Casein kinase 2 (CK2), comprising the catalytic subunits CK2α and CK2α', is a highly conserved and constitutively active serine/threonine kinase that is implicated in oncogenic signaling and tumor maintenance, making it an attractive therapeutic target. We report a medicinal chemistry campaign that delivered an imidazotriazine pan-CK2 series culminating in BMS-135 and its phosphate prodrug BMS-159. Structure-guided design enabled a scaffold hop from imidazopyridazine to imidazotriazine that improved kinome selectivity while preserving critical hinge and Lys68 interactions. Iterative SAR optimization mitigated hERG liability by modulating distal basicity and enhanced metabolic stability via a C8 N-ethyl substitution that blocked N-dealkylation, delivering BMS-135 as a sub-nanomolar CK2 inhibitor with favorable ADMET properties and robust antitumor efficacy across xenograft and patient-derived xenograft models. Subsequent pharmaceutical optimization through a prodrug strategy afforded BMS-159, which markedly improved solubility and enabled oral delivery of the parent with acceptable bioavailability and pharmacokinetic properties suitable for further development.

Abstract UFMylation is an important biological process where proteins are post-translationally modified via the covalent attachment of the ubiquitin-like modifier (UBL), ubiquitin-fold modifier-1 (UFM1). UFMylation is analogous to ubiquitination, occurring via the transfer of UFM1 across E1-, E2-, and E3-like enzymes, represented by ubiquitin-like modifier-activating enzyme 5 (UBA5), ubiquitin-fold modifier conjugating enzyme 1 (UFC1), and UFM1-specific ligase 1 (UFL1), respectively. Recent work has shown that dysregulation of UFMylation is associated with several diseases, sparking interest in characterizing its enzymes as potential drug targets. To date, only inhibitors targeting UBA5 have been identified, but no such ligands exist for UFC1. In this study, we present the structure of UFC1 in complex with the sulfonic acid CAPS, which revealed a novel ligand-binding pocket in UFC1. Using biophysical assays, coupled with X-ray crystallographic studies of UFC1 variants, we show that binding of CAPS to UFC1 appears pH-dependent and is enhanced by Tyr42. Further, our TSA data show that other sulfa- and sulfonate-based compounds induce dose-dependent destabilization of UFC1, consistent with weak but direct interactions with the enzyme. Lastly, using a UFMylation assay, we show that CAPS, along with Tyr42, may have a limited influence on UFM1 transfer to UFC1 and, consequently, downstream UFMylation of protein substrates. Nevertheless, our data indicate that the CAPS-binding pocket may serve as a design scaffold for the development of UFC1 modulators. With UFC1 emerging as a drug target, our study provides a possible avenue for the design and development of novel UFC1-specific modulators with therapeutic potential.

HSP47 (Heat Shock Protein 47) is a SERPIN (H1), which binds to procollagen, and is among several molecular chaperones involved in the assembly of the collagen triple helix. Inhibition of HSP47 function is anticipated to result in reduction in collagen secretion; therefore, hsp47 may be a therapeutic target for the treatment of fibrotic diseases. The structure of HSP47 has previously been determined using a C-terminal His-tag construct in the apo form (4AUA) and with several different collagen model peptides (3ZHA, 4AU2, 4AU3, 4AXY, 7BEE, 7BDU, and 7BFI; Widmer et al., 2012; Abraham et al., 2021). Despite a published protocol for growing HSP47 crystals, we encountered several challenges in obtaining crystals. Issues encountered included the C-terminal His-tag protein tended to precipitate at concentrations described and, moreover, crystallization was difficult. While we were able to obtain crystals, these structures were suboptimal, and we were never able to grow crystals with a C-terminal His-tag construct that replicated 4AU4 either in crystal form or diffraction limit. We describe three new apo crystal forms that we grew with the Widmer et al. 2012 construct, which all show association through the non-cleavable C-terminal-His-tag. Addressing suboptimal crystals, we describe construct design and a crystallization chaperone strategy and show that using a cleavable N-terminal tag led to a more tractable protein and higher resolution diffraction and that using crystallization chaperones in the form of Adnectins led to even better diffracting crystals.

