A first-in-class CDK4 inhibitor demonstrates in vitro, ex-vivo and in vivo efficacy against ovarian cancer.

INTRODUCTION: Cyclin-dependent kinases 4 and 6 (CDK4/6) are fundamental drivers of the cell cycle and are involved in the initiation and progression of various cancers. Deregulation of the CDK4/6-cyclin D-retinoblastoma (Rb) pathway is common in ovarian cancer and is associated with an aggressive phenotype and poor prognosis. Patients with advanced ovarian cancer whose tumor demonstrates Rb-positivity, a low expression of p16 and overexpression of cyclin D1 are most likely to benefit from CDK4/6 inhibition. MATERIALS AND METHOD: Anti-proliferative activity and mechanistic investigations for CDDD2-94, employing palbociclib as comparator, were evaluated by MTT assay, cell cycle and apoptosis analysis, western blotting as well as senescence and colony formation assay. In vivo safety and efficacy studies were done in A2780 tumor-bearing nude mice. Combinations of CDDD2-94 with mTOR, MEK, PI3K or PARP inhibitors were evaluated in A2780 and OVCAR5 ovarian cancer cells. RESULTS: Consistent with a CDK4-targeted mechanism, CDDD2-94 arrested the G1/G0 cell cycle, induced senescence and inhibited the proliferation of Rb-proficient ovarian cancer cells. CDDD2-94 exhibited synergistic anti-proliferative activities with mTOR, MEK, PI3K or PARP inhibitors. Importantly, unlike palbociclib which caused significant reductions in the number of lymphocytes and neutrophils, CDDD2-94 had little effect. CDDD2-94, as single agent and in combination with everolimus, delayed tumor growth and significantly increased survival of mice. CONCLUSION: Given its high specificity in targeting CDK4 and excellent anti-tumor efficacy with low toxicity, CDDD2-94 has potential to be developed as a standalone agent or in combination with targeted therapeutics for the treatment of ovarian cancer.

CDKI-73: an orally bioavailable and highly efficacious CDK9 inhibitor against acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common form of acute leukemia with dismal long-term prognosis with age. The most aggressive subtype of AML is MLL-AML that is characterized by translocations of the mixed-lineage leukemia gene (MLL) and resistance to conventional chemotherapy. Cyclin dependent kinase 9 (CDK9) plays a crucial role in the MLL-driven oncogenic transcription, and hence, inhibiting activity of CDK9 has been proposed as a promising strategy for treatment of AML. We investigated the therapeutic potential of CDKI-73, one of the most potent CDK9 inhibitors, against a panel of AML cell lines and samples derived from 97 patients. CDKI-73 induced cancer cells undergoing apoptosis through transcriptional downregulation of anti-apoptotic proteins Bcl-2, Mcl-1 and XIAP by majorly targeting CDK9. Contrastively, it was relatively low toxic to the bone marrow cells of healthy donors. In MV4-11 xenograft mouse models, oral administration of CDKI-73 resulted in a marked inhibition of tumor growth (p < 0.0001) and prolongation of animal life span (P < 0.001) without causing body weight loss and other overt toxicities. The study suggests that CDKI-73 can be developed as a highly efficacious and orally deliverable therapeutic agent for treatment of AML.

Bioanalysis and Stability of Polymyxins.

Clinical use of the polymyxin antibiotics began approximately 10 years after their discovery in the late 1940s. Their concentrations in biological fluids were measured using microbiological methods. These methods were reasonably accurate for measuring the active polymyxin base, such as polymyxin B and colistin (polymyxin E), but were used inappropriately for measuring the concentrations of "colistin" in humans or animals following the administration of colistimethate, also known as colistin methanesulphonate (CMS). The use of polymyxins for systemic infections waned in the 1970s because of their toxicity and the preference for other antibiotics, but their value for treating infections caused by several important Gram-negative pathogens becoming resistant to other antibiotics was realized in the mid-1990s. The lack of adequate pharmacokinetic and pharmacodynamic knowledge spurred the development of methods more specific for measuring polymyxin B and colistin after their administrations as sulphate salts, and of colistin and CMS after the administration of CMS sodium. These methods have been based on high-performance liquid chromatography, detection and quantification of fluorescent derivatives of the polymyxin bases, or of the bases themselves with detection and quantification by mass spectrometry.

