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JAK1, JAK2, JAK3, and TYK2 belong to the JAK (Janus kinase) family. They play critical roles in cytokine signaling. Constitutive activation of JAK/STAT pathways is associated with a wide variety of...

As an anti-inflammatory strategy, MAPK-activated protein kinase-2 (MK2) inhibition can potentially avoid the clinical failures seen for direct p38 inhibitors, especially tachyphylaxis. CC-99677, a selective targeted covalent MK2 inhibitor, employs a rare chloropyrimidine that bonds to the sulfur of cysteine 140 in the ATP binding site via a nucleophilic aromatic substitutions (S

CDK7 has emerged as an exciting target in oncology due to its roles in two important processes that are misregulated in cancer cells: cell cycle and transcription. This report describes the discovery of SY-5609, a highly potent (sub-nM CDK7 Kd) and selective, orally available inhibitor of CDK7 that entered the clinic in 2020 (ClinicalTrials.gov Identifier: NCT04247126). Structure-based design was leveraged to obtain high selectivity (>4000-times the closest off target) and slow off-rate binding kinetics desirable for potent cellular activity. Finally, incorporation of a phosphine oxide as an atypical hydrogen bond acceptor helped provide the required potency and metabolic stability. The development candidate SY-5609 displays potent inhibition of CDK7 in cells and demonstrates strong efficacy in mouse xenograft models when dosed as low as 2 mg/kg.

Background: CDK12 has emerged as an attractive cancer target due to its role in transcription and DNA damage repair regulation. In pre-clinical models, small molecule CDK12 inhibitors have demonstrated promising tumor killing effect, especially in combination with DNA damaging agents. Here we profiled new oral and selective CDK12 inhibitors. Material and methods: ADP-glo࣪ assays: inhibition of recombinant CDK12/CyclinK, CDK2/CyclinE, CDK7/CyclinH/MAT1 and CDK9/CyclinT2 was measured by assessing ADP converted from ATP via luminescent signal at Km and 20x Km ATP. Kinome screen: 1 µM of Compound A was tested in duplicate against 370 kinases at 10 µM ATP in the radiometric HotSpot™ assay. Cellular assays: p-Ser2, p-Ser5 and total RNA pol II signals were assessed 4hrs after compound treatment while ɣH2AX and BRCA1 signals were measured after 24-48hr compound treatment via Immunofluorescence (IF) assays. Cell proliferation was determined using cell titer glo after 48-72hrs compound treatment. Apoptosis was measured by annexin V/PI staining and flow cytometry analysis after 48-72hrs of treatment. Cell cycle profile was evaluated with Click-iT-EdU and FxCycle violet stain and flow cytometry analysis following 24-48hrs of treatment. Mouse xenograft: balb/c mice were implanted subcutaneously with H1048 or MDA-MB-468 cells and randomized for treatment with test drug or vehicle when tumors reached 150-200mm3. Mice were dosed BID through oral administration for 4 weeks. Combination effect of Compound A and lurbinectedin was tested in vivo. Lurbinectedin and Compound A were dosed QW and BID respectively for 28 days in H1048 CDX model. Results: A series of CDK12 inhibitors were designed and profiled in biochemical and cellular assays. A representative member of the class, Compound A, exhibited selectivity over CDK2, CDK7, and CDK9 of 46-, 27-, and 9-fold, respectively. Compound A inhibited proliferation in a panel of cell lines with EC50 in the low nanomolar range. Compound A treatment led to dose dependent apoptosis in multiple cancer cell lines and induced G2/M arrest in cancer cell lines. In vitro, combination treatment with Compound A and lurbinectedin lead to increased DNA damage accumulation, decreased homologous recombination repair, and overall enhanced antiproliferation. Dose dependent tumor growth inhibition in SCLC and TNBC CDX models was observed with Compound A treatment. Enhanced and durable antitumor effect was observed with lurbinectedin and Compound A combination compared to single agent treatment in vivo. Conclusions: We designed and profiled orally available, CDK12 selective inhibitors with potent activity as single agent in vitro and in vivo in multiple cancer models. Compound A demonstrated enhanced antitumor effect in vitro and in vivo when combined with DNA damaging agents. These data support the rationale for advancing one or more members of this class toward clinical development. Citation Format: Shan hu, David Moebius, Wojciech Dworakowski, Elliott Cooper, Derek LaPlaca, Sydney Alnemy, Phone Perera, Jason Marineau, Claudio Chuaqui, John P. Carulli, Eric Olson. An oral and selective CDK12 inhibitor demonstrates robust anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5393.

Janus kinases (JAKs) have been demonstrated to be critical in cytokine signaling and have thus been implicated in both cancer and inflammatory diseases. The JAK family consists of four highly homologous members: JAK1-3 and TYK2. The development of small-molecule inhibitors that are selective for a specific family member would represent highly desirable tools for deconvoluting the intricacies of JAK family biology. Herein, we report the discovery of a potent JAK1 inhibitor, 24, which displays ∼1000-fold selectivity over the other highly homologous JAK family members (determined by biochemical assays), while also possessing good selectivity over other kinases (determined by panel screening). Moreover, this compound was demonstrated to be orally bioavailable and possesses acceptable pharmacokinetic parameters. In an in vivo study, the compound was observed to dose dependently modulate the phosphorylation of STAT3 (a downstream marker of JAK1 inhibition).