While the progression of STING activators into the clinic has been successful, the discovery and clinical progression of STING inhibitors remain elusive. Questions persist about the molecular properties needed to distinguish between a STING activator and inhibitor, particularly within SAVI disease, a monogenic autoinflammatory disease that renders STING constitutively active, and how different conformations correlate to function. In this work, we use an orthosteric STING activator and inhibitor from the same chemical series to discover that STING M271 is a critical residue for molecular activation that can be leveraged as a unique molecular signature for pharmacological or genetically driven activation and inhibition. Furthermore, we demonstrate how the therapeutic requirements of a molecular corrector of SAVI STING differs from an orthosteric STING inhibitor, and why this is important for the SAVI disease population.

Ubiquitin-like modifier activating enzyme 5 (UBA5), an E1 enzyme in the UFMylation pathway, is of interest as a therapeutic target in diseases such as cancer due to the critical role that UFMylation plays in cellular function. Strategic structure-activity relationship optimization of high-throughput screen hit 6, a Bruton's tyrosine kinase covalent inhibitor, was conducted by using both conventional and microscale library synthesis. This approach identified 49 and 50 as potent and selective noncovalent UBA5 inhibitors with significantly improved surface plasmon resonance and nanodifferential scanning fluorimetry biophysical profiles. Cellular target engagement for 49 and 50 was confirmed by a cellular thermal shift assay and translated to inhibition of UFMylation of UBA5 and UFC1 in retinal pigment epithelial-1 cells as well as reduction of UFMylation of the downstream protein UFMylation substrate RPL26. Furthermore, 49 and 50 demonstrated specificity for UBA5 among the other E1-E2 transesterification pathways. In this communication, we report the discovery and synthesis of potent and selective UBA5 inhibitors, providing valuable tool compounds for studying the UFMylation pathway.

Heat-shock protein 47 (HSP47) is a potential target for inhibitors that ameliorate fibrosis by reducing collagen assembly. In an effort to develop a structure-based drug-design system, it was not possible to replicate a previous literature result (PDB entry 4au4) for apo dog HSP47; instead, crystal forms were obtained in which pairs of dog HSP47 molecules interacted through a noncleavable C-terminal His-tag to build up tetramers, all of which had multiple molecules of HSP47 in the asymmetric unit and none of which diffracted as well as the literature precedent. To overcome these difficulties, a two-pronged approach was followed: (i) the His-tag was moved from the C-terminus to the N-terminus and was made cleavable, and (ii) Adnectin (derived from the tenth domain of human fibronectin type III) crystallization chaperones were developed. Both approaches provided well diffracting crystals, but the latter approach yielded crystal forms with only one or two HSP47 complexes per asymmetric unit, which made model building less onerous.

STING is broadly implicated in diseases ranging from cancer, autoimmune disease, neurodegeneration to rare, monogenic diseases.[1][1] Early drug discovery campaigns focused on STING activation as a promising platform for cancer immunotherapy yet failed in multiple clinical trials due to lack of efficacy thus far.[2][2] Current research and development activities concentrate on STING inhibition for treating autoimmune disease and neuroinflammation. While the progression of STING activators into the clinic has been successful, the discovery and clinical progression of STING inhibitors remain elusive. Questions persist about the molecular properties needed to distinguish between a STING activator and inhibitor, particularly within SAVI disease, a monogenic autoinflammatory disease that renders STING constitutively active.[3][3] Here we leverage an orthosteric STING activator and inhibitor from the same chemical series to discover that STING M271 is a critical residue for molecular activation. The M271CH3 NMR chemical shifts reveal a unique molecular signature for pharmacological or genetically driven activation and inhibition that is not captured by x-ray crystallography. Additionally, M271 directly interacts with the most common SAVI mutation, V155M, and using an orthosteric STING inhibitor, we show partial rescue and molecular correction of STING V155M. Finally, these data present insights into therapeutic STING molecular correction for treating SAVI patients. Our results elucidate an unappreciated structural interaction critical for STING modulation that could be utilized as a molecular diagnostic tool for drug discovery. Furthermore, we demonstrate for the first time how the therapeutic requirements of a molecular corrector differ from an orthosteric STING inhibitor, and why this is important for the SAVI disease population. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3

Kinase inhibition continues to be a major focus of pharmaceutical research and discovery due to the central role of these proteins in the regulation of cellular processes. One family of kinases of pharmacological interest, due to its role in activation of immunostimulatory pathways, is the Janus kinase family. Small molecule inhibitors targeting the individual kinase proteins within this family have long been sought-after therapies. High sequence and structural similarity of the family members makes selective inhibitors difficult to identify but critical because of their inter-related multiple cellular regulatory pathways. Herein, we describe the identification of inhibitors of the important Janus kinase, TYK2, a regulator of type I interferon response. In addition, the biochemical and structural confirmation of the direct interaction of these small molecules with the TYK2 pseudokinase domain is described and a potential mechanism of allosteric regulation of TYK2 activity through stabilization of the pseudokinase domain is proposed.

