Discovery of a Series of 7-Azaindoles as Potent and Highly Selective CDK9 Inhibitors for Transient Target Engagement.

Optimization of a series of azabenzimidazoles identified from screening hit 2 and the information gained from a co-crystal structure of the azabenzimidazole-based lead 6 bound to CDK9 led to the discovery of azaindoles as highly potent and selective CDK9 inhibitors. With the goal of discovering a highly selective and potent CDK9 inhibitor administrated intravenously that would enable transient target engagement of CDK9 for the treatment of hematological malignancies, further optimization focusing on physicochemical and pharmacokinetic properties led to azaindoles 38 and 39. These compounds are highly potent and selective CDK9 inhibitors having short half-lives in rodents, suitable physical properties for intravenous administration, and the potential to achieve profound but transient inhibition of CDK9 in vivo.

Protein Ligand Interactions Using Surface Plasmon Resonance.

Surface Plasmon Resonance (SPR) is a powerful biophysical method for characterizing small molecule binding to proteins. Owing to its ability to characterize binary inteactions between warheads and E3 ligases or substrates, SPR is a useful tool for the development of targeted protein degraders. SPR is also an effective method for optimizing linkers and characterizing ternary complex interactions that are mediated by heterobifunctional ligands (Roy et al. ACS Chem Biol 14:361-368, 2019). Recent advances in the throughput of modern instruments have improved the ability of SPR to rapidly triage ligands based on binding kinetics and affinity, making this technique invaluable for driving degrader optimization. This chapter describes the characterization of ligands binding to the Thalidomide Binding Domain of mouse Cereblon (mCRBN-TBD) using the Biacore 8K+.

Discovery of AZD4573, a Potent and Selective Inhibitor of CDK9 That Enables Short Duration of Target Engagement for the Treatment of Hematological Malignancies.

A CDK9 inhibitor having short target engagement would enable a reduction of Mcl-1 activity, resulting in apoptosis in cancer cells dependent on Mcl-1 for survival. We report the optimization of a series of amidopyridines (from compound 2), focusing on properties suitable for achieving short target engagement after intravenous administration. By increasing potency and human metabolic clearance, we identified compound 24, a potent and selective CDK9 inhibitor with suitable predicted human pharmacokinetic properties to deliver transient inhibition of CDK9. Furthermore, the solubility of 24 was considered adequate to allow i.v. formulation at the anticipated effective dose. Short-term treatment with compound 24 led to a rapid dose- and time-dependent decrease of pSer2-RNAP2 and Mcl-1, resulting in cell apoptosis in multiple hematological cancer cell lines. Intermittent dosing of compound 24 demonstrated efficacy in xenograft models derived from multiple hematological tumors. Compound 24 is currently in clinical trials for the treatment of hematological malignancies.

Development of a Novel B-Cell Lymphoma 6 (BCL6) PROTAC To Provide Insight into Small Molecule Targeting of BCL6.

B-cell lymphoma 6 (BCL6) inhibition is a promising mechanism for treating hematological cancers but high quality chemical probes are necessary to evaluate its therapeutic potential. Here we report potent BCL6 inhibitors that demonstrate cellular target engagement and exhibit exquisite selectivity for BCL6 based on mass spectrometry analyses following chemical proteomic pull down. Importantly, a proteolysis-targeting chimera (PROTAC) was also developed and shown to significantly degrade BCL6 in a number of diffuse large B-cell lymphoma (DLBCL) cell lines, but neither BCL6 inhibition nor degradation selectively induced marked phenotypic response. To investigate, we monitored PROTAC directed BCL6 degradation in DLBCL OCI-Ly1 cells by immunofluorescence and discovered a residual BCL6 population. Analysis of subcellular fractions also showed incomplete BCL6 degradation in all fractions despite having measurable PROTAC concentrations, together providing a rationale for the weak antiproliferative response seen with both BCL6 inhibitor and degrader. In summary, we have developed potent and selective BCL6 inhibitors and a BCL6 PROTAC that effectively degraded BCL6, but both modalities failed to induce a significant phenotypic response in DLBCL despite achieving cellular concentrations.

Structure-Based Design of Selective Noncovalent CDK12 Inhibitors.

Cyclin-dependent kinase (CDK) 12 knockdown via siRNA decreases the transcription of DNA-damage-response genes and sensitizes BRCA wild-type cells to poly(ADP-ribose) polymerase (PARP) inhibition. To recapitulate this effect with a small molecule, we sought a potent, selective CDK12 inhibitor. Crystal structures and modeling informed hybridization between dinaciclib and SR-3029, resulting in lead compound 5 [(S)-2-(1-(6-(((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)amino)-9-ethyl-9H-purin-2-yl)piperidin-2-yl)ethan-1-ol]. Further structure-guided optimization delivered a series of selective CDK12 inhibitors, including compound 7 [(S)-2-(1-(6-(((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)amino)-9-isopropyl-9H-purin-2-yl)piperidin-2-yl)ethan-1-ol]. Profiling of this compound across CDK9, 7, 2, and 1 at high ATP concentration, single-point kinase panel screening against 352 targets at 0.1 µm, and proteomics via kinase affinity matrix technology demonstrated the selectivity. This series of compounds inhibits phosphorylation of Ser2 on the C-terminal repeat domain of RNA polymerase II, consistent with CDK12 inhibition. These selective compounds were also acutely toxic to OV90 as well as THP1 cells.