A novel series of covalent inhibitors of EGFR (epidermal growth factor receptor) kinase was discovered through a combination of subset screening and structure-based design. These compounds preferentially inhibit mutant forms of EGFR (activating mutant and T790M mutant) over wild-type EGFR in cellular assays measuring EGFR autophosphorylation and proliferation, suggesting an improved therapeutic index in non-small cell lung cancer patients would be achievable relative to established EGFR inhibitors. We describe our design approaches, resulting in the identification of the lead compound 5, and our efforts to develop an understanding of the structure-activity relationships within this series. In addition, strategies to overcome challenges around metabolic stability and aqueous solubility are discussed. Despite limitations in its physical properties, 5 is orally bioavailable in mice and demonstrates pronounced antitumor activity in in vivo models of mutant EGFR-driven cancers.

The Wnt pathway is an evolutionarily conserved and tightly regulated signaling network with important roles in embryonic development and adult tissue regeneration. Impaired Wnt pathway regulation, arising from mutations in Wnt signaling components, such as Axin, APC, and β-catenin, results in uncontrolled cell growth and triggers oncogenesis. To explore the reported link between CK2 kinase activity and Wnt pathway signaling, we sought to identify a potent, selective inhibitor of CK2 suitable for proof of concept studies in vivo. Starting from a pyrazolo[1,5-a]pyrimidine lead (2), we identified compound 7h, a potent CK2 inhibitor with picomolar affinity that is highly selectivity against other kinase family enzymes and inhibits Wnt pathway signaling (IC50 = 50 nM) in DLD-1 cells. In addition, compound 7h has physicochemical properties that are suitable for formulation as an intravenous solution, has demonstrated good pharmacokinetics in preclinical species, and exhibits a high level of activity as a monotherapy in HCT-116 and SW-620 xenografts.

We have identified a class of azabenzimidazoles as potent and selective JAK1 inhibitors. Investigations into the SAR are presented along with the structural features required to achieve selectivity for JAK1 versus other JAK family members. An example from the series demonstrated highly selective inhibition of JAK1 versus JAK2 and JAK3, along with inhibition of pSTAT3 in vivo, enabling it to serve as a JAK1 selective tool compound to further probe the biology of JAK1 selective inhibitors.

Inhibition of the protein-protein interaction between B-cell lymphoma 6 (BCL6) and corepressors has been implicated as a therapeutic target in diffuse large B-cell lymphoma (DLBCL) cancers and profiling of potent and selective BCL6 inhibitors are critical to test this hypothesis. We identified a pyrazolo[1,5-a]pyrimidine series of BCL6 binders from a fragment screen in parallel with a virtual screen. Using structure-based drug design, binding affinity was increased 100000-fold. This involved displacing crystallographic water, forming new ligand-protein interactions and a macrocyclization to favor the bioactive conformation of the ligands. Optimization for slow off-rate constant kinetics was conducted as well as improving selectivity against an off-target kinase, CK2. Potency in a cellular BCL6 assay was further optimized to afford highly selective probe molecules. Only weak antiproliferative effects were observed across a number of DLBCL lines and a multiple myeloma cell line without a clear relationship to BCL6 potency. As a result, we conclude that the BCL6 hypothesis in DLBCL cancer remains unproven.

The discovery and optimization of a series of 6-aryl-azabenzimidazole inhibitors of TBK1 and IKK-ε is described. Various internal azabenzimidazole leads and reported TBK1/IKK-ε inhibitors were docked into a TBK1 homology model. The resulting overlays inspired a focused screen of 6-substituted azabenzimidazoles against TBK1/IKK-ε. This screen resulted in initial hit compound 3. The TBK1/IKK-ε enzyme and cell potency of this compound was further improved using structure guided drug design. Systematic exploration of the C6 aryl group led to compound 19, a potent inhibitor of TBK1 with selectivity against cell cycle kinases CDK2 and Aurora B. Further elaboration and optimization gave compound 25, a single digit nM inhibitor of TBK1. These compounds may serve as in vitro probes to evaluate TBK1/IKK-ε as an oncology target.