This annual review is the sixth of its kind since 2016 (see references). Our objective is to explore and share articles which we deem influential and significant in the field of biotransformation and bioactivation. These fields are constantly evolving with new molecular structures and discoveries of corresponding pathways for metabolism that impact relevant drug development with respect to efficacy and safety. Based on the selected articles, we created three sections: (1) drug design, (2) metabolites and drug metabolizing enzymes, and (3) bioactivation and safety (Table 1). Unlike in years past, more biotransformation experts have joined and contributed to this effort while striving to maintain a balance of authors from academic and industry settings.[Table: see text].

Small molecule JAK inhibitors have emerged as a major therapeutic advancement in treating autoimmune diseases. The discovery of isoform selective JAK inhibitors that traditionally target the catalytically active site of this kinase family has been a formidable challenge. Our strategy to achieve high selectivity for TYK2 relies on targeting the TYK2 pseudokinase (JH2) domain. Herein we report the late stage optimization efforts including a structure-guided design and water displacement strategy that led to the discovery of BMS-986165 (11) as a high affinity JH2 ligand and potent allosteric inhibitor of TYK2. In addition to unprecedented JAK isoform and kinome selectivity, 11 shows excellent pharmacokinetic properties with minimal profiling liabilities and is efficacious in several murine models of autoimmune disease. On the basis of these findings, 11 appears differentiated from all other reported JAK inhibitors and has been advanced as the first pseudokinase-directed therapeutic in clinical development as an oral treatment for autoimmune diseases.

In sharp contrast to a previously reported series of 6-anilino imidazopyridazine based Tyk2 JH2 ligands, 6-((2-oxo-N1-substituted-1,2-dihydropyridin-3-yl)amino)imidazo[1,2-b]pyridazine analogs were found to display dramatically improved metabolic stability. The N1-substituent on 2-oxo-1,2-dihydropyridine ring can be a variety of alkyl, aryl, and heteroaryl groups, but among them, 2-pyridyl provided much enhanced Caco-2 permeability, attributed to its ability to form intramolecular hydrogen bonds. Further structure-activity relationship studies at the C3 position led to the identification of highly potent and selective Tyk2 JH2 inhibitor 6, which proved to be highly effective in inhibiting IFNγ production in a rat pharmacodynamics model and fully efficacious in a rat adjuvant arthritis model.

As a member of the Janus (JAK) family of nonreceptor tyrosine kinases, TYK2 plays an important role in mediating the signaling of pro-inflammatory cytokines including IL-12, IL-23, and type 1 interferons. The nicotinamide 4, identified by a SPA-based high-throughput screen targeting the TYK2 pseudokinase domain, potently inhibits IL-23 and IFNα signaling in cellular assays. The described work details the optimization of this poorly selective hit (4) to potent and selective molecules such as 47 and 48. The discoveries described herein were critical to the eventual identification of the clinical TYK2 JH2 inhibitor (see following report in this issue). Compound 48 provided robust inhibition in a mouse IL-12-induced IFNγ pharmacodynamic model as well as efficacy in an IL-23 and IL-12-dependent mouse colitis model. These results demonstrate the ability of TYK2 JH2 domain binders to provide a highly selective alternative to conventional TYK2 orthosteric inhibitors.

In solving the P-gp and BCRP transporter-mediated efflux issue in a series of benzofuran-derived pan-genotypic palm site inhibitors of the hepatitis C virus NS5B replicase, it was found that close attention to physicochemical properties was essential. In these compounds, where both molecular weight (MW >579) and TPSA (>110 Å2) were high, attenuation of polar surface area together with weakening of hydrogen bond acceptor strength of the molecule provided a higher intrinsic membrane permeability and more desirable Caco-2 parameters, as demonstrated by trifluoroacetamide 11 and the benchmark N-ethylamino analog 12. In addition, the tendency of these inhibitors to form intramolecular hydrogen bonds potentially contributes favorably to the improved membrane permeability and absorption. The functional group minimization that resolved the efflux problem simultaneously maintained potent inhibitory activity toward a gt-2 HCV replicon due to a switching of the role of substituents in interacting with the Gln414 binding pocket, as observed in gt-2a NS5B/inhibitor complex cocrystal structures, thus increasing the efficiency of the optimization. Noteworthy, a novel intermolecular S=O···C=O n → π* type interaction between the ligand sulfonamide oxygen atom and the carbonyl moiety of the side chain of Gln414 was observed. The insights from these structure-property studies and crystallography information provided a direction for optimization in a campaign to identify second generation pan-genotypic NS5B inhibitors.