Discovery of Pyrazolo[1,5-a]pyrimidine B-Cell Lymphoma 6 (BCL6) Binders and Optimization to High Affinity Macrocyclic 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.

Target Deconvolution Efforts on Wnt Pathway Screen Reveal Dual Modulation of Oxidative Phosphorylation and SERCA2.

Wnt signaling is critical for development, cell proliferation and differentiation, and mutations in this pathway resulting in constitutive signaling have been implicated in various cancers. A pathway screen using a Wnt-dependent reporter identified a chemical series based on a 1,2,3-thiadiazole-5-carboxamide (TDZ) core with sub-micromolar potency. Herein we report a comprehensive mechanism-of-action deconvolution study toward identifying the efficacy target(s) and biological implication of this chemical series involving bottom-up quantitative chemoproteomics, cell biology, and biochemical methods. Through observing the effects of our probes on metabolism and performing confirmatory cellular and biochemical assays, we found that this chemical series inhibits ATP synthesis by uncoupling the mitochondrial potential. Affinity chemoproteomics experiments identified sarco(endo)plasmic reticulum Ca2+ -dependent ATPase (SERCA2) as a binding partner of the TDZ series, and subsequent validation studies suggest that the TDZ series can act as ionophores through SERCA2 toward Wnt pathway inhibition.

Unified Software Solution for Efficient SPR Data Analysis in Drug Research.

Surface plasmon resonance (SPR) is a powerful method for obtaining detailed molecular interaction parameters. Modern instrumentation with its increased throughput has enabled routine screening by SPR in hit-to-lead and lead optimization programs, and SPR has become a mainstream drug discovery technology. However, the processing and reporting of SPR data in drug discovery are typically performed manually, which is both time-consuming and tedious. Here, we present the workflow concept, design and experiences with a software module relying on a single, browser-based software platform for the processing, analysis, and reporting of SPR data. The efficiency of this concept lies in the immediate availability of end results: data are processed and analyzed upon loading the raw data file, allowing the user to immediately quality control the results. Once completed, the user can automatically report those results to data repositories for corporate access and quickly generate printed reports or documents. The software module has resulted in a very efficient and effective workflow through saved time and improved quality control. We discuss these benefits and show how this process defines a new benchmark in the drug discovery industry for the handling, interpretation, visualization, and sharing of SPR data.

Potent and Selective CK2 Kinase Inhibitors with Effects on Wnt Pathway Signaling in Vivo.

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 ß-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.

Asparagine deamidation reduces DNA-binding affinity of the Drosophila melanogaster Scr homeodomain.

Spontaneous deamidation of asparagine is a non-enzymatic post-translational modification of proteins. Residue Asn 321 is the main site of deamidation of the Drosophila melanogaster Hox transcription factor Sex Combs Reduced (Scr). Formation of iso-aspartate, the major deamidation product, is detected by HNCACB triple-resonance NMR spectroscopy. The rate of deamidation is quantified by fitting the decay of Asn NH2 side-chain signals in a time-series of (15)N-(1)H HSQC NMR spectra. The deamidated form of Scr binds to specific DNA target sequences with reduced affinity as determined by an electrophoretic mobility shift assay.

Quantitative investigation of the affinity of human respiratory syncytial virus phosphoprotein C-terminus binding to nucleocapsid protein.

BACKGROUND: There are no approved small molecule drug therapies for human respiratory syncytial virus (hRSV), a cause of morbidity and mortality in at-risk newborns, the immunocompromised, and the elderly. We have investigated as a potential novel hRSV drug target the protein-protein interaction between the C-terminus of the viral phosphoprotein (P) and the viral nucleocapsid protein (N), components of the ribonucleoprotein complex that contains, replicates, and transcribes the viral RNA genome. Earlier work by others established that the 9 C-terminal residues of P are necessary and sufficient for binding to N. METHODS: We used a fluorescence anisotropy assay, surface plasmon resonance and 2-D NMR to quantify the affinities of peptides based on the C terminus of P for RNA-free, monomeric N-terminal-truncated N(13-391). We calculated the contributions to the free energies of binding of P to N(13-391) attributable to the C-terminal 11 residues, phosphorylation of the C-terminal 2 serine residues, the C-terminal Asp-Phe, and the phenyl ring of the C-terminal Phe. RESULTS: Binding studies confirmed the crucial role of the phosphorylated C-terminal peptide D(pS)DNDL(pS)LEDF for binding of P to RNA-free, monomeric N(13-391), contributing over 90% of the binding free energy at low ionic strength. The phenyl ring of the C-terminal Phe residue contributed an estimated -2.7 kcal/mole of the free energy of binding, the C-terminal Asp-Phe residues contributed -3.8 kcal/mole, the sequence DSDNDLSLE contributed -3.1 kcal/mole, and phosphorylation of the 2 Ser residues contributed -1.8 kcal/mole. Due to the high negative charge of the C-terminal peptide, the affinity of the P C-terminus for N(13-391) decreased as the ionic strength increased. CONCLUSIONS: The results support the idea that the interaction of the C-terminal residues of P with N constitutes a protein-protein interaction hotspot that may be a suitable target for small-molecule drugs that inhibit viral genome replication and transcription.

