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Background:Aberrant expression of eukaryotic translation initiation factor 4E (eIF4E) is common in many types of cancer including acute myeloid leukaemia (AML). Phosphorylation of eIF4E by MAPK-interacting kinases (Mnks) is essential for the eIF4E-mediated oncogenic activity. As such, the pharmacological inhibition of Mnks can be an effective strategy for the treatment of cancer.Methods:A series of N-phenyl-4-(1H-pyrrol-3-yl)pyrimidin-2-amine derivatives was designed and synthesised. The Mnk inhibitory activity of these derivatives as well as their anti-proliferative activity against MV4-11 AML cells was determined.Results:These compounds were identified as potent Mnk2 inhibitors. Most of them demonstrated potent anti-proliferative activity against MV4-11 AML cells. The cellular mechanistic studies of the representative inhibitors revealed that they reduced the level of phosphorylated eIF4E and induced apoptosis by down-regulating the anti-apoptotic protein myeloid cell leukaemia 1 (Mcl-1) and by cleaving poly(ADP-ribose)polymerase (PARP). The lead compound 7k possessed desirable pharmacokinetic properties and oral bioavailability.Conclusion:This work proposes that exploration of the structural diversity in the context of Nphenyl- 4-(1H-pyrrol-3-yl)pyrimidin-2-amine would offer potent and selective Mnk inhibitors.

Here, we report the design, synthesis, and evaluation of a series of inhibitors of Staphylococcus aureus BPL (SaBPL), where the central acyl phosphate of the natural intermediate biotinyl-5′-AMP (1) is replaced by a sulfonamide isostere. Acylsulfamide (6) and amino sulfonylurea (7) showed potent in vitro inhibitory activity (Ki = 0.007 ± 0.003 and 0.065 ± 0.03 μM, respectively) and antibacterial activity against S. aureus ATCC49775 with minimum inhibitory concentrations of 0.25 and 4 μg/mL, respectively. Additionally, the bimolecular interactions between the BPL and inhibitors 6 and 7 were defined by X-ray crystallography and molecular dynamics simulations. The high acidity of the sulfonamide linkers of 6 and 7 likely contributes to the enhanced in vitro inhibitory activities by promoting interaction with SaBPL Lys187. Analogues with alkylsulfamide (8), β-ketosulfonamide (9), and β-hydroxysulfonamide (10) isosteres were devoid of significant activity. Binding free energy estimation using computational methods suggests deprotonated 6 and 7 to be the best binders, which is consistent with enzyme assay results. Compound 6 was unstable in whole blood, leading to poor pharmacokinetics. Importantly, 7 has a vastly improved pharmacokinetic profile compared to that of 6 presumably due to the enhanced metabolic stability of the sulfonamide linker moiety. Refereed/Peer-reviewed

CDK8 is deregulated in multiple types of human cancer and is viewed as a therapeutic target for the treatment of the disease. Accordingly, the search for small-molecule inhibitors of CDK8 is being intensified. Capitalising on our initial discovery of AU1-100, a potent CDK8 inhibitor yet with a limited degree of kinase selectivity, a structure-based optimisation was carried out, with a series of new multi-substituted pyridines rationally designed, chemically prepared and biologically evaluated. Such endeavour has culminated in the identification of 42, a more potent CDK8 inhibitor with superior kinomic selectivity and oral bioavailability. The mechanism underlying the anti-proliferative effect of 42 on MV4-11 cells was studied, revealing that the compound arrested the G1 cell cycle and triggered apoptosis. The low risk of hepato- and cardio-toxicity of 42 was estimated. These findings merit further investigation of 42 as a targeted cancer therapeutic. Refereed/Peer-reviewed