Effect of Garlic, Gingko, and St. John's Wort Extracts on the Pharmacokinetics of Fexofenadine: A Mechanistic Study.

The aim of this study was to determine the effects of garlic and ginkgo herbal extracts on the pharmacokinetics of the P-glycoprotein (P-gp)/organic anion-transporting polypeptides (Oatps) substrate fexofenadine. Male rats were dosed orally with garlic (120 mg/kg), ginkgo (17 mg/kg), St. John's wort (SJW; 1000 mg/kg; positive control), or Milli-Q water for 14 days. On day 15, rats either were administered fexofenadine (orally or i.v.), had their livers isolated and perfused with fexofenadine, or had their small intestines divided into four segments (SI-SIV) and analyzed for P-gp and Oatp1a5. In vivo, SJW increased the clearance of i.v. administered fexofenadine by 28%. Garlic increased the area under the curve0-∞ and maximum plasma concentration of orally administered fexofenadine by 47% and 85%, respectively. Ginkgo and SJW had no effect on the oral absorption of fexofenadine. In the perfused liver, garlic, ginkgo, and SJW increased the biliary clearance of fexofenadine with respect to perfusate by 71%, 121%, and 234%, respectively. SJW increased the biliary clearance relative to the liver concentration by 64%. The ratio of liver to perfusate concentrations significantly increased in all treated groups. The expression of Oatp1a5 in SI was increased by garlic (88%) and SJW (63%). There were no significant changes in the expression of P-gp. Induction of intestinal Oatp1a5 by garlic may explain the increased absorption of orally administered fexofenadine. Ginkgo had no effect on the expression of intestinal P-gp or Oatp1a5. A dual inductive effect by SJW on opposing intestinal epithelial transport by Oatp1a5 and P-gp remains a possibility.

The association between total phthalate concentration and non-communicable diseases and chronic inflammation in South Australian urban dwelling men.

OBJECTIVE: To investigate associations between urinary total phthalate concentration, chronic low-grade inflammation and non-communicable diseases in a cohort of South Australian men. METHODS: 1504 men aged 39-84 years who provided a urinary sample at the follow-up visit of the Men Androgen Inflammation Lifestyle Environment and Stress (MAILES) study, a randomly-selected group of urban-dwelling, community-based men from Adelaide, Australia (n = 2038; study participation rate: 78.1%). Total phthalate concentration was quantified in fasting morning urine samples. Chronic diseases were assessed through self-report questionnaire or directly measured using standardised clinical and laboratory procedures. Inflammatory biomarkers were assayed by ELISA or spectroscopy. Multivariable linear and logistic regression models were applied to determine associations of log-transformed urinary phthalate concentration with inflammation and chronic disease. RESULTS: Total phthalates were detected in 99.6% of urinary samples; geometric mean (95% CI) was 114.1 (109.5-118.9)µg/g creatinine. Higher total phthalate levels were associated with higher levels of hs-CRP, IL-6 (all p < 0.05) and TNF-α but not MPO. Urinary total phthalate concentrations were positively associated with cardiovascular disease, type-2-diabetes and hypertension. Comparing extreme quartiles of total phthalate, prevalence ratios were 1.78 (95% CI 1.17 - 2.71, p-trend = 0.001) for cardiovascular disease and 1.84 (95%CI 1.34 - 2.51, p-trend = 0.001) for type-2-diabetes and 1.14 (95%CI 1.01 - 1.29, p-trend = 0.013) for hypertension. Total phthalates and asthma and depression were not significantly associated. CONCLUSION: A positive association between total phthalates and cardiovascular disease, type-2-diabetes, hypertension and increased levels of chronic low-grade inflammatory biomarkers was observed in urban-dwelling Australian men.