Previously, we reported on a series of highly potent, selective, and non-covalent CDK7 inhibitors that demonstrated antiproliferative activity against triple-negative breast cancer (TNBC) and ovarian cancer (OVA) cell lines and tumor growth inhibition in cell line-derived (CDX) and patient-derived (PDX) mouse xenograft models. Here, we report on the in vitro and in vivo profile of our development candidate, SY-5609. Methods: Kinase inhibition assays at both Km and 2 mM [ATP] were used to assess inhibition of CDK2, CDK7, CDK9, and CDK12. SPR was used to determine the Kd, kon, and koff binding characteristics of SY-5609 to immobilized CDK7/Cyclin H dimer. CDK7 compound occupancy was determined using a biotinylated small molecule probe to pull down free CDK7 following incubating of HL60 cells with SY-5609. Inhibition of tumor cell line growth was assessed following 72 hrs of incubation with SY-5609. Flow cytometry was used to assess apoptosis and cell cycle modulation after 48 hrs of treatment. Effects on DNA damage and repair were assessed by immunofluorescence staining for γH2AX and RAD51 proteins. To assess in vivo effects, mice were implanted subcutaneously and randomized for treatment when tumors reached 150-200 mm3 and dosed orally for 3 weeks by both QD and BID dosing regimens. Collected tumor tissue samples were analyzed for protein levels of MCL1, pCDK2, MYC, and RNA Pol II CTD pSer5 by western blot. Results: SY-5609 bound CDK7/Cyclin H with a Kd of 0.059 nM and occupied CDK7 in HL60 cells with an EC50 of 33 nM. Cell growth inhibition EC50 values were 6-17 nM in a panel of solid tumor cell lines. Selectivity of SY-5609 over CDK12, CDK9, and CDK2 was 2492-, 2508-, and 8068-fold, respectively. SY-5609 led to induction of apoptosis, cell cycle arrest, and inhibition of DNA damage repair in tumor cell lines. Dose-dependent tumor growth inhibition was observed in a panel of CDX and PDX solid tumor models with both QD and BID dosing of SY-5609 with resulting decreases in direct (pCDK2, RNA Pol II CTD pSer5) and indirect (MCL1, MYC) protein biomarkers. In summary, we describe SY-5609, an orally available, potent, and selective CDK7 inhibitor that drives strong responses in CDX and PDX tumor models. These data support the rationale for advancing SY-5609 into IND-enabling studies. Citation Format: Shanhu Hu, Jason Marineau, Kristin Hamman, Michael Bradley, Anneli Savinainen, Sydney Alnemy, Nisha Rajagopal, David Orlando, Claudio Chuaqui, Eric Olson. SY-5609, an orally available selective CDK7 inhibitor demonstrates broad anti-tumor activity in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4421.

Structure based design, synthesis, and biological evaluation of a novel series of 1-methyl-1H-imidazole, as potent Jak2 inhibitors to modulate the Jak/STAT pathway, are described. Using the C-ring fragment from our first clinical candidate AZD1480 (24), optimization of the series led to the discovery of compound 19a, a potent, orally bioavailable Jak2 inhibitor. Compound 19a displayed a high level of cellular activity in hematopoietic cell lines harboring the V617F mutation and in murine BaF3 TEL-Jak2 cells. Compound 19a demonstrated significant tumor growth inhibition in a UKE-1 xenograft model within a well-tolerated dose range.

A novel series of small-molecule inhibitors has been developed to target the double mutant form of the epidermal growth factor receptor (EGFR) tyrosine kinase, which is resistant to treatment with gefitinib and erlotinib. Our reported compounds also show selectivity over wild-type EGFR. Guided by molecular modeling, this series was evolved to target a cysteine residue in the ATP binding site via covalent bond formation and demonstrates high levels of activity in cellular models of the double mutant form of EGFR. In addition, these compounds show significant activity against the activating mutations, which gefitinib and erlotinib target and inhibition of which gives rise to their observed clinical efficacy. A glutathione (GSH)-based assay was used to measure thiol reactivity toward the electrophilic functionality of the inhibitor series, enabling both the identification of a suitable reactivity window for their potency and the development of a reactivity quantitative structure-property relationship (QSPR) to support design.

Centrosome amplification is observed in many human cancers and has been proposed to be a driver of both genetic instability and tumorigenesis. Cancer cells have evolved mechanisms to bundle multiple centrosomes into two spindle poles to avoid multipolar mitosis that can lead to chromosomal segregation defects and eventually cell death. KIFC1, a kinesin-14 family protein, plays an essential role in centrosomal bundling in cancer cells, but its function is not required for normal diploid cell division, suggesting that KIFC1 is an attractive therapeutic target for human cancers. To this end, we have identified the first reported small molecule inhibitor AZ82 for KIFC1. AZ82 bound specifically to the KIFC1/microtubule (MT) binary complex and inhibited the MT-stimulated KIFC1 enzymatic activity in an ATP-competitive and MT-noncompetitive manner with a Ki of 0.043 μM. AZ82 effectively engaged with the minus end-directed KIFC1 motor inside cells to reverse the monopolar spindle phenotype induced by the inhibition of the plus end-directed kinesin Eg5. Treatment with AZ82 caused centrosome declustering in BT-549 breast cancer cells with amplified centrosomes. Consistent with genetic studies, our data confirmed that KIFC1 inhibition by a small molecule holds promise for targeting cancer cells with amplified centrosomes and provided evidence that functional suppression of KIFC1 by inhibiting its enzymatic activity could be an effective means for developing cancer therapeutics.

In this letter, we describe the design, synthesis, and structure-activity relationship of 5-anilinopyrazolo[1,5-a]pyrimidine inhibitors of CK2 kinase. Property-based optimization of early leads using the 7-oxetan-3-yl amino group led to a series of matched molecular pairs with lower lipophilicity, decreased affinity for human plasma proteins, and reduced binding to the hERG ion channel. Agents in this study were shown to modulate pAKT(S129), a direct substrate of CK2, in vitro and in vivo, and exhibited tumor growth inhibition when administered orally in a murine DLD-1 xenograft.