Aims: Long-term use of the immunosuppressant tacrolimus is limited by nephrotoxicity. Following renal transplantation, the risk of nephrotoxicity may be determined more by allograft than by blood tacrolimus concentrations, and thus may be affected by donor CYP3A5 and ABCB1 genetics. Little is known regarding factors that determine tacrolimus intrarenal exposure. Methods: This study investigated the relationship between trough blood (C0Blood) and allograft (CGraft) tacrolimus concentrations and tacrolimus dose, haematocrit, genetics, acute nephrotoxicity, rejection status, delayed graft function, and time post-transplant. C0Blood and CGraft were quantified in 132 renal transplant recipients together with recipient and donor CYP3A5 (rs776746) and ABCB1 3435 (rs1045642) genotypes. Results: C0Blood ranged from 2.6 to 52.3 ng/mL and CGraft from 33 to 828 pg/mg tissue. Adjusting for dose, recipients who were CYP3A5 expressors had lower C0Blood compared to nonexpressors, whilst delayed graft function was associated with higher C0Blood. Linear regression showed that the significant predictors of CGraft were C0Blood (point-wise P = 7 × 10−10), dose (P = .004) acute nephrotoxicity (P = .002) and an interaction between C0Blood and acute tacrolimus nephrotoxicity (P = .0002), with an adjusted r2 = 0.35 and no contribution from donor or recipient CYP3A5 or ABCB1 genotype. The association between CGraft and acute nephrotoxicity depended on one very high CGraft (828 pg/mg tissue). Conclusions: Recipient and donor CYP3A5 and ABCB1 3435C>T genotypes are not determinants of allograft tacrolimus exposure in kidney transplant recipients. However, tacrolimus dose and C0Blood were significant predictors of CGraft, and the relationship between C0Blood and CGraft appeared to differ in the presence or absence of acute nephrotoxicity. Refereed/Peer-reviewed

Background and purpose Cyclin D-dependent kinases 4 and 6 (CDK4/6) are crucial regulators of the G1 to S phase transition of the cell cycle and are actively pursued as therapeutic targets in cancer. We sought to discover a novel series of orally bioavailable and highly selective small molecule inhibitors of CDK4/6. Experimental approach The discovery of pharmacological inhibitors and optimization for potency, selectivity and drug properties were achieved by iterative chemical synthesis, biochemical screening against a panel of kinases, cell-based assays measuring cellular viability, cell cycle distribution, induction of apoptosis and the level of retinoblastoma tumour suppressor protein (Rb) phosphorylation and E2 factor (E2F)-regulated gene expression and in vitro biopharmaceutical and in vivo pharmacokinetic profiling. Key results We discovered several lead compounds that displayed >1000-fold selectivity for CDK4/6 over other members of the CDK family. The lead compounds, 82, 91 and 95, potently inhibited the growth of cancer cells by inducing G1 arrest with a concomitant reduction in the phosphorylation of Rb at S780 and in E2F-regulated gene expression. With a remarkable selectivity for CDK4 over 369 human protein kinases, 91 was identified as a highly potent and orally bioavailable drug candidate. Conclusions and implications We have identified unique and new inhibitors of CDK4/6 as potential drug candidates. Compound 91 represents an ideal candidate for further development as targeted cancer therapy.

Cyclin D dependent kinases (CDK4 and CDK6) regulate entry into S phase of the cell cycle and are validated targets for anticancer drug discovery. Herein we detail the discovery of a novel series of 4-thiazol-N-(pyridin-2-yl)pyrimidin-2-amine derivatives as highly potent and selective inhibitors of CDK4 and CDK6. Medicinal chemistry optimization resulted in 83, an orally bioavailable inhibitor molecule with remarkable selectivity. Repeated oral administration of 83 caused marked inhibition of tumor growth in MV4-11 acute myeloid leukemia mouse xenografts without having a negative effect on body weight and showing any sign of clinical toxicity. The data merit 83 as a clinical development candidate. Refereed/Peer-reviewed