Comparison of CYP2D metabolism and hepatotoxicity of the myocardial metabolic agent perhexiline in Sprague-Dawley and Dark Agouti rats.

1. Perhexiline, a chiral anti-anginal agent, may be useful to develop new cardiovascular therapies, despite its potential hepatotoxicity. 2. This study compared Dark Agouti (DA) and Sprague-Dawley (SD) rats, as models of perhexiline's metabolism and hepatotoxicity in humans. Rats (n = 4/group) received vehicle or 200 mg/kg/d of racemic perhexiline maleate for 8 weeks. Plasma and liver samples were collected to determine concentrations of perhexiline and its metabolites, hepatic function and histology. 3. Median (range) plasma and liver perhexiline concentrations in SD rats were 0.09 (0.04-0.13) mg/L and 5.42 (0.92-8.22) ng/mg, respectively. In comparison, DA rats showed higher (p < 0.05) plasma 0.50 (0.16-1.13) mg/L and liver 24.5 (9.40-54.7) ng/mg perhexiline concentrations, respectively, 2.5- and 3.7-fold higher cis-OH-perhexiline concentrations, respectively (p < 0.05), and lower plasma metabolic ratio (0.89 versus 1.55, p < 0.05). In both strains, the (+):(-) enantiomer ratio was 2:1. Perhexiline increased plasma LDH concentrations in DA rats (p < 0.05), but had no effect on plasma biochemistry in SD rats. Liver histology revealed lower glycogen content in perhexiline-treated SD rats (p < 0.05), but no effects on lipid content in either strain. 4. DA rats appeared more similar to humans with respect to plasma perhexiline concentrations, metabolic ratio, enantioselective disposition and biochemical changes suggestive of perhexiline-induced toxicity.

Weather variability permitted within amphibian monitoring protocol and affects on calling Hylidae.

Anuran populations are sensitive to changing environmental conditions and act as useful indicators. Presently, much information collected concerning frog populations comes from volunteers following the North American Amphibian Monitoring Protocol. Does weather variability allowed within protocol affect the abundance of calling frogs? For 10 years, Credit Valley Conservation (Ontario, Canada) has been collecting anuran data concerning nine frog species employing three frog monitoring runs. Records include frog abundance by protocol code and five weather variables. Antecedent precipitation and temperature were determined from the nearest weather station. Locations with large source populations of two Hylidae species were selected (spring peeper calling in April and gray tree frog in May). Spearman correlations suggested there were no significant relationships between calling abundance of Hylidae species and ambient wind speed or humidity. However, gray tree frogs were temperature sensitive and calling was significantly related to increased water and air temperatures as well as day time high temperatures over the previous 2 weeks. Both species of calling Hylidae were affected by the volume and timing of precipitation (though, in different ways). Gray tree frogs seem to prefer drier conditions (when temperatures are significantly warmer) while spring peepers prefer to call during, or closely following, precipitation. Monitors targeting gray tree frog should track local weather conditions and focus on evenings when it is (a) warmer than the minimum temperatures and (b) drier than suggested by the protocol. It is recommended that an additional monitoring run could be added to reduce detection variability of this species.

Comparative analysis of recombinant Human Papillomavirus 8 L1 production in plants by a variety of expression systems and purification methods.