Group I p21-activated kinase (PAK) inhibitors are indicated as important in cancer progression, but achieving high kinase selectivity has been challenging. A bis-anilino pyrimidine PAK1 inhibitor was identified and optimized through structure-based drug design to improve PAK1 potency and achieve high kinase selectivity, giving in vitro probe compound AZ13705339 (18). Reduction of lipophilicity to lower clearance afforded AZ13711265 (14) as an in vivo probe compound with oral exposure in mouse. Such probes will allow further investigation of PAK1 biology.

The design, synthesis and biological evaluation of a series of azabenzimidazole derivatives as TBK1/IKKε kinase inhibitors are described. Starting from a lead compound 1a, iterative design and SAR exploitation of the scaffold led to analogues with nM enzyme potencies against TBK1/IKKε. These compounds also exhibited excellent cellular activity against TBK1. Further structure-based design to improve selectivity over CDK2 and Aurora B resulted in compounds such as 5b-e. These probe compounds will facilitate study of the complex cancer biology of TBK1 and IKKε.

Interruption of TGFbeta signaling through inhibition of the TGFbetaR1 kinase domain may prove to have beneficial effect in both fibrotic and oncological diseases. Herein we describe the SAR of a novel series of TGFbetaR1 kinase inhibitors containing a pyrazolone core. Most TGFbetaR1 kinase inhibitors described to date contain a core five-membered ring bearing N as H-bond acceptor. Described herein is a novel strategy to replace the core structure with pyrazolone ring, in which the carbonyl group is designed as an H-bond acceptor to interact with catalytic Lys 232.

The desire to exploit structural information to aid structure based design and virtual screening led to the development of the interaction fingerprint for analyzing, mining, and filtering the binding patterns underlying the complex 3D data. In this paper we introduce a new approach, weighted SIFt (or w-SIFt), extending the concept of SIFt to capture the relative importance of different binding interactions. The methodology presented here for determining the weights in w-SIFt involves utilizing a dimensionality reduction technique for eliminating linear redundancies in the data followed by a stochastic optimization. We find that the relative weights of the fingerprint bits provide insight into what interactions are critical in determining inhibitor potency. Moreover, the weighted interaction fingerprint can serve as an interpretable position dependent scoring function for ligand protein interactions.

Objective— TGF-β plays a significant role in vascular injury-induced stenosis. This study evaluates the efficacy of a novel, small molecule inhibitor of ALK5/ALK4 kinase, in the rat carotid injury model of vascular fibrosis. Methods and Results— The small molecule, SM16, was shown to bind with high affinity to ALK5 kinase ATP binding site using a competitive binding assay and biacore analysis. SM16 blocked TGF-β and activin-induced Smad2/3 phosphorylation and TGF-β-induced plasminogen activator inhibitor (PAI)-luciferase activity in cells. Good overall selectivity was demonstrated in a large panel of kinase assays, but SM16 also showed nanomolar inhibition of ALK4 and weak (micromolar) inhibition of Raf and p38. In the rat carotid injury model, SM16 dosed once daily orally at 15 or 30 mg/kg SM16 for 14 days caused significant inhibition of neointimal thickening and lumenal narrowing. SM16 also prevented induction of adventitial smooth muscle α-actin–positive myofibroblasts and the production of intimal collagen, but did not decrease the percentage of proliferative cells. Conclusion— These results are the first to demonstrate the efficacy of an orally active, small-molecule ALK5/ALK4 inhibitor in a vascular fibrosis model and suggest the potential therapeutic application of these inhibitors in vascular fibrosis.

Targeted covalent inhibition of disease-associated proteins has become a powerful methodology in the field of drug discovery, leading to the approval of new therapeutics. Nevertheless, current approaches are often limited owing to their reliance on a cysteine residue to generate the covalent linkage. Here we used aryl boronic acid carbonyl warheads to covalently target a noncatalytic lysine side chain, and generated to our knowledge the first reversible covalent inhibitors for Mcl-1, a protein-protein interaction (PPI) target that has proven difficult to inhibit via traditional medicinal chemistry strategies. These covalent binders exhibited improved potency in comparison to noncovalent congeners, as demonstrated in biochemical and cell-based assays. We identified Lys234 as the residue involved in covalent modification, via point mutation. The covalent binders discovered in this study will serve as useful starting points for the development of Mcl-1 therapeutics and probes to interrogate Mcl-1-dependent biological phenomena.

