role in ovarian cancer development. Utilising an unparalleled large cohort of 623 patients, Wnt5a protein expression was shown to be significantly higher in ovarian cancer patients when compared to benign and borderline ovarian tumours and healthy control patients. In addition, we have utilised in vitro models to show for the first time in ovarian cancer that Wnt5a driven non-canonical pathways can alter epithelial to mesenchymal transition (EMT).

RYR3 gene polymorphisms and cardiovascular disease outcomes in the context of antihypertensive treatment.

Nearly one-third of adults in the United States have hypertension, which is associated with increased cardiovascular disease (CVD) morbidity and mortality. The goal of antihypertensive pharmacogenetic research is to enhance understanding of drug response based on the interaction of individual genetic architecture and antihypertensive therapy to improve blood pressure control and ultimately prevent CVD outcomes. In the context of the Genetics of Hypertension Associated Treatment study and using a case-only design, we examined whether single-nucleotide polymorphisms in RYR3 interact with four classes of antihypertensive drugs, particularly the calcium channel blocker amlodipine versus other classes, to modify the risk of coronary heart disease (CHD; fatal CHD and non-fatal myocardial infarction combined) and heart failure (HF) in high-risk hypertensive individuals. RYR3 mediates the mobilization of stored Ca(+2) in cardiac and skeletal muscle to initiate muscle contraction. There was suggestive evidence of pharmacogenetic effects on HF, the strongest of which was for rs877087, with the smallest P-value=0.0005 for the codominant model when comparing amlodipine versus all other treatments. There were no pharmacogenetic effects observed for CHD. The findings reported here for the case-only analysis of the antihypertensive pharmacogenetic effect of RYR3 among 3058 CHD cases and 1940 HF cases show that a hypertensive patient's genetic profile may help predict which medication(s) might better lower CVD risk.

Ngnochannel array glass.

The fabrication and characterization of a glass containing a regular parallel array of submicrometer channels or capillaries are described. The capillaries are arranged in a two-dimensional hexagonal close packing configuration with channel diameters as small as 33 nanometers and packing densities as high as 3 x 10(10) channels per square centimeter. The high-temperature stability of the nanochannel glass array is well suited as a host or template for the formation of quantum confined semiconductor structures or as a mask for massively parallel patterned lithographic applications.

Molecular basis for interactions of G protein betagamma subunits with effectors.

Both the alpha and betagamma subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) communicate signals from receptors to effectors. Gbetagamma subunits can regulate a diverse array of effectors, including ion channels and enzymes. Galpha subunits bound to guanine diphosphate (Galpha-GDP) inhibit signal transduction through Gbetagamma subunits, suggesting a common interface on Gbetagamma subunits for Galpha binding and effector interaction. The molecular basis for interaction of Gbetagamma with effectors was characterized by mutational analysis of Gbeta residues that make contact with Galpha-GDP. Analysis of the ability of these mutants to regulate the activity of calcium and potassium channels, adenylyl cyclase 2, phospholipase C-beta2, and beta-adrenergic receptor kinase revealed the Gbeta residues required for activation of each effector and provides evidence for partially overlapping domains on Gbeta for regulation of these effectors. This organization of interaction regions on Gbeta for different effectors and Galpha explains why subunit dissociation is crucial for signal transmission through Gbetagamma subunits.

Distinct Patterns of Stromal and Tumor Expression of ROR1 and ROR2 in Histological Subtypes of Epithelial Ovarian Cancer.

OBJECTIVE: The ROR1 and ROR2 receptor tyrosine kinases have both been implicated in ovarian cancer progression and have been shown to drive migration and invasion. There is an increasing importance of the role of stroma in ovarian cancer metastasis; however, neither ROR1 nor ROR2 expression in tumor or stromal cells has been analyzed in the same clinical cohort. AIM: To determine ROR1 and ROR2 expression in ovarian cancer and surrounding microenvironment and examine associations with clinicopathological characteristics. METHODS: Immunohistochemistry for ROR1 and ROR2 was used to assess receptor expression in a cohort of epithelial ovarian cancer patients (n=178). Results were analyzed in relation to clinical and histopathological characteristics and survival. Matched patient sample case studies of normal, primary, and metastatic lesions were used to examine ROR expression in relation to ovarian cancer progression. RESULTS: ROR1 and ROR2 are abnormally expressed in malignant ovarian epithelium and stroma. Higher ROR2 tumor expression was found in early-stage, low-grade endometrioid carcinomas. ROR2 stromal expression was highest in the serous subtype. In matched patient case studies, metastatic samples had higher expression of ROR2 in the stroma, and a recurrent sample had the highest expression of ROR2 in both tumor and stroma. CONCLUSION: ROR1 and ROR2 are expressed in tumor-associated stroma in all histological subtypes of ovarian cancer and hold potential as therapeutic targets which may disrupt tumor and stroma interactions.

