The search for novel proline analogs for viscosity reduction and stabilization of highly concentrated monoclonal antibody solutions.

Administration of monoclonal antibodies (mAbs) is currently focused on subcutaneous injection associated with increased patient adherence and reduced treatment cost, leading to sustainable healthcare. The main bottleneck is low volume that can be injected, requiring highly concentrated mAb solutions. The latter results in increased solution viscosity with pronounced mAb aggregation propensity because of intensive protein-protein interactions. Small molecule excipients have been proposed to restrict the protein-protein interactions, contributing to reduced viscosity. The aim of the study was to discover novel compounds that reduce the viscosity of highly concentrated mAb solution. First, the chemical space of proline analogs was explored and 35 compounds were determined. Viscosity measurements revealed that 18 proline analogs reduced the mAb solution viscosity similar to or more than proline. The compounds forming both electrostatic and hydrophobic interactions with mAb reduced the viscosity of the formulation more efficiently without detrimentally effecting mAb physical stability. A correlation between the level of interaction and viscosity-reducing effect was confirmed with molecular dynamic simulations. Structure rigidity of the compounds and aromaticity contributed to their viscosity-reducing effect, dependent on molecule size. The study results highlight the novel proline analogs as an effective approach in viscosity reduction in development of biopharmaceuticals for subcutaneous administration.

Leveraging Ligand Affinity and Properties: Discovery of Novel Benzamide-Type Cereblon Binders for the Design of PROTACs.

Immunomodulatory imide drugs (IMiDs) such as thalidomide, pomalidomide, and lenalidomide are the most common cereblon (CRBN) recruiters in proteolysis-targeting chimera (PROTAC) design. However, these CRBN ligands induce the degradation of IMiD neosubstrates and are inherently unstable, degrading hydrolytically under moderate conditions. In this work, we simultaneously optimized physiochemical properties, stability, on-target affinity, and off-target neosubstrate modulation features to develop novel nonphthalimide CRBN binders. These efforts led to the discovery of conformationally locked benzamide-type derivatives that replicate the interactions of the natural CRBN degron, exhibit enhanced chemical stability, and display a favorable selectivity profile in terms of neosubstrate recruitment. The utility of the most potent ligands was demonstrated by their transformation into potent degraders of BRD4 and HDAC6 that outperform previously described reference PROTACs. Together with their significantly decreased neomorphic ligase activity on IKZF1/3 and SALL4, these ligands provide opportunities for the design of highly selective and potent chemically inert proximity-inducing compounds.

Tunable Heteroaromatic Nitriles for Selective Bioorthogonal Click Reaction with Cysteine.

The binucleophilic properties of 1,2-aminothiol and its rare occurrence in nature make it a useful reporter for tracking molecules in living systems. The 1,2-aminothiol moiety is present in cysteine, which is a substrate for a biocompatible click reaction with heteroaromatic nitriles. Despite the wide range of applications for this reaction, the scope of nitrile substrates has been explored only to a limited extent. In this study, we expand the chemical space of heteroaromatic nitriles for bioconjugation under physiologically relevant conditions. We systematically assembled a library of 116 2-cyanobenzimidazoles, 1-methyl-2-cyanobenzimidazoles, 2-cyanobenzothiazoles, and 2-cyanobenzoxazoles containing electron-donating and electron-withdrawing substituents at all positions of the benzene ring. The compounds were evaluated for their stability, reactivity, and selectivity toward the N-terminal cysteine of model oligopeptides. In comparison to the benchmark 6-hydroxy-2-cyanobenzothiazole or 6-amino-2-cyanobenzothiazole, we provide highly selective and moderately reactive nitriles as well as highly reactive yet less selective analogs with a variety of enabling attachment chemistries to aid future applications in bioconjugation, chemical biology, and nanomaterial science.

Optimization of triazolo[4,5-d]pyrimidines towards human CC chemokine receptor 7 (CCR7) antagonists.

CCR7 signaling directs the migration of both immune cells and cancer cells to the lymph nodes, is involved in numerous chronic inflammatory disorders and lymph node metastases. Despite the therapeutic promise of CCR7 antagonists, no potent and selective small molecule CCR7 antagonists have been reported to date. Since most human chemokine G protein-coupled receptors (GPCRs) share a conserved intracellular allosteric binding site, new CCR7 antagonist chemotypes may be identified by screening small molecules that are known to target this site in other chemokine GPCRs. In this work, our previously prepared series of 14 scaffold-modified analogues of a known thiazolo[4,5-d]pyrimidine CXCR2 antagonist were screened as potential CCR7 antagonists. This resulted in the discovery of a triazolo[4,5-d]pyrimidine analogue with an IC50 of 2.43 µM against CCR7 and 0.66 µM against CXCR2. Exploration of the structure-activity relationship (SAR) for the 3-, 5- and 7-position substituents of this triazolo[4,5-d]pyrimidine resulted in improved potency and selectivity, with an IC50 of 0.43 µM and 11.02 µM against CCR7 and CXCR2, respectively, for the most selective derivative. Molecular docking showed that the binding mode of these triazolo[4,5-d]pyrimidines in CCR7 and CXCR2 corresponds with those of previously co-crystallized ligands.

Discovery of a fragment hit compound targeting D-Ala:D-Ala ligase of bacterial peptidoglycan biosynthesis.

