Targeted degradation of MERTK and other TAM receptor paralogs by heterobifunctional targeted protein degraders.

TAM receptors (TYRO3, AXL, and MERTK) comprise a family of homologous receptor tyrosine kinases (RTK) that are expressed across a range of liquid and solid tumors where they contribute to both oncogenic signaling to promote tumor proliferation and survival, as well as expressed on myeloid and immune cells where they function to suppress host anti-tumor immunity. In recent years, several strategies have been employed to inhibit TAM kinases, most notably small molecule tyrosine kinase inhibitors and inhibitory neutralizing monoclonal antibodies (mAbs) that block receptor dimerization. Targeted protein degraders (TPD) use the ubiquitin proteasome pathway to redirect E3 ubiquitin ligase activity and target specific proteins for degradation. Here we employ first-in-class TPDs specific for MERTK/TAMs that consist of a cereblon E3 ligase binder linked to a tyrosine kinase inhibitor targeting MERTK and/or AXL and TYRO3. A series of MERTK TPDs were designed and investigated for their capacity to selectively degrade MERTK chimeric receptors, reduce surface expression on primary efferocytic bone marrow-derived macrophages, and impact on functional reduction in efferocytosis (clearance of apoptotic cells). We demonstrate proof-of-concept and establish that TPDs can be tailored to either selectivity degrades MERTK or concurrently degrade multiple TAMs and modulate receptor expression in vitro and in vivo. This work demonstrates the utility of proteome editing, enabled by tool degraders developed here towards dissecting the therapeutically relevant pathway biology in preclinical models, and the ability for TPDs to degrade transmembrane proteins. These data also provide proof of concept that TPDs may serve as a viable therapeutic strategy for targeting MERTK and other TAMs and that this technology could be expanded to other therapeutically relevant transmembrane proteins.

Identification and Characterization of Von Hippel-Lindau-Recruiting Proteolysis Targeting Chimeras (PROTACs) of TANK-Binding Kinase 1.

Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that recruit an E3 ligase to a target protein to facilitate ubiquitination and subsequent degradation of that protein. While the field of targeted degraders is still relatively young, the potential for this modality to become a differentiated and therapeutic reality is strong, such that both academic and pharmaceutical institutions are now entering this interesting area of research. In this article, we describe a broadly applicable process for identifying degrader hits based on the serine/threonine kinase TANK-binding kinase 1 (TBK1) and have generalized the key structural elements associated with degradation activities. Compound 3i is a potent hit (TBK1 DC50 = 12 nM, Dmax = 96%) with excellent selectivity against a related kinase IKKε, which was further used as a chemical tool to assess TBK1 as a target in mutant K-Ras cancer cells.

Discovery of tricyclic indoles that potently inhibit Mcl-1 using fragment-based methods and structure-based design.

Myeloid cell leukemia-1 (Mcl-1) is an antiapoptotic member of the Bcl-2 family of proteins that is overexpressed and amplified in many cancers. Overexpression of Mcl-1 allows cancer cells to evade apoptosis and contributes to the resistance of cancer cells to be effectively treated with various chemotherapies. From an NMR-based screen of a large fragment library, several distinct chemical scaffolds that bind to Mcl-1 were discovered. Here, we describe the discovery of potent tricyclic 2-indole carboxylic acid inhibitors that exhibit single digit nanomolar binding affinity to Mcl-1 and greater than 1700-fold selectivity over Bcl-xL and greater than 100-fold selectivity over Bcl-2. X-ray structures of these compounds when complexed to Mcl-1 provide detailed information on how these small-molecules bind to the target, which was used to guide compound optimization.

Discovery of potent myeloid cell leukemia 1 (Mcl-1) inhibitors using fragment-based methods and structure-based design.

Myeloid cell leukemia 1 (Mcl-1), a member of the Bcl-2 family of proteins, is overexpressed and amplified in various cancers and promotes the aberrant survival of tumor cells that otherwise would undergo apoptosis. Here we describe the discovery of potent and selective Mcl-1 inhibitors using fragment-based methods and structure-based design. NMR-based screening of a large fragment library identified two chemically distinct hit series that bind to different sites on Mcl-1. Members of the two fragment classes were merged together to produce lead compounds that bind to Mcl-1 with a dissociation constant of <100 nM with selectivity for Mcl-1 over Bcl-xL and Bcl-2. Structures of merged compounds when complexed to Mcl-1 were obtained by X-ray crystallography and provide detailed information about the molecular recognition of small-molecule ligands binding Mcl-1. The compounds represent starting points for the discovery of clinically useful Mcl-1 inhibitors for the treatment of a wide variety of cancers.