Presence of mouse mammary tumour-like virus gene sequences may be associated with morphology of specific human breast cancer.

BACKGROUND: Mouse mammary tumour virus (MMTV) has a proven role in breast carcinogenesis in wild mice and genetically susceptible in-bred mice. MMTV-like env gene sequences, which indicate the presence of a replication-competent MMTV-like virus, have been identified in some human breast cancers, but rarely in normal breast tissues. However, no evidence for a causal role of an MMTV-like virus in human breast cancer has emerged, although there are precedents for associations between specific histological characteristics of human cancers and the presence of oncogenic viruses. AIM: To investigate the possibility of an association between breast cancer and MMTV-like viruses. METHODS: Histological characteristics of invasive ductal human breast cancer specimens were compared with archival MMTV-associated mammary tumours from C3H experimental mice. The presence of MMTV-like env DNA sequences in the human breast cancer specimens was determined by polymerase chain reaction and confirmed by Southern hybridisation. RESULTS: MMTV-like env gene sequences were identified in 22 of 59 (37.3%) human breast cancer specimens. Seventeen of 43 (39.5%) invasive ductal carcinoma breast cancer specimens and 4 of 16 (25%) ductal carcinoma in situ specimens had some histological characteristics, which were similar to MMTV-associated mouse mammary tumours. However, these similarities were not associated with the presence or absence of MMTV-like gene sequences in the human breast tumour specimens. A significant (p = 0.05) correlation was found between the grade of the human breast cancer and similarity to the mouse mammary tumours. The lower the grade, the greater the similarity. CONCLUSION: Some human breast cancer specimens, in which MMTV-like env DNA sequences have been identified, were shown to have histological characteristics (morphology) similar to MMTV-associated mouse mammary tumours. These observations are compatible with, but not conclusive of, an association between the presence of MMTV-like env DNA sequences and some human breast cancers.

Effects of oral androstenedione on phospholipid fatty acids, ATP, caspase-3, prostaglandin E(2) and C-reactive protein in serum and livers of pregnant and non-pregnant female rats.

Androstenedione, a steroidal dietary supplement taken to enhance athletic performance, could affect serum and liver lipid metabolism, induce liver toxicity or alter inflammatory response depending on dose and duration of exposure. Pregnancy could further exaggerate these effects. To examine this, mature female rats were gavaged with 0, 5, 30 or 60 mg/kg/day androstenedione beginning two weeks prior to mating and continuing through gestation day 19. Non-pregnant female rats were gavaged over the same time frame with 0 or 60 mg/kg/day androstenedione. Serum was collected and livers were removed from dams on gestation day 20 and from non-pregnant rats after 5 weeks of treatment. Androstenedione had no effect on serum total cholesterol, triglycerides or HDL-cholesterol, but significantly decreased C-reactive protein in pregnant rats and prostaglandin E(2) in serum of both pregnant and non-pregnant rats. There were treatment related decreases in liver ATP and, to a lesser degree, caspase-3 and no change in alkaline phosphatase of pregnant female rats. Androstenedione decreased docosahexaenoic acid in both serum and liver phospholipids of pregnant female rats. In conclusion, oral androstenedione did not result in overt hepatotoxicity in pregnant female rats, but produced modest changes in lipid metabolism and may impair regeneration of injured hepatic cells or tissue.

Migration and invasion is inhibited by silencing ROR1 and ROR2 in chemoresistant ovarian cancer.