Human papillomavirus 8 (HPV-8), one of the high-risk cutaneous papillomaviruses (cHPVs), is associated with epidermodysplasia verruciformis and nonmelanoma skin cancer in immuno-compromised individuals. Currently, no vaccines against cHPVs have been reported; however, recent studies on cross-neutralizing properties of their capsid proteins (CP) have fostered an interest in vaccine production against these viruses. We examined the potential of producing HPV-8 major CP L1 in Nicotiana benthamiana by agroinfiltration of different transient expression vectors: (i) the binary vector pBIN19 with or without silencing suppressor constructs, (ii) the nonreplicating Cowpea mosaic virus-derived expression vector pEAQ-HT and (iii) a replicating Tobacco mosaic virus (TMV)-based vector alone or with signal peptides. Although HPV-8 L1 was successfully expressed using pEAQ-HT and TMV, a 15-fold increase was obtained with pEAQ-HT. In contrast, no L1 protein could be immune detected using pBIN19 irrespective of whether silencing suppressors were coexpressed, although such constructs were required for identifying L1-specific transcripts. A fourfold yield increase in L1 expression was obtained when 22 C-terminal amino acids were deleted (L1ΔC22), possibly eliminating a nuclear localization signal. Electron microscopy showed that plant-made HPV-8 L1 proteins assembled in appropriate virus-like particles (VLPs) of T = 1 or T = 7 symmetry. Ultrathin sections of L1ΔC22-expressing cells revealed their accumulation in the cytoplasm in the form of VLPs or paracrystalline arrays. These results show for the first time the production and localization of HPV-8 L1 protein in planta and its assembly into VLPs representing promising candidate for potential vaccine production.

Liposomal 5-Fluorouracil Polymer Complexes Facilitate Tumor-Specific Delivery: Pharmaco-Distribution Kinetics Using Microdialysis.

Liposomes are widely used as carriers for anticancer drugs due to their ability to prolong the retention of encapsulated drugs in blood plasma while directing their distribution increasingly into tumor tissue. We report on the development of stealth liposomal formulations for the common chemotherapy drug 5-fluorouracil, where pharmacokinetic studies were undertaken using a microdialysis probe to specifically quantify drug accumulation in tumor, which was contrasted to drug exposure to healthy tissue. Greater accumulation of the drug into the tumor than into healthy subcutaneous tissue was observed for neutral and cationic liposomal 5-fluorouracil polymer complexes in comparison to the conventional delivery by an injected solution. Increased drug accumulation in tumor also correlated to reduced tumor growth. This research has generated new mechanistic insight into liposomal-specific delivery to tumors with potential to improve the efficacy and reduce the toxicity of chemotherapy.

An Orally Bioavailable and Highly Efficacious Inhibitor of CDK9/FLT3 for the Treatment of Acute Myeloid Leukemia.

Mutations in FMS-like tyrosine kinase 3 (FLT3) occur in approximately one-third of AML patients and are associated with a particularly poor prognosis. The most common mutation, FLT3-ITD, is a self-activating internal tandem duplication (ITD) in the FLT3 juxtamembrane domain. Many FLT3 inhibitors have shown encouraging results in clinical trials, but the rapid emergence of resistance has severely limited sustainable efficacy. Co-targeting of CDK9 and FLT3 is a promising two-pronged strategy to overcome resistance as the former plays a role in the transcription of cancer cell-survival genes. Most prominently, MCL-1 is known to be associated with AML tumorigenesis and drug resistance and can be down-regulated by CDK9 inhibition. We have developed CDDD11-8 as a potent CDK9 inhibitor co-targeting FLT3-ITD with Ki values of 8 and 13 nM, respectively. The kinome selectivity has been confirmed when the compound was tested in a panel of 369 human kinases. CDDD11-8 displayed antiproliferative activity against leukemia cell lines, and particularly potent effects were observed against MV4-11 and MOLM-13 cells, which are known to harbor the FLT3-ITD mutation and mixed lineage leukemia (MLL) fusion proteins. The mode of action was consistent with inhibition of CDK9 and FLT3-ITD. Most importantly, CDDD11-8 caused a robust tumor growth inhibition by oral administration in animal xenografts. At 125 mg/kg, CDDD11-8 induced tumor regression, and this was translated to an improved survival of animals. The study demonstrates the potential of CDDD11-8 towards the future development of a novel AML treatment.

Using primary care prescribing databases for pharmacovigilance.