The immunosuppressant cyclosporin is a P‐glycoprotein (P‐gp) substrate whose impaired function has been associated with an increased risk of cyclosporin‐induced nephrotoxicity following renal transplantation. This study investigated the relationship between blood and allograft cyclosporin concentration, and the effect of P‐gp expression.Fifty biopsy samples were obtained from 39 renal transplant recipients who received cyclosporin as part of maintenance immunosuppression. Blood cyclosporin concentrations (2 hours postdose) were obtained from clinical records, matching allograft cyclosporin concentrations were measured in frozen biopsy tissue by liquid chromatography–tandem mass spectrometry, and allograft P‐gp expression was assessed by immunohistochemistry. Blood and allograft cyclosporin concentrations in the 1st month post‐transplantation ranged from 505–2005 μg/L and0.01–16.7 ng/mg tissue, respectively. Dose was the only significant predictor of allograft cyclosporin concentrations (adjusted R² = .24, F ‐statistic = 11.52, P = .0019),with no effect of P‐gp expression or blood cyclosporin concentrations. P‐gp expression is not the major determinant of allograft cyclosporin concentrations. Refereed/Peer-reviewed

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. Refereed/Peer-reviewed

Here we report adamantyl cyanoguanidine compounds based on hybrids of the adamantyl amide scaffold reported by AstraZeneca and cyanoguanidine scaffold reported by Abbott Laboratories. Compound 27 displayed five-fold greater inhibitory potency than the lead compound 2 in both pore-formation and interleukin-1β release assays, while 35-treated mice displayed an antidepressant phenotype in behavioral studies. This SAR study provides a proof of concept for hybrid compounds, which will help in the further development of P2X

Here we report adamantyl cyanoguanidine compounds based on hybrids of the adamantyl amide scaffold reported by AstraZeneca and cyanoguanidine scaffold reported by Abbott Laboratories. Compound 27 displayed five-fold greater inhibitory potency than the lead compound 2 in both pore-formation and interleukin-1β release assays, while 35-treated mice displayed an antidepressant phenotype in behavioral studies. This SAR study provides a proof of concept for hybrid compounds, which will help in the further development of P2X7R antagonists. Refereed/Peer-reviewed

Mycophenolic acid (MPA) has a low therapeutic index and large inter-individual pharmacokinetic variability necessitating therapeutic drug monitoring to individualise dosing after transplantation. There is an ongoing discrepancy as to whether plasma MPA concentrations sufficiently predict kidney rejection or toxicity and whether immunosuppressant concentrations within the graft tissue may better predict transplant outcomes. The aim of the study was to develop an LC-MS/MS method for the quantification of MPA concentrations in human kidney biopsies taken as part of routine clinical procedures. A total of 4 surplus human kidney biopsies obtained from 4 different kidney transplant recipients were available to use for this study. MPA was also quantified in 2 kidney samples from rats administered MPA to assess tissue extraction reproducibility. Human kidney biopsies and rat kidneys were homogenised mechanically and underwent liquid-liquid extraction before analysis by LC-MS/MS. MPA-free human kidney tissue was used in calibrators and quality control samples. Analyte detection was achieved from multiple reaction monitoring of the ammonium adducts of both MPA (m/z 321.1 -> 207.3) and N-phthaloyl-L-phenylalanine (PPA, internal standard, m/z 296.2 -> 250.2) using positive electrospray ionisation. The method was linear (calibration curves R-2 > 0.99, n = 10), precise, and accurate with coefficients of variation and bias less than 15%. Extraction efficiencies for MPA and PPA were approximately 97% and 86%, respectively, and matrix effects were minimal. In 4 kidney transplant recipients, tissue MPA concentrations ranged from 1.3 to 7.7 ng/mg of tissue, however, the correlation between blood (C-o) and tissue MPA concentrations could not be established. The method was successfully applied to the quantification of MPA in human kidney biopsies without the need to alter current clinical protocols. (C) 2013 Elsevier B.V. All rights reserved.

