Discovery of quinazoline HPK1 inhibitors with high cellular potency.

Hematopoietic progenitor kinase 1 (HPK1) is regarded as a highly validated target in pre-clinical immune oncology. HPK1 has been described as regulating multiple critical signaling pathway in both adaptive and innate cells. In support of this role, HPK1 KO T cells show enhanced sensitivity to TCR activation and HPK1 KO mice display enhanced anti-tumor activity. Taken together, inhibition of HPK1 has the potential to induce enhanced anti-tumor immune response. Herein, we described the discovery of highly potent HPK1 inhibitors starting form a weak HTS hit. Using a structure-based drug design, HPK1 inhibitors exhibiting excellent cellular single-digit nanomolar potency in both proximal (pSLP76) and distal (IL-2) biomarkers along with sustained elevation of IL-2 cytokine secretion were discovered.

Large-Scale Assessment of Binding Free Energy Calculations in Active Drug Discovery Projects.

Accurate ranking of compounds with regards to their binding affinity to a protein using computational methods is of great interest to pharmaceutical research. Physics-based free energy calculations are regarded as the most rigorous way to estimate binding affinity. In recent years, many retrospective studies carried out both in academia and industry have demonstrated its potential. Here, we present the results of large-scale prospective application of the FEP+ method in active drug discovery projects in an industry setting at Merck KGaA, Darmstadt, Germany. We compare these prospective data to results obtained on a new diverse, public benchmark of eight pharmaceutically relevant targets. Our results offer insights into the challenges faced when using free energy calculations in real-life drug discovery projects and identify limitations that could be tackled by future method development. The new public data set we provide to the community can support further method development and comparative benchmarking of free energy calculations.

Synthesis and SAR development of quinoline analogs as novel P2X7 receptor antagonists.

The P2X7 receptor (P2X7R) plays an important role in diverse conditions associated with tissue damage and inflammation, suggesting that the human P2X7R (hP2X7R) is an attractive therapeutic target. In the present study, the synthesis and structure-activity relationship (SAR) of a novel series of quinoline derivatives as P2X7R antagonists are described herein. These compounds exhibited mechanistic activity (YO PRO) in an engineered HEK293 expressing hP2X7R as well as a functional response (IL-1ß) in human THP-1 (hTHP-1) cellular assays. Compound 19 was identified as the most promising compound in this series with excellent cellular potency, low liver microsomal clearance, good permeability and low efflux ratio. In addition, this compound also displayed good pharmacokinetic properties and acceptable brain permeability (Kp,uu of 0.37).

Trends in Neosubstrate Degradation by Cereblon-Based Molecular Glues and the Development of Novel Multiparameter Optimization Scores.

Molecular glues enable the degradation of previously "undruggable" proteins via the recruitment of cereblon (CRBN) to the target. One major challenge in designing CRBN E3 ligase modulating compounds (CELMoDs) is the selectivity profile toward neosubstrates, proteins recruited by CRBN E3 ligase agents for degradation. Common neosubstrates include Aiolos, Ikaros, GSPT1, CK1α, and SALL4. Unlike achieving potency and selectivity for traditional small molecule inhibitors, reducing the degradation of these neosubstrates is complicated by the ternary nature of the complex formed between the protein, CRBN, and CELMoD. The standard guiding principles of medicinal chemistry, such as enforcing hydrogen bond formation, are less predictive of degradation efficiency and selectivity. Disclosed is an analysis of our glutarimide CELMoD library to identify interpretable chemical features correlated to selectivity profiles and general cytotoxicity. Included is a simple multiparameter optimization function using only three parameters to predict whether molecules will have undesired neosubstrate activity.

Electrostatic docking of a supramolecular host-guest assembly to cytochrome c probed by bidirectional photoinduced electron transfer.

A water-soluble octacarboxyhemicarcerand was used as a shuttle to transport redox-active substrates across the aqueous medium and deliver them to the target protein. The results show that weak multivalent interactions and conformational flexibility can be exploited to reversibly bind complex supramolecular assemblies to biological molecules. Hydrophobic electron donors and acceptors were encapsulated within the hemicarcerand, and photoinduced electron transfer (ET) between the Zn-substituted cytochrome c (MW = 12.3 kD) and the host-guest complexes (MW = 2.2 kD) was used to probe the association between the negatively charged hemicarceplex and the positively charged protein. The behavior of the resulting ternary protein-hemicarcerand-guest assembly was investigated in two binding limits: (1) when K(encaps) ≫ K(assoc), the hemicarcerand transports the ligand to the protein while protecting it from the aqueous medium; and (2) when K(assoc) > K(encaps), the hemicarcerand-protein complex is formed first, and the hemicarcerand acts as an artificial receptor site that intercepts ligands from solution and positions them close to the active site of the metalloenzyme. In both cases, ET mediated by the protein-bound hemicarcerand is much faster than that due to diffusional encounters with the respective free donor or acceptor in solution. The measured ET rates suggest that the dominant binding region of the host-guest complex on the surface of the protein is consistent with the docking area of the native redox partner of cytochrome c. The strong association with the protein is attributed to the flexible conformation and adaptable charge distribution of the hemicarcerand, which allow for surface-matching with the cytochrome.