A subset of the compound repository for lead identification at Biogen Idec was characterized for its chemical stability over a 3-year period. Compounds were stored at 4 °C as 10 mM DMSO stocks, and a small subset of compounds was stored as lyophilized dry films. Compound integrity of 470 discrete compounds (Compound Set I) and 1917 combinatorial chemistryderived compounds (Compound Set II) was evaluated by liquid chromatography/mass spectrometry from the time of acquisition into the library collection and after 3 years of storage. Loss of compound integrity over the 3 years of storage was observed across the 2 subsets tested. Of Compound Set I, 63% of samples retained > 80% purity, whereas 57% of samples from Compound Set II had purity greater than 60%. The stability of the lyophilized samples was superior to the samples stored as DMSO solution. Although storage at 4 °C as DMSO solution was adequate for the majority of compounds, the authors observed and quantified the level of degradation within the compound collection. Their study provides general insight into compound storage and selection of library subsets for future lead identification activities. (Journal of Biomolecular Screening 2006:828-835)

The combination of advances in structure-based drug design efforts in the pharmaceutical industry in parallel with structural genomics initiatives in the public domain has led to an explosion in the number of structures of protein-small molecule complexes structures. This information has critical importance to both the understanding of the structural basis for molecular recognition in biological systems and the design of better drugs. A significant challenge exists in managing this vast amount of data and fully leveraging it. Here, we review our work to develop a simple, fast way to store, organize, mine, and analyze large numbers of protein-small molecule complexes. We illustrate the utility of the approach to the management of inhibitor complexes from the protein kinase family. Finally, we describe our recent efforts in applying this method to the design of target-focused chemical libraries.

KIFC1 (HSET), a member of the kinesin-14 family of motor proteins, plays an essential role in centrosomal bundling in cancer cells, but its function is not required for normal diploid cell division. To explore the potential of KIFC1 as a therapeutic target for human cancers, a series of potent KIFC1 inhibitors featuring a phenylalanine scaffold was developed from hits identified through high-throughput screening (HTS). Optimization of the initial hits combined both design-synthesis-test cycles and an integrated high-throughput synthesis and biochemical screening method. An important aspect of this integrated method was the utilization of DMSO stock solutions of compounds registered in the corporate compound collection as synthetic reactants. Using this method, over 1500 compounds selected for structural diversity were quickly assembled in assay-ready 384-well plates and were directly tested after the necessary dilutions. Our efforts led to the discovery of a potent KIFC1 inhibitor, AZ82, which demonstrated the desired centrosome declustering mode of action in cell studies.

Here we present a new strategy for designing and filtering potentially massive combinatorial libraries using structural information of a binding site. We have developed a variation of the structural interaction fingerprint (SIFt) named r-SIFt, which incorporates the binding interactions of variable fragments in a combinatorial library. This method takes into account the 3D structure of the active site of the target molecule and translates desirable ligand-target binding interactions into library filtering constraints. We show using the MAP kinase p38 as a test case that we can efficiently analyze and classify compounds on the basis of their abilities to interact with the target in the desired binding mode. On the basis of these classifications, decision tree models were generated using the molecular descriptors of the compounds as predictor variables. Our results suggest that r-SIFt coupled with the classification models should be a valuable tool for structure-based focusing of combinatorial chemical libraries.

We describe the discovery, using shape-based virtual screening, of a potent, ATP site-directed inhibitor of the TbetaRI kinase, an important and novel drug target for fibrosis and cancer. The first detailed report of a TbetaRI kinase small molecule co-complex confirms the predicted binding interactions of our small molecule inhibitor, which stabilizes the inactive kinase conformation. Our results validate shape-based screening as a powerful tool to discover useful leads against a new drug target.

Janus kinases are a family of four enzymes; JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) that are critical in cytokine signalling, with constitutive activation of JAK/STAT pathways associated with a wide variety of malignancies. Elevated JAK/STAT signalling leading to increased activation of STAT3 is reported in a wide variety of cancers, including breast, liver, prostate, colorectal, head and neck, oesophageal, pancreatic, bladder, and non-small cell lung, and is implicated in the pathogenesis of diffuse large B-cell lymphoma and nasopharyngeal carcinomas. Overall, up to 70% of human tumours are linked to persistent elevated STAT3 activity which can be associated with poorer prognosis in many of these settings. In addition, elevated pSTAT3 is observed in response to chemotherapy treatment, and also in response to treatment with inhibitors of oncogenic signalling pathways such as EGFR, MAPK and AKT, and is associated with resistance or poorer response to agents targeting these pathways. In many of these cases, JAK1 is believed to be a primary driver of STAT3 phosphorylation and signalling, suggesting inhibition of JAKs as a therapeutic approach to treat these potential resistance mechanisms. The mixed JAK1/2 kinase inhibitor ruxolitinib is approved for the treatment of myeloproliferative neoplasms including intermediate or high risk myelofibrosis and polycythemia vera and has been tested in a variety of tumor settings. Since JAK2 is essential for the signal transduction downstream of erythropoietin, thrombopoietin and related receptors that control erythrocyte and megakaryocyte expansion, dosing of inhibitors that target JAK2 can be limited by toxicities such as thrombocytopenia and anaemia. Starting from a non-kinome selective screening hit, structure-based design was used to optimise a series of aminopyrimidines that led to JAK1-selective candidate drug AZD4205. This compound demonstrates ATP competitive binding with IC50’s in a high ATP concentration enzyme assay against JAK1 of 73 nM (Ki = 2.8 nM), with high selectivity against JAK2 and JAK3 with IC50’s of 13,233 nM and >30,000 nM respectively. In addition it showed potent inhibition of p-STAT3 in a cell based assay of JAK1 activity with an IC50 of 128 nM and excellent selectivity across the kinome. In summary, AZD4205 is a highly potent JAK1-selective kinase inhibitor with excellent preclinical pharmacokinetics with potential for further clinical development. The optimization from screening hit to first disclosure of this candidate drug will be presented. Citation Format: Jason G. Kettle, Qibin Su, Neil Grimster, Sameer Kawatkar, Scott Throner, Richard Woessner, Huawei Chen, Geraldine Bebernitz, Kristen Bell, Erica Anderson, Linette Ruston, Jon Winter-Holt, Paul Lyne, Melissa Vasbinder, Claudio Chuaqui. Discovery of the JAK1 selective kinase inhibitor AZD4205 [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 979. doi:10.1158/1538-7445.AM2017-979