There is an urgent need for better therapeutic options for advanced melanoma patients, particularly those without the BRAFV600E/K mutation. In melanoma cells, loss of TRIM16 expression is a marker of cell migration and metastasis, while the BRAF inhibitor, vemurafenib, induces melanoma cell growth arrest in a TRIM16-dependent manner. Here we identify a novel small molecule compound which sensitized BRAF wild-type melanoma cells to vemurafenib. High throughput, cell-based, chemical library screening identified a compound (C012) which significantly reduced melanoma cell viability, with limited toxicity for normal human fibroblasts. When combined with the BRAFV600E/K inhibitor, vemurafenib, C012 synergistically increased vemurafenib potency in 5 BRAFWT and 4 out of 5 BRAFV600E human melanoma cell lines (Combination Index: CI < 1), and, dramatically reduced colony forming ability. In addition, this drug combination was significantly anti-tumorigenic in vivo in a melanoma xenograft mouse model. The combination of vemurafenib and C012 markedly increased expression of TRIM16 protein, and knockdown of TRIM16 significantly reduced the growth inhibitory effects of the vemurafenib and C012 combination. These findings suggest that the combination of C012 and vemurafenib may have therapeutic potential for the treatment of melanoma, and, that reactivation of TRIM16 may be an effective strategy for patients with this disease. Refereed/Peer-reviewed

Therapeutic drug monitoring is used to individualize cyclosporine A (CsA) dosing after transplantation. However, immunosuppressant concentrations within the graft may better predict clinical outcomes, including toxicity. This study aimed to develop a method suitable for CsA measurement using routine fine-needle biopsy samples. CsA was quantified retrospectively in kidney and liver tissues from 10 rats administered CsA, and 21 core needle kidney biopsies taken from renal transplant patients with suspected graft dysfunction. Dried biopsies were weighed (mean +/- SD weights of 0.22 +/- 0.18 mg), enzymatically solubilized, and then CsA was extracted and quantified using online 2-dimensional liquid chromatography-tandem mass spectrometry. The method was linear (r(2)>0.997, n = 10), accurate, and precise (quality control and calibrator coefficient of variation and bias 0.05). Similarly, in 16 transplant patients, for whom blood CsA concentrations (C2) were available within 1 day of the renal biopsy being performed, there was no significant correlation between CsA concentrations in blood and kidney tissue (Spearman r = 0.168, P>0.05). In situ CsA measurements acquired using this method could make an easy transition into clinical use due to their retrospective nature and minimal disruption to current clinical protocols and could provide an additional tool for optimizing clinical outcomes in the future.

Ovarian cancer is one of the most common malignancies, accounting for more deaths than any other cancer of the female reproductive system. Cyclin-dependent kinases 4 and 6 (CDK4/6) are fundamental drivers of the cell cycle and are required for the initiation and progression of various cancers. Over-expression of CDK4/6 &/or cyclin D1 is commonly observed in ovarian cancer and is associated with an aggressive phenotype and poor prognosis. Patients with advanced ovarian cancer whose tumor demonstrated Rb-positive & a low expression of p16INK4a have the worst clinical outcome, but these patients are likely to gain benefit from CDK4/6 inhibition. Here, we describe the preclinical development of 2-94; a potent, highly selective and orally bioavailable CDK4 inhibitor for the treatment of ovarian cancer. Specifically, 2-94 exhibits high selectivity for CDK4/D1 (Ki = 2 nM) over CDK6/D3 (Ki = 279 nM). Consistent with a CDK4-targeted mechanism, 2-94 potently inhibited proliferation of Rb proficient ovarian cancer cells. It induced G1 cell cycle arrest by reducing Rb phosphorylation at S780, S795 and S807/11 residues in a dose dependent manner. Apoptosis was induced with evident increase of caspase 3/7 activity and reduction of Bcl-2 protein levels in A2780 cells. In addition, 2-94 has shown synergistic activity with mTOR, MEK, PI3K and PARP inhibitors. In contrast, no apparent cell cycle effect, senescence or apoptosis was observed in human bone marrow mononuclear (hBMNCs) cells by 2-94 treatment unlike palbociclib and ribociclib which increased the G1 population of hBMNCs, suggesting the low toxicity of 2-94 compared to clinical compounds. Consistent with its favourable pharmacokinetic properties and high oral bioavailability, 2-94 delayed tumor growth and significantly increased the survival of A2780 tumor bearing nude mice by oral administration. 2-94 was more efficacious when compared to palbociclib, resulting in a T/C = 22% for 2-94 vs T/C = 31% for palbociclib. Moreover, 2-94 showed little or no effect on lymphocyte and neutrophil counts whilst palbociclib caused their reduction. This lower effect of 2-94 on the neutrophils and lymphocytes might be due to its lesser effect on CDK6-cyclin D3 compared to palbociclib. Both 2-94 and palbociclib didn't cause histopathological changes in the bone marrow, intestine, liver, heart and kidney of the mice. Pharmacodynamical studies confirmed that 2-94 reduced the Rb phosphorylation at S780, S795 and S807/811 in the tumour models. In conclusion, 2-94 is a first-in-class CDK4 inhibitor with high selectivity against a panel of 360 human kinases. This selectivity can be taken as a reason for the observed better safety of 2-94 compared to palbociclib. The favourable oral pharmacokinetics, robust antitumor efficacy and excellent safety profiles make 2-94 a highly attractive candidate for development towards the clinic. Citation Format: Laychiluh B. Mekonnen, Solomon Zeleke, Jimma Lenjisa, Mingfeng Yu, Benjamin Noll, Gary Heinemann, Robert Milne, Hugo Albrecht, Martin K. Oehler, Shudong Wang. A first-in-class CDK4 inhibitor shows excellent in vitro and in vivo efficacy against ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1524.