NIPP1 maintains EZH2 phosphorylation and promoter occupancy at proliferation-related target genes.

The histone methyltransferase EZH2 regulates cell proliferation and differentiation by silencing Polycomb group target genes. NIPP1, a nuclear regulator of serine/threonine protein phosphatase 1 (PP1), has been implicated in the regulation of EZH2 occupancy at target loci, but the underlying mechanism is not understood. Here, we demonstrate that the phosphorylation of EZH2 by cyclin-dependent kinases at Thr416 creates a docking site for the ForkHead-associated domain of NIPP1. Recruited NIPP1 enables the net phosphorylation of EZH2 by inhibiting its dephosphorylation by PP1. Accordingly, a NIPP1-binding mutant of EZH2 is hypophosphorylated, and the knockdown of NIPP1 results in a reduced phosphorylation of endogenous EZH2. Conversely, the loss of PP1 is associated with a hyperphosphorylation of EZH2. A genome-wide promoter-binding profiling in HeLa cells revealed that the NIPP1-binding mutant shows a deficient association with about a third of the Polycomb target genes, and these are enriched for functions in proliferation. Our data identify PP1 as an EZH2 phosphatase and demonstrate that the phosphorylation-regulated association of EZH2 with proliferation-related targets depends on associated NIPP1.

The molecular basis for substrate specificity of the nuclear NIPP1:PP1 holoenzyme.

Regulation of protein phosphatase 1 (PP1) is controlled by a diverse array of regulatory proteins. However, how these proteins direct PP1 specificity is not well understood. More than one-third of the nuclear pool of PP1 forms a holoenzyme with the nuclear inhibitor of PP1, NIPP1, to regulate chromatin remodeling, among other essential biological functions. Here, we show that the PP1-binding domain of NIPP1 is an intrinsically disordered protein, which binds PP1 in an unexpected manner. NIPP1 forms an α helix that engages PP1 at a unique interaction site, using polar rather than hydrophobic contacts. Importantly, the structure also reveals a shared PP1 interaction site outside of the RVxF motif, the ΦΦ motif. Finally, we show that NIPP1:PP1 substrate selectivity is determined by altered electrostatics and enhanced substrate localization. Together, our results provide the molecular basis by which NIPP1 directs PP1 substrate specificity in the nucleus.

Phi-value analysis for ultrafast folding proteins by NMR relaxation dispersion.

Proteins that fold rapidly, on the (sub-) microsecond time scale, offer the prospect of direct comparison between experimental data and molecular dynamics simulations. However, experimental studies for such proteins frequently are hindered because folding rates are too fast to measure using conventional stopped-flow methods. To overcome this impediment, NMR spin relaxation dispersion experiments are used to quantify mutational effects on kinetics (DeltaDeltaG(o)), stability (DeltaDeltaG(o)), and phi-values (DeltaDeltaG(dagger)/DeltaDeltaG(o)) for proteins exhibiting chemical exchange line broadening that is fast on the NMR chemical shift time scale. The accuracy of phi-value analysis is enhanced because mutational effects on denatured or intermediate states can be detected through changes in line broadening. The transition and intermediate states of the villin headpiece domain, HP67, are characterized in varying solvent conditions to validate the method.

Partially folded equilibrium intermediate of the villin headpiece HP67 defined by 13C relaxation dispersion.

Identification and characterization of ensembles of intermediate states remains an important objective in describing protein folding in atomic detail. The 67-residue villin headpiece, HP67, consists of an N-terminal subdomain (residues 10-42) that transiently unfolds at equilibrium under native-like conditions and a highly stable C-terminal subdomain (residues 43-76). The transition between folded and unfolded states of the N-terminal domain has been characterized previously by (15)N NMR relaxation dispersion measurements (Grey et al. in J Mol Biol 355:1078, 2006). In the present work, (13)C spin relaxation was used to further characterize backbone and hydrophobic core contributions to the unfolding process. Relaxation of (13)C(alpha) spins was measured using the Hahn echo technique at five static magnetic fields (11.7, 14.1, 16.4, 18.8, and 21.1 T) and the Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion method at a static magnetic field of 14.1 T. Relaxation of methyl (13)C spins was measured using CPMG relaxation dispersion experiments at static magnetic fields of 14.1 and 18.8 T. Results for (13)C and (15)N spins yielded a consistent model in which the partially unfolded intermediate state of the N-terminal subdomain maintains residual structure for residues near the unprotonated His41 imidazole ring and in the interface between the N- and C-terminal subdomains. In addition, a second faster process was detected that appears to represent local dynamics within the folded state of the molecule and is largely confined to the hydrophobic interface between the N- and C-terminal subdomains.