The pressure broadening and shifting coefficients for pure rotational transitions of CO in a He bath gas at very low temperatures are calculated from the best available potential energy surfaces, and compared with very recent measurements by M. M. Beaky, T. M. Goyette, and F. C. De Lucia [J. Chem. Phys. 105, 3994 (1996)]. The results obtained for two recent empirical potentials determined from fits to Van der Waals spectra, and for a recent high quality purely ab initio surface, are consistent with one another. The best of the spectroscopic potentials also yields good agreement with high temperature virial coefficients and transport properties. Predictions from all three of these potentials agree with linebroadening and shifting measurements at temperatures above ∼20 K, but are in substantial disagreement with the measurements at temperatures below 4 K. At present, the source of this discrepancy is not known.

A reliable new three‐dimensional potential energy surface is obtained for the H2–Ar system using an exchange‐coulomb potential model with five parameters determined empirically from a least‐squares fit to experimental data. This surface fully accounts for new high resolution IR data, virial coefficients, and vibrational transition pressure‐shifting coefficients used in the analysis, and yields excellent predictions of elastic and inelastic scattering cross sections and hyperfine transition intensities not included in the analysis. Quantitative comparisons with the best previous empirical potential and a high quality fully ab initio potential are also presented.

For 3He–CO and 4He–CO van der Waals bimers, fully resolved infrared spectra in the 4.7 μm region near the fundamental band origin of the CO monomer have been measured for the first time. Only a small fraction of the observed lines could be assigned using conventional empirical spectroscopic techniques, and little additional insight was gained from synthetic spectra generated from a published ab initio potential for this system. However, a complete set of unique assignments was made on the basis of comparisons with synthetic spectra generated from a variety of trial potential energy surfaces, and least‐squares fits to the observed transition frequencies were used to determine a new anisotropic potential energy surface for this system. This new surface is much deeper and has a much stronger well depth anisotropy than the best previous one, and its predictions of very low temperature microwave line broadening cross sections raise serious questions regarding the methodology for calculating that property.

In this paper we describe a series of 3-cyano-5-aryl-7-aminopyrazolo[1,5-a]pyrimidine hits identified by kinase-focused subset screening as starting points for the structure-based design of conformationally constrained 6-acetamido-indole inhibitors of CK2. The synthesis, SAR, and effects of this novel series on Akt signaling and cell proliferation in vitro are described.

Accurate calculations of the energies of all bound rovibrational states, of the energies and lifetimes of some metastable states, and of the resulting rovibrational spectrum are performed for the helium-acetylene Van der Waals complex using an iterative secular equation method. The calculations are carried out both on an empirical potential surface which has a single ‘T shaped’ minimum, and on a potential surface based on ab initio electronic structure calculations which has a single minimum at a linear geometry. The two surfaces differ in the strength of the anisotropy, as well as its sign. A comparison of the spectra generated by the two surfaces allows spectroscopic manifestations of the shape of the potential energy surface to be discerned, results which should help guide analyses of the spectra of similar very weakly bound complexes.

Accurate new C6 dispersion energy coefficients, and their dependence on the diatom orientation and bond length, are calculated for molecular hydrogen interacting with an atom of H, Li, Be, He, Ne, Ar, Kr or Xe. They are generated from accurateab initio pseudo dipole oscillator strength distributions (DOSD) for H2, H, He and Be, and reliable semiempirical ones for Li, Ne, Ar, Kr and Xe. Compact power series expansions for the diatom bond-length dependence of these coefficients, suitable for incorporation into representations of full potential energy surfaces for these systems, are determined and assessed.

The 4-(5-fluoro-6-methyl-pyridin-2-yl)-5-quinoxalin-6-yl-1H-imidazol-2-ylamine 3 is a potent and selective inhibitor of TGF-betaR1. Substitution of the amino group of 3 typically led to a slight decrease in the affinity for the receptor and in TGF-beta-inducted PAI-luciferase reporter activity. However, 2-acetamidoimidazoles were identified as attractive candidates for further optimization as a result of their significant activity combined to their superior pharmacokinetic profile.

The multifunctional cytokine, TGF-β, is often overexpressed in human tumors and in preclinical studies has been demonstrated to have autocrine and paracrine protumor activities including immune evasion, invasiveness, epithelial to mesenchymal transition, angiogenesis, tumor-stromal interactions, survival, induction of tumor interstitial pressure, and decreased drug penetration. These findings suggest that antagonism of the TGF-β pathway may be of benefit in the treatment of cancer. One attractive target, the type I TGF-β receptor (ALK5) has an intracellular serine/threonine kinase, which is required for TGF-β signaling and is amenable to inhibition by small molecule, ATP binding site-targeted kinase inhibitors.