Cyclin-dependent kinases 4 and 6 (CDK4/6) in concert with D-type cyclins, are vital drivers of the cell cycle. Activated CDK4/6-cyclin D complexes regulate cell proliferation, senescence and apoptosis via phosphorylation of proteins including Rb, FOXM1 and SMAD3. Deregulation of the CDK4/6-cyclin D-Rb-E2F pathway has been found in over 90% of human cancers, and amplification of genes coding for cyclin D1 (CCND1) and CDK4/6, or deletions of the locus encoding p16INK4a are the major mechanisms. For instance, CCDN1 amplification and cyclin D protein overexpression are seen in ~ 35% and ≥ 50% of breast cancer cases, respectively. Similarly, ≥ 40% of prostate cancers overexpress CDK6 while loss of p16INK4a occurs in ~ 80% of head and neck squamous cell carcinoma. Specific mutations or translocations in the genes encoding CDK4/6-cyclin D-Rb-E2F pathway are aberrantly activated in melanoma and mantle cell lymphoma. Therefore, CDK4/6 inhibition can be an effective treatment strategy for these cancers. With our extensive medicinal chemistry, biochemical and cellular assays, in vivo pharmacokinetic, safety and efficacy studies, we have identified a highly potent dual inhibitor of CDK4/6, 3-14, that showed excellent selectivity over a panel of >360 kinases. 3-14 is more potent anti-proliferative agent than palbociclib against a range of cancer cell lines including breast, ovarian, colon, prostate, pancreatic, lung and melanoma cancer cells. 3-14 arrested cells in G1 phase of the cell cycle, prevented colony formation and induced senescence in MCF7 breast cancer cells. 3-14 caused dose dependent inhibition of Rb phosphorylation at S780, S795 and S807/11 residues, and this was translated to the reduction in the levels of E2F regulated proteins such as cyclin E2, A2. Moreover, 3-14 possessed excellent pharmaceutical properties, and pharmacokinetics with high oral bioavailability in rodents. 3-14 (MTD = 250 mg/kg) is better tolerated than palbociclib (MTD = 150 mg/kg) in mice. In A2780 ovarian cancer xenograft, 3-14 (100 mg/kg p.o. daily) delayed tumor growth (i.e. T/C = 24.4 %) and increased animal life span (90%), and was more efficacious compared to palbociclib (i.e. T/C = 38% and ILS = 73%). Both 3-14 and palbociclib didn't cause histopathological changes in the bone marrow, intestine, liver, heart and kidney except for the non-significant reduction in the neutrophils. Given its excellent efficacy and safety as well as pharmacokinetic profiles, 3-14 is warranted as a development candidate for further preclinical and clinical studies. Citation Format: Laychiluh Bantie, Solomon Tadesse, Jimma Likisa, Mingfeng Yu, Benjamin Noll, Hugo Albrecht, Robert Milne, Shudong Wang. Preclinical development of a highly potent and selective dual inhibitor of CDK4/6 for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2314.