AIMS: In the UK, adverse drug reactions (ADRs) are responsible for over 6.5% of all hospital admissions, representing a significant morbidity and cost burden to the health service. We aimed to develop an ADR monitoring system capable of identifying the reasons for patient discontinuation of drug therapy within 6 months of the index prescription. METHODS: Patients first prescribed amlodipine between 1 March 2004 and 28 February 2007 who discontinued their amlodipine medication within 6 months of the index prescription were identified from the practice team information (PTI) database. Once identified, reasons for amlodipine discontinuation were assessed by an electronic database search using relevant Readcodes and key words and by a direct approach to the primary care medical records. RESULTS: The PTI database identified 995 patients [61.4% females, mean age 65.9 years (SD 12.4 years)] who discontinued amlodipine within 6 months of an index prescription. An electronic search of the database, using Readcodes, identified that 19.4% (193) of patients who discontinued their medication had an ADR recorded in the database. Six (20%) of 30 participating primary care practices, contributing to the PTI database, agreed to be approached directly and supply the reasons for discontinuation for the 51 patients identified as having discontinued amlodipine in their practices. Completed data were returned for all 51 patients, 98% of whom discontinued amlodipine due to an ADR or adverse drug event. CONCLUSIONS: The results of this study confirm that primary care prescribing databases can be easily used to identify the frequency and nature of ADRs occurring in an ADR-enriched population identified through medication discontinuation.

CDK9: A Comprehensive Review of Its Biology, and Its Role as a Potential Target for Anti-Cancer Agents.

Cyclin-dependent kinases (CDKs) are proteins pivotal to a wide range of cellular functions, most importantly cell division and transcription, and their dysregulations have been implicated as prominent drivers of tumorigenesis. Besides the well-established role of cell cycle CDKs in cancer, the involvement of transcriptional CDKs has been confirmed more recently. Most cancers overtly employ CDKs that serve as key regulators of transcription (e.g., CDK9) for a continuous production of short-lived gene products that maintain their survival. As such, dysregulation of the CDK9 pathway has been observed in various hematological and solid malignancies, making it a valuable anticancer target. This therapeutic potential has been utilized for the discovery of CDK9 inhibitors, some of which have entered human clinical trials. This review provides a comprehensive discussion on the structure and biology of CDK9, its role in solid and hematological cancers, and an updated review of the available inhibitors currently being investigated in preclinical and clinical settings.

Potent and orally bioavailable CDK8 inhibitors: Design, synthesis, structure-activity relationship analysis and biological evaluation.

CDK8 regulates transcription either by phosphorylation of transcription factors or, as part of a four-subunit kinase module, through a reversible association of the kinase module with the Mediator complex, a highly conserved transcriptional coactivator. Deregulation of CDK8 has been found in various types of human cancer, while the role of CDK8 in supressing anti-cancer response of natural killer cells is being understood. Currently, CDK8-targeting cancer drugs are highly sought-after. Herein we detail the discovery of a series of novel pyridine-derived CDK8 inhibitors. Medicinal chemistry optimisation gave rise to 38 (AU1-100), a potent CDK8 inhibitor with oral bioavailability. The compound inhibited the proliferation of MV4-11 acute myeloid leukaemia cells with the kinase activity of cellular CDK8 dampened. No systemic toxicology was observed in the mice treated with 38. These results warrant further pre-clinical studies of 38 as an anti-cancer agent.

Elucidation of the pharmacokinetic/pharmacodynamic determinant of colistin activity against Pseudomonas aeruginosa in murine thigh and lung infection models.