Lead identification by high-throughput screening of large compound libraries has been supplemented with virtual screening and focused compound libraries. To complement existing approaches for lead identification at Biogen Idec, a kinase-focused compound collection was designed, developed and validated. Two strategies were adopted to populate the compound collection: a ligand shape-based virtual screening and a receptor-based approach (structural interaction fingerprint). Compounds selected with the two approaches were cherry-picked from an existing high-throughput screening compound library, ordered from suppliers and supplemented with specific medicinal compounds from internal programs. Promising hits and leads have been generated from the kinase-focused compound collection against multiple kinase targets. The principle of the collection design and screening strategy was validated and the use of the kinase-focused compound collection for lead identification has been added to existing strategies.

A major challenge facing structure-based drug discovery efforts is how to leverage the massive amount of experimental (X-ray and NMR) and virtual structural information generated from drug discovery projects. Many important drug targets have large numbers of protein-inhibitor complexes, necessitating tools to compare and contrast their similarities and differences. This information would be valuable for understanding potency and selectivity of inhibitors and could be used to define target constraints to assist virtual screening. We describe a profile-based approach that enables us to capture the conservation of interactions between a set of protein-ligand receptor complexes. The use of profiles provides a sensitive means to compare multiple inhibitors binding to a drug target. We demonstrate the utility of profile-based analysis of small molecule complexes from the protein-kinase family to identify similarities and differences in binding of ATP, p38, and CDK2 compounds to kinases and how these profiles can be applied to differentiate the selectivity of these inhibitors. Importantly, our virtual screening results demonstrate superior enrichment of kinase inhibitors using profile-based methods relative to traditional scoring functions. Interaction-based analysis should provide a valuable tool for understanding inhibitor binding to other important drug targets.

Representing and understanding the three-dimensional (3D) structural information of protein-ligand complexes is a critical step in the rational drug discovery process. Traditional analysis methods are proving inadequate and inefficient in dealing with the massive amount of structural information being generated from X-ray crystallography, NMR, and in silico approaches such as structure-based docking experiments. Here, we present SIFt (structural interaction fingerprint), a novel method for representing and analyzing 3D protein-ligand binding interactions. Key to this approach is the generation of an interaction fingerprint that translates 3D structural binding information from a protein-ligand complex into a one-dimensional binary string. Each fingerprint represents the "structural interaction profile" of the complex that can be used to organize, analyze, and visualize the rich amount of information encoded in ligand-receptor complexes and also to assist database mining. We have applied SIFt to tackle three common tasks in structure-based drug design. The first involved the analysis and organization of a typical set of results generated from a docking study. Using SIFt, docking poses with similar binding modes were identified, clustered, and subsequently compared with conventional scoring function information. A second application of SIFt was to analyze approximately 90 known X-ray crystal structures of protein kinase-inhibitor complexes obtained from the Protein Databank. Using SIFt, we were able to organize the structures and reveal striking similarities and diversity between their small molecule binding interactions. Finally, we have shown how SIFt can be used as an effective molecular filter during the virtual chemical library screening process to select molecules with desirable binding mode(s) and/or desirable interaction patterns with the protein target. In summary, SIFt shows promise to fully leverage the wealth of information being generated in rational drug design.

Small molecule inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase such as gefitinib and erlotinib have been employed successfully in the treatment of non-small cell lung cancer (NSCLC) patients harboring an activating mutation (EGFRm+). However, resistance to these inhibitors in the form of additional mutations in the kinase domain such as T790M is emerging as a growing clinical issue. This presentation will describe the discovery of AZD9291, an orally bioavailable, irreversible EGFR inhibitor of both the resistance (NCI-H1975, cell phosphorylation IC50 0.5 μM). Wild type EGFR inhibition is believed to drive the observed dose limiting toxicities (such as skin rash and diarrhea) for these first generation therapies in the clinic. New data will be discussed for the first time including the medicinal chemistry program that led to the identification of AZD9291, details of significant in vivo oral activity in pre-clinical xenograft models (including tumor regression in the L858R/T790M double mutant setting at a dose of 5 mpk) and the first disclosure of the candidate drug structure. The pre-clinical findings from this work strongly supported selection of AZD9291 as a clinical candidate, and first dose in man was achieved with AZD9291 in March 2013. L858R/T790M Double Mutant (resistance) cell IC50 (μM)EGFRm+ Single Mutant (activating) cell IC50 (μM)EGFR Wild Type cell IC50 (μM)Aqueous solubility, (salt) (pH=6.8, μg/mL)L858R/T790M Double Mutant efficacy (% TGI at 5mpk PO QD for 14 days)AZD92910.5>490119 Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B94. Citation Format: M. Raymond V. Finlay, Mark Anderton, Susan Ashton, Peter G. Ballard, Rob H. Bradbury, Sam Butterworth, Nicola Colclough, Darren A. E. Cross, Heather L. McFarland, Martine J. Mellor, Richard A. Ward, Mike J. Waring. Discovery of and first disclosure of the clinical candidate AZD9291, a potent and selective third-generation EGFR inhibitor of both activating and T790M resistant mutations that spares the wild type form of the receptor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B94.