Cyclin D dependent kinases CDK4 & CDK6 are crucial regulators of the G1 to S phase transition of the cell cycle. The facts that myriad cancer types show aberrance in INK4-CDK4/6-cyclin D-Rb-E2F pathway, & the rapidly emerging positive clinical data of pharmacological inhibitors have validated CDK4/6 as anticancer drug targets. As the first commercialized CDK inhibitor, palbociclib in combination with letrozole or fulvestrant has received regulatory approval for the treatment of breast cancer. This represents an important scientific advance in the field. However, the limited structural diversity & undesired side effects due to broader kinase interactions of existing inhibitors mean that the hunt for new & highly selective CDK4/6 inhibitor drug candidates continues. Using our advanced medicinal chemistry, targeted biochemical & cell-based assays, & animal pharmacology, we synthesized & evaluated a novel series of inhibitors. Many compounds were highly potent & selective against CDK4/6, & exhibited low nanomolar potency against a range of human cancer cell lines. Notably, inhibition of CDK4D1 by compound CDKI-15 (Ki = 4 nM) was over 3 orders of magnitude greater than CDK1B, CDK2A, CDK7H & CDK9T1. Interrogation of a panel of 369 protein kinases revealed CDKI-15 to be highly selective for CDK4/6 with only 3 other kinases inhibited potently. CDKI-15 reduced the level of Rb phosphorylation & induced G1 cell cycle arrest, confirming cellular inhibition of CDK4/6 in cancer cells. Moreover, CDKI-15 possesses superior pharmacokinetic profile with oral bioavailability of 100% in mice. Treatment of nude BALB/c mice bearing human MV4-11 acute myeloid leukemia cells with CDKI-15 at daily dose of 100 mg/kg by oral administration resulted in a robust inhibition of tumor growth compared to vehicle treated animals (T/C = 30%, p < 0.00001). Strikingly, CDKI-15 caused a complete & sustained tumor regression in one-third of the animals. No detectable toxicity was observed in the animals during & post treatment. Taken together, our data provide a rationale for CDKI-15 to be developed towards clinic for cancer therapy. Citation Format: Solomon Tadesse, Laychiluh Bantie, Khamis Tomusange, Saiful Islam, Muhammed H. Rahaman, Benjamin Noll, Frankie Lam, Mingfeng Yu, Shudong Wang. CDKI-15, a novel and highly selective CDK4/6 inhibitor: discovery, in vitro and in vivo anticancer efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2353. doi:10.1158/1538-7445.AM2017-2353

Objectives An LC-MS/MS method was developed for simultaneous quantitation of tacrolimus, sirolimus and everolimus in whole blood, and compared to HPLC-UV and immunoassay methods. Design and methods Blood (0.1 mL) was analysed following solid-phase extraction and chromatographic resolution using a C18 column (45 °C) and mobile phase of methanol/40 mM ammonium acetate/glacial acetic acid (83/17/0.1) at 200 μL/min, with positive electrospray ionisation and multiple reaction monitoring. Results Intra- and inter-day imprecision and inaccuracy were ≤ 12.2% over a 1.5–40 μg/L calibration range. An external quality assurance programme confirmed acceptable inaccuracy and imprecision of the LC-MS/MS method, but highlighted problems with immunoassay quantitation, particularly for everolimus, showing a > 30% bias in FPIA everolimus concentrations measured in pooled patient samples versus spiked drug-free whole blood. Conclusions LC-MS/MS provides significant accuracy and precision advantages compared to HPLC and immunoassays. Discrepancies in everolimus concentrations measured by the Seradyn FPIA immunoassay require further investigation.