Colistin is increasingly used as last-line therapy against Gram-negative pathogens. The pharmacokinetic (PK)/pharmacodynamic (PD) index that best correlates with the efficacy of colistin remains undefined. The activity of colistin against three strains of Pseudomonas aeruginosa was studied in neutropenic mouse thigh and lung infection models. The PKs of unbound colistin were determined from single-dose PK studies together with extensive plasma protein binding analyses. Dose-fractionation studies were conducted over 24 h with a dose range of 5 to 160 mg/kg of body weight/day. The bacterial burden in the thigh or lung was measured at 24 h after the initiation of treatment. Relationships between antibacterial effect and measures of exposure to unbound (f) colistin (area under the concentration-time curve [fAUC/MIC], maximum concentration of drug in plasma [fC(max)]/MIC, and the time that the concentration in plasma is greater than the MIC [fT > MIC]) were examined by using an inhibitory sigmoid maximum-effect model. Nonlinearity in the PKs of colistin, including its plasma protein binding, was observed. The PK/PD index that correlated best with its efficacy was fAUC/MIC in both the thigh infection model (R(2) = 87%) and the lung infection model (R(2) = 89%). The fAUC/MIC targets required to achieve 1-log and 2-log kill against the three strains were 15.6 to 22.8 and 27.6 to 36.1, respectively, in the thigh infection model, while the corresponding values were 12.2 to 16.7 and 36.9 to 45.9 in the lung infection model. The findings of this in vivo study indicate the importance of achieving adequate time-averaged exposure to colistin. The results will facilitate efforts to define the more rational design of dosage regimens for humans.

Inhibition of morphine metabolism by ketamine.

Clinical observation of a synergistic effect of ketamine on morphine analgesia remains controversial. Although a pharmacodynamic basis for an interaction has been explored in animal and clinical studies, the possibility of a pharmacokinetic mechanism has not been investigated. Whereas both morphine and morphine-6-glucuronide are effective analgesics, morphine-3-glucuronide (M3G) lacks activity. Thus, changes in the metabolism and disposition of morphine may result in an altered response. First, we investigated the interaction between morphine and ketamine in the isolated perfused rat liver preparation. The clearance of morphine was decreased from 16.8 +/- 4.6 ml/min in the control period to 7.7 +/- 2.8 ml/min in the ketamine-treatment period, with the formation clearance of M3G decreasing from 8.0 +/- 4.1 ml/min to 2.1 +/- 1.1 ml/min. Fractional conversion of morphine to M3G was significantly decreased from 0.46 +/- 0.17 in the control period to 0.28 +/- 0.14 upon the addition of ketamine. The possible mechanism of the interaction was further investigated in vitro with rat liver microsomes as the enzyme source. The formation of M3G followed single-enzyme Michaelis-Menten kinetics, with a mean apparent K(m) of 2.18 +/- 0.45 mM and V(max) of 8.67 +/- 0.59 nmol/min/mg. Ketamine inhibited morphine 3-glucuronidation noncompetitively, with a mean K(i) value of 33.3 +/- 7.9 microM. The results demonstrate that ketamine inhibits the glucuronidation of morphine in a rat model.

Renal disposition of colistin in the isolated perfused rat kidney.

Nephrotoxicity is an important limitation to the clinical use of colistin against Pseudomonas aeruginosa and other gram-negative pathogens. Previous work reported net tubular reabsorption of colistin by the kidney in vivo, but there is no knowledge of its disposition within the kidney. This study investigated the renal disposition and potential transport mechanisms of colistin in the isolated perfused rat kidney (IPK) model by perfusing with colistin sulfate alone (2 microg/ml) or in the presence of potential inhibitors (tetraethylammonium [TEA], glycine-glycine [Gly-Gly], or hydrochloric acid [HCl]) at three different concentrations. When perfused alone, the renal clearances (CL(R)) for colistin A and B (the major components of colistin) in control kidneys were constant and low (mean values < 0.05 ml/min throughout the perfusion). The mean clearance ratios [CR, defined as CL(R)/(f(u) x GFR), where f(u) is the fraction of drug unbound in perfusate and GFR is the glomerular filtration rate] were significantly less than 1. It was concluded that there is net tubular reabsorption of colistin, and this exceeded the reabsorption of water. Less than 10% eliminated from perfusate was recovered in urine, suggesting considerable renal accumulation of colistin. The CR values for colistin were significantly increased when perfused with TEA (500 microM), Gly-Gly (833 microM), and HCl (2,500, 5,000, and 10,000 microM). It is proposed that renal reabsorption of colistin may involve organic cation transporters (inhibited by TEA) and peptide transporters (inhibited by Gly-Gly) and that the process is sensitive to the pH of urine.