Inhibitors of the ROCK serine/threonine kinases: key effectors of the RhoA small GTPase.

Aberrant activation of the RhoA small GTPase has been implicated in cancer and other human diseases. Therefore, inhibitors of RhoA may have important therapeutic value. However, similar to the Ras small GTPases, RhoA itself is not considered a tractable target and is currently considered to be "undruggable." While recent efforts suggest that direct inhibitors of the Ras oncoprotein may yet be developed, the most promising directions for anti-Ras inhibitors involve inhibitors of protein kinases that are activated downstream of Ras. By analogy, protein kinases activated downstream of RhoA may provide more attractive directions for the development of anti-RhoA inhibitors. Among the multitude of RhoA effectors, the ROCK serine/threonine kinases have emerged as attractive targets for anti-RhoA drug discovery. In this review, we summarize the current status of the development of small molecule inhibitors of ROCK.

ROCK1 and ROCK2 are required for non-small cell lung cancer anchorage-independent growth and invasion.

Evidence is emerging that the closely related ROCK1 and ROCK2 serine/threonine kinases support the invasive and metastatic growth of a spectrum of human cancer types. Therefore, inhibitors of ROCK are under preclinical development. However, a key step in their development involves the identification of genetic biomarkers that will predict ROCK inhibitor antitumor activity. One identified mechanism for ROCK activation in cancer involves the loss of function of the DLC1 tumor suppressor gene, which encodes a GTPase activating protein (RhoGAP) for the RhoA and RhoC small GTPases. DLC-1 loss may lead to hyperactivation of RhoA/C and its downstream effectors, the ROCK kinases. We therefore determined whether loss of DLC-1 protein expression identifies non-small cell lung carcinoma (NSCLC) cell lines whose growth and invasion phenotypes are sensitive to ROCK inhibition. We identified and characterized a novel small molecule pharmacologic inhibitor of ROCK and additionally applied genetic approaches to impair ROCK1 and/or ROCK2 activity, and we determined that although NSCLC anchorage-dependent growth was ROCK-independent, both anchorage-independent growth and Matrigel invasion were ROCK-dependent. However, loss of DLC-1 expression did not correlate with ROCK activation or with OXA-06 sensitivity. Unexpectedly, suppression of ROCK1 or ROCK2 expression alone was sufficient to impair anchorage-independent growth, supporting their nonoverlapping roles in oncogenesis. Mechanistically, the block in anchorage-independent growth was associated with accumulation of cells in the G(0)-G(1) phase of the cell cycle, but not increased anoikis. We conclude that ROCK may be a useful therapeutic target for NSCLC.

RhoGDI2 antagonizes ovarian carcinoma growth, invasion and metastasis.

Previous studies described functional roles for Rho GDP dissociation inhibitor 2 (RhoGDI2) in bladder, gastric and breast cancers. However, only limited expression and no functional analyses have been done for RhoGDI2 in ovarian cancer. We determined RhoGDI2 protein expression and function in ovarian cancer. First, protein gel blot analysis was performed to determine the expression levels of RhoGDI2 in ovarian cells lines. RhoGDI2 but not RhoGDI1 protein expression levels varied widely in ovarian carcinoma cell lines, with elevated levels seen in Ras-transformed ovarian epithelial cells. Next, immunohistochemistry was performed to detect RhoGDI2 expression in patient samples of ovarian cysts and ovarian cancer with known histological subtype, stage, grade and outcome. RhoGDI2 protein was significantly overexpressed in high-grade compared with low-grade ovarian cancers, correlated with histological subtype, and did not correlate with stage of ovarian cancer nor between carcinomas and benign cysts. Unexpectedly, stable suppression of RhoGDI2 protein expression in HeyA8 ovarian cancer cells increased anchorage-independent growth and Matrigel invasion in vitro and in tail-vein lung colony metastatic growth in vivo. Finally, we found that RhoGDI2 stably-associated preferentially with Rac1 and suppression of RhoGDI2 expression resulted in decreased Rac1 activity and Rac-associated JNK and p38 mitogenactivated protein kinase signaling. RhoGDI2 antagonizes the invasive and metastatic phenotype of HeyA8 ovarian cancer cells. In summary, our results suggest significant cell context differences in RhoGDI2 function in cancer cell growth.