Discovery of a class of potent gap-junction modifiers as novel antiarrhythmic agents.

In an effort to discover potent, orally bioavailable compounds for the treatment of atrial fibrillation (AF) and ventricular tachycardia (VT), we developed a class of gap-junction modifiers typified by GAP-134 (1, R(1)=OH, R(2)=NH(2)), a compound currently under clinical evaluation. Selected compounds with the desired in-vitro profile demonstrated positive in vivo results in the mouse CaCl(2) arrhythmia model upon oral administration.

Arylphthalazines as potent, and orally bioavailable inhibitors of VEGFR-2.

A series of arylphthalazine derivatives were synthesized and evaluated as antagonists of VEGF receptor II (VEGFR-2). IM-094482 57, which was prepared in two steps from commercially available starting materials, was found to be a potent inhibitor of VEGFR-2 in enzymatic, cellular and mitogenic assays (comparable activity to ZD-6474). Additionally, 57 inhibited the related receptor, VEGF receptor I (VEGFR-1), and showed excellent exposure when dosed orally to female CD-1 mice.

Synthesis and evaluation of heteroaryl-ketone derivatives as a novel class of VEGFR-2 inhibitors.

We have discovered novel inhibitors of VEGFR-2 kinase with low nanomolar potency in both enzymatic and cell-based assays. Active series are heteroaryl-ketone compounds containing a central aromatic ring with either an indazolyl or indolyl keto group in the ortho orientation to the benzylic amine group (Fig. 1). The best compounds were demonstrated to be inactive against a small select panel of tyrosine and serine/threonine kinases with the exception of VEGFR-1 kinase, a close family member. In addition, the lead candidate 8 displayed acceptable exposure levels when administered orally to mice.

Structure-Based Design of Highly Selective Inhibitors of the CREB Binding Protein Bromodomain.

Chemical probes are required for preclinical target validation to interrogate novel biological targets and pathways. Selective inhibitors of the CREB binding protein (CREBBP)/EP300 bromodomains are required to facilitate the elucidation of biology associated with these important epigenetic targets. Medicinal chemistry optimization that paid particular attention to physiochemical properties delivered chemical probes with desirable potency, selectivity, and permeability attributes. An important feature of the optimization process was the successful application of rational structure-based drug design to address bromodomain selectivity issues (particularly against the structurally related BRD4 protein).

Structure-Based Approach To Identify 5-[4-Hydroxyphenyl]pyrrole-2-carbonitrile Derivatives as Potent and Tissue Selective Androgen Receptor Modulators.

In an effort to find new and safer treatments for osteoporosis and frailty, we describe a novel series of selective androgen receptor modulators (SARMs). Using a structure-based approach, we identified compound 7, a potent AR (ARE EC50 = 0.34 nM) and selective (N/C interaction EC50 = 1206 nM) modulator. In vivo data, an AR LBD X-ray structure of 7, and further insights from modeling studies of ligand receptor interactions are also presented.

Perturbation of cellular proteostasis networks identifies pathways that modulate precursor and intermediate but not mature levels of frataxin.

Friedreich's Ataxia is a genetic disease caused by expansion of an intronic trinucleotide repeat in the frataxin (FXN) gene yielding diminished FXN expression and consequently disease. Since increasing FXN protein levels is desirable to ameliorate pathology, we explored the role of major cellular proteostasis pathways and mitochondrial proteases in FXN processing and turnover. We targeted p97/VCP, the ubiquitin proteasome pathway (UPP), and autophagy with chemical inhibitors in cell lines and patient-derived cells. p97 inhibition by DBeQ increased precursor FXN levels, while UPP and autophagic flux modulators had variable effects predominantly on intermediate FXN. Our data suggest that these pathways cannot be modulated to influence mature functional FXN levels. We also targeted known mitochondrial proteases by RNA interference and discovered a novel protease PITRM1 that regulates intermediate FXN levels. Treatment with the aforementioned chemical and genetic modulators did not have a differential effect in patient cells containing lower amounts of FXN. Interestingly, a number of treatments caused a change in total amount of FXN protein, without an effect on mature FXN. Our results imply that regulation of FXN protein levels is complex and that total amounts can be modulated chemically and genetically without altering the absolute amount of mature FXN protein.