Ovarian cancer survival remains poor despite recent advances in our understanding of genetic profiles. Unfortunately, the majority of ovarian cancer patients have recurrent disease after chemotherapy and lack other treatment options. Wnt signalling has been extensively implicated in cancer progression and chemoresistance. Therefore, we investigated the previously described Wnt receptors ROR1 and ROR2 as regulators of epithelial-to-mesenchymal transition (EMT) in a clinically relevant cell line model. The parental A2780- and cisplatin-resistant A2780-cis cell lines were used as a model of ovarian cancer chemoresistance. Proliferation, adhesion, migration and invasion were measured after transient overexpression of ROR1 and ROR2 in the parental A2780 cell line, and silencing of ROR1 and ROR2 in the A2780-cis cell line. Here we show that ROR1 and ROR2 expression is increased in A2780-cis cells, alongside ß-catenin-independent Wnt targets. Knockdown of ROR1 and ROR2 significantly inhibited cell migration and invasion and simultaneous knockdown of ROR1 and ROR2 significantly sensitised cells to cisplatin, whilereas ROR overexpression in the parental cell line increased cell invasion. Therefore, ROR1 and ROR2 have the potential as novel drug targets in metastatic and recurrent ovarian cancer patients.

The effects of genes implicated in cardiovascular disease on blood pressure response to treatment among treatment-naive hypertensive African Americans in the GenHAT study.

African Americans have the highest prevalence of hypertension in the United States. Blood pressure (BP) control is important to reduce cardiovascular disease-related morbidity and mortality in this ethnic group. Genetic variants have been found to be associated with BP response to treatment. Previous pharmacogenetic studies of BP response to treatment in African Americans suffer limitations of small sample size as well as a limited number of candidate genes, and often focused on one antihypertensive treatment. Using 1131 African-American treatment-naive participants from the Genetics of Hypertension Associated Treatment Study, we examined whether variants in 35 candidate genes might modulate BP response to four different antihypertensive medications, including an angiotensin-converting enzyme inhibitor (lisinopril), a calcium channel blocker (amlodipine), and an a-adrenergic blocker (doxazosin) as compared with a thiazide diuretic (chlorthalidone) after 6 months of follow-up. Several suggestive gene by treatment interactions were identified. For example, among participants with two minor alleles of renin rs6681776, diastolic BP response was much improved on doxazosin compared with chlorthalidone (on average -9.49 mm Hg vs -1.70 mm Hg) (P=0.007). Although several suggestive loci were identified, none of the findings passed significance criteria after correction for multiple testing. Given the impact of hypertension and its sequelae in this population, this research highlights the potential for genetic factors to contribute to BP response to treatment. Continued concerted research efforts focused on genetics are needed to improve treatment response in this high-risk group.

Expression of the novel Wnt receptor ROR2 is increased in breast cancer and may regulate both ß-catenin dependent and independent Wnt signalling.

PURPOSE: Wnt signalling has been implicated in breast cancer, and in particular aberrant ß-catenin-independent Wnt signalling has been associated with breast cancer metastasis and Tamoxifen resistance. Despite Wnt pathway involvement in many human cancers, attempts to target the pathway therapeutically have been disappointing. The recent discovery that the receptor tyrosine kinase-like orphan receptor 2 (ROR2) is a novel Wnt receptor provides a potential new therapeutic and diagnostic target. METHODS: To clarify the role of ROR2 in breast cancer, we investigated its expression via ROR2 immunohistochemistry in a clinical cohort of breast cancer patients, and via in vitro studies incorporating both overexpression and knock-down of ROR2. RESULTS: ROR2 was expressed in the majority of breast cancer patients (87%), including those classed as triple negative. Breast cancer patients expressing ROR2 had a significantly shorter overall survival than those lacking ROR2 expression (P < 0.05). Overexpression of ROR2 in the mammary epithelial cell line, MCF10A, increased both ß-catenin-dependent and ß-catenin-independent targets and decreased cell adhesion. Knock-down of ROR2 in the breast cancer cell lines, MDA-MB-453 and HCC1143, decreased both ß-catenin-dependent and ß-catenin-independent targets and increased cell adhesion. Treatment of ROR2-expressing breast cancer cells with the novel berberine derivative, NAX53, significantly inhibited cell proliferation and migration. CONCLUSIONS: This is the first study to report the expression of ROR2 in breast cancer. Breast cancer patients expressing ROR2 had a significantly worse prognosis than those lacking ROR2. ROR2 may regulate both ß-catenin-dependent and ß-catenin-independent Wnt signalling pathways, and represents a potential diagnostic and therapeutic target.