Effect of St John's wort on the disposition of fexofenadine in the isolated perfused rat liver.

OBJECTIVES: This study examined the effects of St John's wort (Hypericum perforatum) on the disposition of fexofenadine, a substrate of P-glycoprotein/organic anion transporting polypeptide, in the isolated perfused rat liver. METHODS: Male Sprague-Dawley rats were given St John's wort, 1000 mg/kg, by intragastric gavage once daily for 14 days. On day 15, livers were isolated surgically and perfused in a recirculating system with fexofenadine (2 microg/ml), either alone or following addition of ciclosporin (0.5 microg/ml) 5 min before the addition of fexofenadine. Perfusate samples and bile were collected for 60 min. Fexofenadine in perfusate, bile and the homogenised livers was measured by HPLC. KEY FINDINGS: Administration of St John's wort significantly increased biliary clearance with respect to perfusate and biliary clearance with respect to the concentration in the liver, by 74% and 71%, respectively. This was reversed by ciclosporin. CONCLUSIONS: St John's wort enhanced the elimination of fexofenadine into the bile. This could be because it increases the activity of P-glycoprotein on the canalicular membrane of hepatocytes.

Targeting CDK9 for treatment of colorectal cancer.

Colorectal cancer (CRC) remains one of the most lethal human malignancies, and pursuit of new therapeutic targets for treatment has been a major research focus. Cyclin-dependent kinase 9 (CDK9), which plays a crucial role in transcription, has emerged as a target for cancer treatment. CDKI-73, one of the most potent and pharmacologically superior CDK9 inhibitors, has demonstrated excellent anti-tumour efficacy against several types of cancers. In this study, we evaluated its therapeutic potential against CRC. CDKI-73 elicited high cytotoxicity against all colon cancer cell lines tested. Cell cycle and apoptosis analysis in HCT 116 and HT29 cells revealed that CDKI-73 induced cell death without accumulation of DNA at any phase of the cell cycle. Moreover, it caused depolarisation of mitochondrial membrane, leading to caspase-independent apoptosis. Knockdown by shRNA demonstrated the CDK9-targeted mechanism of CDKI-73, which also affected the Mnk/eIF4E signalling axis. In addition, RT-qPCR analysis showed that CDKI-73 down-regulated multiple pro-survival factors at the mRNA level. Its in vivo anti-tumour efficacy was further evaluated in Balb/c nude mice bearing HCT 116 xenograft tumours. CDKI-73 significantly inhibited tumour growth (***P < 0.001) without overt toxicity. Analysis of the tumour tissues collected from the xenografted animals confirmed that the in vivo anti-tumour efficacy was associated with CDK9 targeting of CDKI-73. Overall, this study provides compelling evidence that CDKI-73 is a promising drug candidate for treating colorectal cancer.

Discovery of CDK5 Inhibitors through Structure-Guided Approach.

Specific abrogation of cyclin-dependent kinase 5 (CDK5) activity has been validated as a viable approach for the development of anticancer agents. However, no selective CDK5 inhibitor has been reported to date. Herein, a structure-based in silico screening was employed to identify novel scaffolds from a library of compounds to identify potential CDK5 inhibitors that would be relevant for drug discovery. Hits, representatives of three chemical classes, were identified as inhibitors of CDK5. Structural modification of hit-1 resulted in 29 and 30. Compound 29 is a dual inhibitor of CDK5 and CDK2, whereas 30 preferentially inhibits CDK5. Both leads exhibited anticancer activity against acute myeloid leukemia (AML) cells via a mechanism consistent with targeting cellular CDK5. This study provides an effective strategy for discovery of CDK5 inhibitors as potential antileukemic agents.