Transcriptional Profiling of a Selective CREB Binding Protein Bromodomain Inhibitor Highlights Therapeutic Opportunities.

Bromodomains are involved in transcriptional regulation through the recognition of acetyl lysine modifications on diverse proteins. Selective pharmacological modulators of bromodomains are lacking, although the largely hydrophobic nature of the pocket makes these modules attractive targets for small-molecule inhibitors. This work describes the structure-based design of a highly selective inhibitor of the CREB binding protein (CBP) bromodomain and its use in cell-based transcriptional profiling experiments. The inhibitor downregulated a number of inflammatory genes in macrophages that were not affected by a selective BET bromodomain inhibitor. In addition, the CBP bromodomain inhibitor modulated the mRNA level of the regulator of G-protein signaling 4 (RGS4) gene in neurons, suggesting a potential therapeutic opportunity for CBP inhibitors in the treatment of neurological disorders.

1-(2-Hydroxy-2-methyl-3-phenoxypropanoyl)indoline-4-carbonitrile derivatives as potent and tissue selective androgen receptor modulators.

We present a novel series of selective androgen receptor modulators (SARMs) which shows excellent biological activity and physical properties. 1-(2-Hydroxy-2-methyl-3-phenoxypropanoyl)-indoline-4-carbonitriles showed potent binding to the androgen receptor (AR) and activated AR-mediated transcription in vitro. Representative compounds demonstrated diminished activity in promoting the intramolecular interaction between the AR carboxyl (C) and amino (N) termini. This N/C-termini interaction is a biomarker assay for the undesired androgenic responses in vivo. In orchidectomized rats, daily administration of a lead compound from this series showed anabolic activity by increasing levator ani muscle weight. Importantly, minimal androgenic effects (increased tissue weights) were observed in the prostate and seminal vesicles, along with minimal repression of circulating luteinizing hormone (LH) levels and no change in the lipid and triglyceride levels. This lead compound completed a two week rat toxicology study, and was well tolerated at doses up to 100 mg/kg/day, the highest dose tested, for 14 consecutive days.

Discovery of Dual VEGFR-2 and Tubulin Inhibitors with in Vivo Efficacy.

In an effort to develop potent, orally bioavailable compounds for the treatment of neoplastic diseases, we developed a class of dual VEGFR-2 kinase and tubulin inhibitors. Targeting the VEGFR receptor kinase and tubulin structure allows for inhibition of both tumor cells and tumor vasculature. Previously, a combination of two compounds, a VEGF receptor tyrosine kinase inhibitor and tubulin agent, was demonstrated to produce an enhanced antitumor response in animal studies. We have reaffirmed their results, with the added benefit that both activities are found in one compound.

Antitumor Activity of IMC-038525, a Novel Oral Tubulin Polymerization Inhibitor.

Microtubules are a well-validated target for anticancer therapy. Molecules that bind tubulin affect dynamic instability of microtubules causing mitotic arrest of proliferating cells, leading to cell death and tumor growth inhibition. Natural antitubulin agents such as taxanes and Vinca alkaloids have been successful in the treatment of cancer; however, several limitations have encouraged the development of synthetic small molecule inhibitors of tubulin function. We have previously reported the discovery of two novel chemical series of tubulin polymerization inhibitors, triazoles (Ouyang et al. Synthesis and structure-activity relationships of 1,2,4-triazoles as a novel class of potent tubulin polymerization inhibitors. Bioorg Med Chem Lett. 2005; 15:5154-5159) and oxadiazole derivatives (Ouyang et al. Oxadiazole derivatives as a novel class of antimitotic agents: synthesis, inhibition of tubulin polymerization, and activity in tumor cell lines. Bioorg Med Chem Lett. 2006; 16:1191-1196). Here, we report on the anticancer effects of a lead oxadiazole derivative in vitro and in vivo. In vitro, IMC-038525 caused mitotic arrest at nanomolar concentrations in epidermoid carcinoma and breast tumor cells, including multidrug-resistant cells. In vivo, IMC-038525 had a desirable pharmacokinetic profile with sustained plasma levels after oral dosing. IMC-038525 reduced subcutaneous xenograft tumor growth with significantly greater efficacy than the taxane paclitaxel. At efficacious doses, IMC-038525 did not cause substantial myelosuppression or peripheral neurotoxicity, as evaluated by neutrophil counts and changes in myelination of the sciatic nerve, respectively. These data indicate that IMC-038525 is a promising candidate for further development as a chemotherapeutic agent.