Ras superfamily GEFs and GAPs: validated and tractable targets for cancer therapy?

There is now considerable and increasing evidence for a causal role for aberrant activity of the Ras superfamily of small GTPases in human cancers. These GTPases function as GDP-GTP-regulated binary switches that control many fundamental cellular processes. A common mechanism of GTPase deregulation in cancer is the deregulated expression and/or activity of their regulatory proteins, guanine nucleotide exchange factors (GEFs) that promote formation of the active GTP-bound state and GTPase-activating proteins (GAPs) that return the GTPase to its GDP-bound inactive state. In this Review, we assess the association of GEFs and GAPs with cancer and their druggability for cancer therapeutics.

Aberrant overexpression of the Rgl2 Ral small GTPase-specific guanine nucleotide exchange factor promotes pancreatic cancer growth through Ral-dependent and Ral-independent mechanisms.

Our recent studies established essential and distinct roles for RalA and RalB small GTPase activation in K-Ras mutant pancreatic ductal adenocarcinoma (PDAC) cell line tumorigencity, invasion, and metastasis. However, the mechanism of Ral GTPase activation in PDAC has not been determined. There are four highly related mammalian RalGEFs (RalGDS, Rgl1, Rgl2, and Rgl3) that can serve as Ras effectors. Whether or not they share distinct or overlapping functions in K-Ras-mediated growth transformation has not been explored. We found that plasma membrane targeting to mimic persistent Ras activation enhanced the growth-transforming activities of RalGEFs. Unexpectedly, transforming activity did not correlate directly with total cell steady-state levels of Ral activation. Next, we observed elevated Rgl2 expression in PDAC tumor tissue and cell lines. Expression of dominant negative Ral, which blocks RalGEF function, as well as interfering RNA suppression of Rgl2, reduced PDAC cell line steady-state Ral activity, growth in soft agar, and Matrigel invasion. Surprisingly, the effect of Rgl2 on anchorage-independent growth could not be rescued by constitutively activated RalA, suggesting a novel Ral-independent function for Rgl2 in transformation. Finally, we determined that Rgl2 and RalB both localized to the leading edge, and this localization of RalB was dependent on endogenous Rgl2 expression. In summary, our observations support nonredundant roles for RalGEFs in Ras-mediated oncogenesis and a key role for Rgl2 in Ral activation and Ral-independent PDAC growth.

Functional and structural characterization of a thiol peroxidase from Mycobacterium tuberculosis.

A thiol peroxidase (Tpx) from Mycobacterium tuberculosis was functionally analyzed. The enzyme shows NADPH-linked peroxidase activity using a thioredoxin-thioredoxin reductase system as electron donor, and anti-oxidant activity in a thiol-dependent metal-catalyzed oxidation system. It reduces H2O2, t-butyl hydroperoxide, and cumene hydroperoxide, and is inhibited by sulfhydryl reagents. Mutational studies revealed that the peroxidatic (Cys60) and resolving (Cys93) cysteine residues are critical amino acids for catalytic activity. The X-ray structure determined to a resolution of 1.75 A shows a thioredoxin fold similar to that of other peroxiredoxin family members. Superposition with structural homologues in oxidized and reduced forms indicates that the M. tuberculosis Tpx is a member of the atypical two-Cys peroxiredoxin family. In addition, the short distance that separates the Calpha atoms of Cys60 and Cys93 and the location of these cysteine residues in unstructured regions may indicate that the M. tuberculosis enzyme is oxidized, though the side-chain of Cys60 is poorly visible. It is solely in the reduced Streptococcus pneumoniae Tpx structure that both residues are part of two distinct helical segments. The M. tuberculosis Tpx is dimeric both in solution and in the crystal structure. Amino acid residues from both monomers delineate the active site pocket.

Solution scattering reveals large differences in the global structures of type II protein kinase A isoforms.