Why do lean hypertensives have higher mortality rates than other hypertensives? Findings of the Hypertension Detection and Follow-up Program.

Specific causes of death were analyzed for 10,908 participants in the Hypertension Detection and Follow-up Program, to explore possible explanations for the observed excess 8.3-year mortality from all causes in hypertensives with low body mass. Although the cardiovascular mortality rate among men in the lowest decile of body mass (body mass index 21.96 or less) was 50% higher than that of men in the median class (body mass index 26.4-28.8), death rate for noncardiovascular deaths was more than 2 1/2 times higher in men with lean versus median body mass. The pattern was similar among women. Among noncardiovascular causes, striking differences in mortality rates between lean hypertensives and those of average body mass were observed for cirrhotic death (relative risk of 12+ in men and 11+ in women), for nonmalignant respiratory disease in men (relative risk of 7+), for violent death (both sexes), and for malignant neoplasms in men. Prevalence of smoking was almost twice as high in the lowest compared with the median body mass group; among the lean, excess deaths, particularly noncardiovascular deaths, were concentrated among smokers. Thus, male smokers in the lowest decile of body mass constituted only 3% of the study population, but contributed 8% of all deaths, 11% of all noncardiovascular deaths, and 22% of all cirrhotic deaths. A larger proportion of deaths occurred early in follow-up in the lean versus other hypertensives, suggesting occult disease among the lean at baseline. There was no evidence that more severe or treatment-resistant hypertension was present in or could explain excess mortality among the hypertensives with low body mass. The inference from the findings is not that overweight is protective for hypertensives nor that excess risk is due to leanness per se. Rather, a reasonable hypothesis, particularly from findings on specific causes of death, is that excess mortality in lean hypertensives is due to deleterious lifestyles, particularly smoking and excess alcohol intake, contributing to both leanness and risk of death.

Prognostic value of serum creatinine and effect of treatment of hypertension on renal function. Results from the hypertension detection and follow-up program. The Hypertension Detection and Follow-up Program Cooperative Group.

The Hypertension Detection and Follow-up Program followed up 10,940 persons for 5 years in a community-based, randomized, controlled trial of treatment for hypertension. Participants were randomized to one of two treatment groups, stepped care and referred care. The primary end point of the study was all-cause mortality, with morbid events involving the heart, brain, and kidney as secondary end points. Loss of renal function, ascertained by a change in serum creatinine, was among these secondary events. Baseline serum creatinine concentration had a significant prognostic value for 8-year mortality. For persons with a serum creatinine concentration greater than or equal to 1.7 mg/dl, 8-year mortality was more than three times that of all other participants. The estimated 5-year incidence of substantial decline in renal function was 21.7/1,000 in the stepped-care group and 24.6/1,000 in the referred-care group. Among persons with a baseline serum creatinine level between 1.5 and 1.7 mg/dl, the 5-year incidence of decline was 113.3/1,000 (stepped care) and 226.6/1,000 (referred care) (p less than 0.01). The incidence of decline in renal function was greater in men, blacks, and older adults, as well as in those with higher entry diastolic blood pressure. Among persons with a baseline serum creatinine level greater than or equal to 1.7 mg/dl, serum creatinine concentration declined by 25% or more in 28.6% of stepped-care and 25.2% of referred-care participants. Although the incidence of clinically significant hypercreatininemia in a hypertensive population is low, an elevated serum creatinine concentration is a very potent independent risk factor for mortality. The slightly lower rate of development of hypercreatininemia and the higher rate of improvement in stepped-care compared with referred-care participants is consistent with the belief that aggressive treatment of hypertension may reduce renal damage and the associated increased risk of death.

Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 3. Structure-activity studies of ketomethylene-containing peptidomimetics.

The structure-based design, chemical synthesis, and biological evaluation of various ketomethylene-containing human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds are comprised of a peptidomimetic binding determinant and an ethyl propenoate Michael acceptor moiety which forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. The ketomethylene-containing inhibitors typically display slightly reduced 3CP inhibition activity relative to the corresponding peptide-derived molecules, but they also exhibit significantly improved antiviral properties. Optimization of the ketomethylene-containing compounds is shown to provide several highly active 3C protease inhibitors which function as potent antirhinoviral agents (EC90 = <1 microM) against multiple virus serotypes in cell culture.