Constitutively activated pY705-STAT3 is oncogenic and prevalent in many tumor types, and has been shown to be driven in large part by constitutive autocrine and paracrine cytokine signaling. JAK kinases are critical mediators of cytokine signaling, and JAK1 has been shown to play an essential role downstream of IL-6 family cytokines, the upregulation of which has been documented in many tumor types. A JAK1 selective inhibitor should provide a therapeutic means of suppressing Y705 phosphorylation of STAT3 without incurring the hematological toxicity associated with inhibition of JAK2 and JAK3 activity, allowing for more intensive dosing and greater combinability with other agents, particularly those with hematological toxicities. We have developed highly selective inhibitors of JAK1 and used them to address the hypothesis that JAK1 is the primary driver of STAT3 mediated signaling in cancer cells. The compounds are highly selective in enzyme assays, with 1000-fold selectivity for JAK1 vs. other JAK family members, and minimal activity against other kinases at 1uM. In cell lines, they are over 500-fold selective for JAK1 vs. JAK2, measured using assays specific for JAK1-mediated STAT3 vs. JAK2-mediated STAT5 phosphorylation. In vivo, the compounds are also highly selective for JAK1 mediated signaling, measured by comparing inhibition of STAT3 phosphorylation in H1975 subcutaneous xenografts vs. inhibition of STAT5 phosphorylation in a disseminated TEL-JAK2 transformed BaF3 model. At doses where inhibition of STAT3 phosphorylation was greater than 90%, only minimal inhibition of STAT5 phosphorylation was observed. In a wide variety of human tumor lines (CRC, NSCLC, pancreatic, DLBCL) with a variety of genetic backgrounds (K-ras, b-raf, PI3K, EGFR mutant, PTEN null), complete knockdown of pSTAT3 (Y705) was observed with JAK1 selective compounds, at concentrations achievable in vivo. These effects are consistent with experiments where inhibition of Y705 phosphorylation of STAT3 was achieved with a JAK1-specific siRNA. At doses where in vivo activity was observed, the compounds had only minimal effect in an erythropoietin-driven mouse model of polycythemia and extramedullary erythropoiesis. These data support the hypotheses that in a variety of tumor types, pSTAT3-mediated signaling is dependent on JAK1 kinase activity, and that a selective JAK1 inhibitor can suppress STAT3 activation at doses that avoid the hematological toxicity associated with JAK2 and JAK3 inhibition. Citation Format: Richard Woessner, Geraldine Bebernitz, Kirsten Bell, Robert Caccese, Nakpangi Johnson, Jason Kettle, Caroline Rivard, Patricia Schroeder, Qibin Su, Haixia Wang, Minwei Ye, Weijia Zheng, Michael Zinda, Paul Lyne, Melissa Vasbinder, Dennis Huszar, Claudio Chuaqui. Highly selective JAK1 kinase inhibitors suppress Y705-mediated STAT3 activation in a wide range of tumor types, with reduced hematological toxicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 931. doi:10.1158/1538-7445.AM2013-931

Corrigendum to ‘‘Successful shape-based virtual screening: the discovery of a potent inhibitor of the type I TGFb receptor kinase (TbRI)’’ [Bioorg. Med. Chem. Lett. 13 (2003) 4355] Juswinder Singh,* Claudio E. Chuaqui, P. Ann Boriack-Sjodin, Wen-Cherng Lee, Timothy Pontz, Michael J. Corbley, H.-Kam Cheung, Robert M. Arduini, Jonathan N. Mead, Miki N. Newman, James L. Papadatos, Scott Bowes, Serene Josiah and Leona E. Ling

A kinetic spectrophotometric method for evaluation of pKa has been developed. In the present study this has been utilized towards the pKa determination, in methanol, of the conjugate acids of 4-picoline, aniline, hydroxylamine, methoxyamine, and hydrazinium ion. For the first two cases the pKa values determined by the present method could be compared with the potentiometrically measured values, in a methanol solvent, with satisfactory agreement. Our proposed method involves essentially the spectrophotometric determination of the rate of the acid catalyzed conversion of p-nitrophenyl sulfate to p-nitrophenol in methanol in the presence of the acidic species whose pKa in this medium is required.

A series of alkyl aryl sulfates has been investigated as inhibitors of papain and ficin activity. The results show that the percentage of residual enzymatic activity depends on several factors, including the solvolytic reactivity of the alkylating agent. It is possible to correlate the reactivities of the sulfates with a selectivity parameter which is based on product ratio results. The resulting correlations indicate that the reactivity-selectivity principle is applicable to this system. It is shown, however, from the results of amino acid analyses that specific methylation of active-site cysteine or histidine residues is not effective with the enzymes in the native or denatured state. This phenomenon contrasts with reported work on methyl p -nitrobenzenesulfonate in which extensive methylation of papain in the denatured state was observed. The finding that the nonspecific methylation of the enzymes by the alkyl aryl sulfates leads to inhibition of enzymic activity is discussed in terms of conformational and other effects.