Isoform diversity within the protein kinase A (PKA) family is achieved by catalytic (C) subunits binding to different isoforms of regulatory subunit homodimers (R2). In a previous small-angle X-ray scattering study, we showed that the type Ialpha R2 homodimer has a distinctive Y-shaped structure, while the IIalpha and IIbeta homodimers are highly flexible and extended in solution. Here we present the results of X-ray scattering experiments on different isoforms of the PKA holoenzyme (R2C2) and show that the type IIbeta R2 homodimer undergoes a dramatic compaction upon binding C subunits that involves a 10A reduction in radius of gyration (from 56 to 46 A) and a 35 A shortening of the maximum linear dimension (from 180-145 A). In contrast, the type IIalpha R2 homodimer shows very little change in these structural parameters and remains extended upon C-subunit binding. This large difference is surprising given the highly conserved sequence and domain organization for the different R isoforms. A mutant RIIbeta holoenzyme and an RIIalpha/RIIbeta chimera were used to explore the role of the sequence linking different functional domains within RIIbeta in the observed C subunit-induced compaction. Structural modeling was used to aid in interpreting the scattering results in terms of the role of inter-domain and inter-subunit contacts in determining the global conformations of the different isoforms. The results provide an important structural foundation for understanding isoform-specific PKA localization and signaling.

A simple electrostatic switch important in the activation of type I protein kinase A by cyclic AMP.

Cyclic AMP activates protein kinase A by binding to an inhibitory regulatory (R) subunit and releasing inhibition of the catalytic (C) subunit. Even though crystal structures of regulatory and catalytic subunits have been solved, the precise molecular mechanism by which cyclic AMP activates the kinase remains unknown. The dynamic properties of the cAMP binding domain in the absence of cAMP or C-subunit are also unknown. Here we report molecular-dynamics simulations and mutational studies of the RIalpha R-subunit that identify the C-helix as a highly dynamic switch which relays cAMP binding to the helical C-subunit binding regions. Furthermore, we identify an important salt bridge which links cAMP binding directly to the C-helix that is necessary for normal activation. Additional mutations show that a hydrophobic "hinge" region is not as critical for the cross-talk in PKA as it is in the homologous EPAC protein, illustrating how cAMP can control diverse functions using the evolutionarily conserved cAMP-binding domains.

Dynamics of signaling by PKA.

The catalytic and regulatory subunits of cAMP-dependent protein kinase (PKA) are highly dynamic signaling proteins. In its dissociated state the catalytic subunit opens and closes as it moves through its catalytic cycle. In this subunit, the core that is shared by all members of the protein kinase family is flanked by N- and C-terminal segments. Each are anchored firmly to the core by well-defined motifs and serve to stabilize the core. Protein kinases are not only catalysts, they are also scaffolds. One of their major functions is to bind to other proteins. In addition to its interactions with the N- and C- termini, the catalytic subunit interacts with its inhibitor proteins, PKI and the regulatory subunits. Both bind with subnanomolar affinity. To achieve this tight binding requires docking of a substrate mimetic to the active site cleft as well as a peripheral docking site. The peripheral site used by PKI is distinct from that used by RIalpha as revealed by a recent structure of a C:RIalpha complex. Upon binding to the catalytic subunit, the linker region of RIalpha becomes ordered. In addition, cAMP-binding domain A undergoes major conformational changes. RIalpha is a highly malleable protein. Using small angle X-ray scattering, the overall shape of the regulatory subunits and corresponding holoenzymes have been elucidated. These studies reveal striking and surprising isoform differences.

The conformationally dynamic C helix of the RIalpha subunit of protein kinase A mediates isoform-specific domain reorganization upon C subunit binding.

Different isoforms of the full-length protein kinase A (PKA) regulatory subunit homodimer (R2) and the catalytic (C) subunit-bound holoenzyme (R2C2) have very different global structures despite similar molecular weights and domain organization within their primary sequences. To date, it has been the linker sequence between the R subunit dimerization/docking domain and cAMP-binding domain A that has been implicated in modulating domain interactions to give rise to these differences in global structure. The small angle solution scattering data presented here for three different isoforms of PKA heterodimer (deltaR-C) complexes reveal a role for another conformationally dynamic sequence in modulating inter-subunit and domain interactions, the C helix that connects the cAMP-binding domains A and B of the R subunit. The deltaR-C heterodimer complexes studied here were each formed with a monomeric N-terminal deletion mutant of the R subunit (deltaR) that contains the inhibitor sequence and both cAMP-binding domains. The scattering data show that type IIalpha and type IIbeta deltaR-C heterodimers are relatively compact and globular, with the C subunit contacting the inhibitor sequence and both cAMP-binding domains. In contrast, the type Ialpha heterodimer is significantly more extended, with the C subunit interacting with the inhibitor sequence and cAMP-binding domain A, whereas domain B extends out such that its surface is almost completely solvent exposed. These data implicate the C helix of RIalpha in modulating isoform-specific interdomain communication in the PKA holoenzyme, adding another layer of structural complexity to our understanding of signaling dynamics in this multisubunit, multidomain protein kinase.