Design, synthesis, and evaluation of nonpeptidic inhibitors of human rhinovirus 3C protease.

The design, synthesis, and biological evaluation of reversible, nonpeptidic inhibitors of human rhinovirus (HRV) 3C protease (3CP) are reported. A novel series of 2,3-dioxindoles (isatins) were designed that utilized a combination of protein structure-based drug design, molecular modeling, and structure-activity relationship (SAR). The C-2 carbonyl of isatin was envisioned to react in the active site of HRV 3CP with the cysteine responsible for catalytic proteolysis, thus forming a stabilized transition state mimic. Molecular-modeling experiments using the apo crystal structure of human rhinovirus-serotype 14 (HRV-14) 3CP and a peptide substrate model allowed us to design recognition features into the P1 and P2 subsites, respectively, from the 5- and 1-positions of isatin. Attempts to optimize recognition properties in the P1 subsite using SAR at the 5-position were performed. In addition, a series of ab initio calculations were carried out on several 5-substituted isatins to investigate the stability of sulfide adducts at C-3. The inhibitors were prepared by general synthetic methods, starting with commercially available 5-substituted isatins in nearly every case. All compounds were tested for inhibition of purified HRV-14 3CP. Compounds 8, 14, and 19 were found to have excellent selectivity for HRV-14 3CP compared to other proteolytic enzymes, including chymotrypsin and cathepsin B. Selected compounds were assayed for antiviral activity against HRV-14-infected HI-HeLa cells. A 2.8 A cocrystal structure of derivative 19 covalently bound to human rhinovirus-serotype 2 (HRV-2) 3CP was solved and revealed that the isatin was situated in essentially the same conformation as modeled.

Substituted benzamide inhibitors of human rhinovirus 3C protease: structure-based design, synthesis, and biological evaluation.

A series of nonpeptide benzamide-containing inhibitors of human rhinovirus (HRV) 3C protease was identified using structure-based design. The design, synthesis, and biological evaluation of these inhibitors are reported. A Michael acceptor was combined with a benzamide core mimicking the P1 recognition element of the natural 3CP substrate. alpha,beta-Unsaturated cinnamate esters irreversibly inhibited the 3CP and displayed antiviral activity (EC(50) 0.60 microM, HRV-16 infected H1-HeLa cells). On the basis of cocrystal structure information, a library of substituted benzamide derivatives was prepared using parallel synthesis on solid support. A 1.9 A cocrystal structure of a benzamide inhibitor in complex with the 3CP revealed a binding mode similar to that initially modeled wherein covalent attachment of the nucleophilic cysteine residue is observed. Unsaturated ketones displayed potent reversible inhibition but were inactive in the cellular antiviral assay and were found to react with nucleophilic thiols such as DTT.

Tripeptide aldehyde inhibitors of human rhinovirus 3C protease: design, synthesis, biological evaluation, and cocrystal structure solution of P1 glutamine isosteric replacements.

The investigation of tripeptide aldehydes as reversible covalent inhibitors of human rhinovirus (HRV) 3C protease (3CP) is reported. Molecular models based on the apo crystal structure of HRV-14 3CP and other trypsin-like serine proteases were constructed to approximate the binding of peptide substrates, generate transition state models of P1-P1' amide cleavage, and propose novel tripeptide aldehydes. Glutaminal derivatives have limitations since they exist predominantly in the cyclic hemiaminal form. Therefore, several isosteric replacements for the P1 carboxamide side chain were designed and incorporated into the tripeptide aldehydes. These compounds were found to be potent inhibitors of purified HRV-14 3CP with Kis ranging from 0.005 to 0.64 microM. Several have low micromolar antiviral activity when tested against HRV-14-infected H1-HeLa cells. The N-acetyl derivative 3 was also shown to be active against HRV serotypes 2, 16, and 89. High-resolution cocrystal structures of HRV-2 3CP, covalently bound to compounds 3, 15, and 16, were solved. These cocrystal structures were analyzed and compared with our original HRV-14 3CP-substrate and inhibitor models.