The design and synthesis of potent, tripeptidic acylsulfonamide inhibitors of HCV NS3 protease that contain a difluoromethyl cyclopropyl amino acid at P1 are described. A cocrystal structure of 18 with a NS3/4A protease complex suggests the presence of a H-bond between the polarized C-H of the CHF2 moiety and the backbone carbonyl of Leu135 of the enzyme. Structure-activity relationship studies indicate that this H-bond enhances enzyme inhibitory potency by 13- and 17-fold compared to the CH3 and CF3 analogues, respectively, providing insight into the deployment of this unique amino acid.

The development of a series of novel 7-azabenzofurans exhibiting pan-genotype inhibition of HCV NS5B polymerase via binding to the primer grip site is presented. Many challenges, including poor oral bioavailability, high clearance, bioactivation, high human serum shift, and metabolic stability were encountered and overcome through SAR studies. This work culminated in the selection of BMS-986139 (43) as a preclinical candidate.

Kynurenine aminotransferases convert kynurenine to kynurenic acid and play an important role in the tryptophan degradation pathway. Kynurenic acid levels in brain have been hypothesized to be linked to a number of central nervous system (CNS) disorders. Kynurenine aminotransferase II (KATII) has proven to be a key modulator of kynurenic acid levels in brain and, thus, is an attractive target to treat CNS diseases. A sensitive, high-throughput, label-free RapidFire mass spectrometry assay has been developed for human KATII. Unlike other assays, this method is directly applicable to KATII enzymes from different animal species, which allows us to select proper animal model(s) to evaluate human KATII inhibitors. We also established a coupled fluorescence assay for human KATII. The short assay time and kinetic capability of the fluorescence assay provide a useful tool for orthogonal inhibitor validation and mechanistic studies.

The discovery of a back-up to the hepatitis C virus NS3 protease inhibitor asunaprevir (2) is described. The objective of this work was the identification of a drug with antiviral properties and toxicology parameters similar to 2, but with a preclinical pharmacokinetic (PK) profile that was predictive of once-daily dosing. Critical to this discovery process was the employment of an ex vivo cardiovascular (CV) model which served to identify compounds that, like 2, were free of the CV liabilities that resulted in the discontinuation of BMS-605339 (1) from clinical trials. Structure-activity relationships (SARs) at each of the structural subsites in 2 were explored with substantial improvement in PK through modifications at the P1 site, while potency gains were found with small, but rationally designed structural changes to P4. Additional modifications at P3 were required to optimize the CV profile, and these combined SARs led to the discovery of BMS-890068 (29).

Inhibition of signal transduction downstream of the IL-23 receptor represents an intriguing approach to the treatment of autoimmunity. Using a chemogenomics approach marrying kinome-wide inhibitory profiles of a compound library with the cellular activity against an IL-23-stimulated transcriptional response in T lymphocytes, a class of inhibitors was identified that bind to and stabilize the pseudokinase domain of the Janus kinase tyrosine kinase 2 (Tyk2), resulting in blockade of receptor-mediated activation of the adjacent catalytic domain. These Tyk2 pseudokinase domain stabilizers were also shown to inhibit Tyk2-dependent signaling through the Type I interferon receptor but not Tyk2-independent signaling and transcriptional cellular assays, including stimulation through the receptors for IL-2 (JAK1- and JAK3-dependent) and thrombopoietin (JAK2-dependent), demonstrating the high functional selectivity of this approach. A crystal structure of the pseudokinase domain liganded with a representative example showed the compound bound to a site analogous to the ATP-binding site in catalytic kinases with features consistent with high ligand selectivity. The results support a model where the pseudokinase domain regulates activation of the catalytic domain by forming receptor-regulated inhibitory interactions. Tyk2 pseudokinase stabilizers, therefore, represent a novel approach to the design of potent and selective agents for the treatment of autoimmunity.