Differential effects of substrate on type I and type II PKA holoenzyme dissociation.

It has been widely accepted that cAMP activates the protein kinase A (PKA) holoenzyme by dissociating the regulatory and catalytic subunits, thus freeing the catalytic subunit to phosphorylate its targets. However, recent experiments suggest that cAMP does not fully dissociate the holoenzyme. Here, we investigate this mechanism further by using small-angle X-ray scattering to study, at physiological enzyme concentrations, the type Ialpha and type IIbeta holoenzyme structures under equilibrium solution conditions without any labeling of the protein subunits. We observe that while the addition of a molar excess of cAMP to the type Ialpha PKA holoenzyme causes partial dissociation, it is only upon addition of a PKA peptide substrate together with cAMP that full dissociation occurs. Similarly, addition of excess cAMP to the type IIbeta holoenzyme causes only a partial dissociation. However, while the addition of peptide substrate as well as excess cAMP causes somewhat more dissociation, a significant percentage of intact type IIbeta holoenzyme remains. These results confirm that both the type Ialpha and the type IIbeta holoenzymes are more stable in the presence of cAMP than previously thought. They also demonstrate that substrate plays a differential role in the activation of type I versus type II holoenzymes, which could explain some important functional differences between PKA isoforms. On the basis of these data and other recently published data, we propose a structural model of type I holoenzyme activation by cAMP.

Conformational differences among solution structures of the type Ialpha, IIalpha and IIbeta protein kinase A regulatory subunit homodimers: role of the linker regions.

The regulatory (R) subunits of the cAMP-dependent protein kinase (protein kinase A or PKA) are multi-domain proteins responsible for conferring cAMP-dependence and localizing PKA to specific subcellular locations. There are four isoforms of the R subunit in mammals that are similar in molecular mass and domain organization, but clearly serve different biological functions. Although high-resolution structures are available for the cAMP-binding domains and dimerization/docking domains of two isoforms, there are no high-resolution structures of any of the intact R subunit homodimer isoforms. The results of small-angle X-ray scattering studies presented here indicate that the RIalpha, RIIalpha, and RIIbeta homodimers differ markedly in overall shape, despite extensive sequence homology and similar molecular masses. The RIIalpha and RIIbeta homodimers have very extended, rod-like shapes, whereas the RIalpha homodimer likely has a compact Y-shape. Based on a comparison of the R subunit sequences, we predict that the linker regions are the likely cause of these large differences in shape among the isoforms. In addition, we show that cAMP binding does not cause large conformational changes in type Ialpha or IIalpha R subunit homodimers, suggesting that the activation of PKA by cAMP involves only local conformational changes in the R subunits.

C subunits binding to the protein kinase A RI alpha dimer induce a large conformational change.

We present structural data on the RI alpha isoform of the cAMP-dependent protein kinase A that reveal, for the first time, a large scale conformational change within the RI alpha homodimer upon catalytic subunit binding. This result infers that the inhibition of catalytic subunit activity is not the result of a simple docking process but rather is a multi-step process involving local conformational changes both in the cAMP-binding domains as well as in the linker region of the regulatory subunit that impact the global structure of the regulatory homodimer. The results were obtained using small-angle neutron scattering with contrast variation and deuterium labeling. From these experiments we derived information on the shapes and dispositions of the catalytic subunits and regulatory homodimer within a holoenzyme reconstituted with a deuterated regulatory subunit. The scattering data also show that, despite extensive sequence homology between the isoforms, the overall structure of the type I alpha holoenzyme is significantly more compact than the type II alpha isoform. We present a model of the type I alpha holoenzyme, built using available high-resolution structures of the component subunits and domains, which best fits the neutron-scattering data. In this model, the type I alpha holoenzyme forms a flattened V shape with the RI alpha dimerization domain at the point of the V and the cAMP-binding domains of the RI alpha subunits with their bound catalytic subunits at the ends.