Bacterial resistance is an increasing threat to healthcare systems, highlighting the need for discovering new antibacterial agents. An established technique, fragment-based drug discovery, was used to target a bacterial enzyme Ddl involved in the biosynthesis of peptidoglycan. We assembled general and focused fragment libraries that were screened in a biochemical inhibition assay. Screening revealed a new fragment-hit inhibitor of DdlB with a Ki value of 20.7 ± 4.5 µM. Binding to the enzyme was confirmed by an orthogonal biophysical method, surface plasmon resonance, making the hit a promising starting point for fragment development.

Fragment-Sized Thiazoles in Fragment-Based Drug Discovery Campaigns: Friend or Foe?

Thiazoles exhibit a wide range of biological activities and therefore represent useful and attractive building blocks. To evaluate their usefulness and pinpoint their liabilities in fragment screening campaigns, we assembled a focused library of 49 fragment-sized thiazoles and thiadiazoles with various substituents, namely amines, bromides, carboxylic acids, and nitriles. The library was profiled in a cascade of biochemical inhibition assays, redox activity, thiol reactivity, and stability assays. Our study indicates that when thiazole derivatives are identified as screening hits, their reactivity should be carefully addressed and correlated with specific on-target engagement. Importantly, nonspecific inhibition should be excluded using experimental approaches and in silico predictions. To help with validation of hits identified in fragment screening campaigns, we can apply our high-throughput profiling workflow to focus on the most tractable compounds with a clear mechanism of action.

Discovery of compounds with viscosity-reducing effects on biopharmaceutical formulations with monoclonal antibodies.

For the development of concentrated monoclonal antibody formulations for subcutaneous administration, the main challenge is the high viscosity of the solutions. To compensate for this, viscosity reducing agents are commonly used as excipients. Here, we applied two computational chemistry approaches to discover new viscosity-reducing agents: fingerprint similarity searching, and physicochemical property filtering. In total, 94 compounds were selected and experimentally evaluated on two model monoclonal antibodies, which led to the discovery of 44 new viscosity-reducing agents. Analysis of the results showed that using a simple filter that selects only compounds with three or more charge groups is a good 'rule of thumb' for selecting potential viscosity-reducing agents for two model monoclonal antibody formulations.

Covalent inhibitors of bacterial peptidoglycan biosynthesis enzyme MurA with chloroacetamide warhead.

MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) catalyzes the first committed step in the cytoplasmic part of peptidoglycan biosynthesis and is a validated target enzyme for antibacterial drug discovery; the inhibitor fosfomycin has been used clinically for decades. Like fosfomycin, most MurA inhibitors are small heterocyclic compounds that inhibit the enzyme by forming a covalent bond with the active site cysteine. The reactive chloroacetamide group was selected from a series of suitable electrophilic thiol-reactive warheads. The predominantly one-step synthesis led to the construction of the final library of 47 fragment-sized chloroacetamide compounds. Several new E. coli MurA inhibitors were identified, with the most potent compound having an IC50 value in the low micromolar range. The electrophilic reactivity of all chloroacetamide fragments in our library was evaluated by a high-throughput spectrophotometric assay using the reduced Ellman reagent as a surrogate for the cysteine thiol. LC-MS/MS experiments confirmed the covalent binding of the most potent inhibitor to Cys115 of the digested MurA enzyme. The covalent binding was further investigated by a biochemical time-dependent assay and a dilution assay, which confirmed the irreversible and time-dependent mode of action. The efficacy of chloroacetamide derivatives against MurA does not correlate with their thiol reactivity, making the active fragments valuable starting points for fragment-based development of new antibacterial agents targeting MurA.

New inhibitors of cathepsin V impair tumor cell proliferation and elastin degradation and increase immune cell cytotoxicity.

Cathepsin V is a human lysosomal cysteine peptidase with specific functions during pathological processes and is as such a promising therapeutic target. Peptidase inhibitors represent powerful pharmacological tools for regulating excessive proteolytic activity in various diseases. Cathepsin V is highly related to cathepsin L but differs in tissue distribution, binding site morphology, substrate specificity, and function. To validate its therapeutic potential and extend the number of potent and selective cathepsin V inhibitors, we used virtual high-throughput screening of commercially available compound libraries followed by an evaluation of kinetic properties to identify novel potent and selective cathepsin V inhibitors. We identified the ureido methylpiperidine carboxylate derivative, compound 7, as a reversible, selective, and potent inhibitor of cathepsin V. It also exhibited the most preferable characteristics for further evaluation with in vitro functional assays that simulate the processes in which cathepsin V is known to play an important role. Compound 7 exerted significant effects on cell proliferation, elastin degradation, and immune cell cytotoxicity. The latter was increased because compound 7 impaired conversion of immunosuppressive factor cystatin F to its active monomeric form. Taken together, our results present novel potent inhibitors of cathepsin V and provide new hit compounds for detailed development and optimization. Further, we demonstrate that cathepsin V is a potential target for new approaches to cancer therapy.

DNA-encoded library screening on two validated enzymes of the peptidoglycan biosynthetic pathway.

Screening of DNA-encoded libraries is an emerging technology for discovering hits against protein targets. With the recent launch of the DELopen platform, a facile screening of 4.4 billion compounds is available to accelerate the drug discovery process. Here we report an affinity-based screening of the DELopen library for the first time. The screening was performed against two bacterial enzymes of the peptidoglycan biosynthetic pathway, N-acetylglucosamine-enolpyruvyl transferase (MurA) and d-alanine:d-alanine ligase (DdlB). Several binders were obtained and selected for off-DNA synthesis. Hits with confirmed inhibitory potency were deconstructed into smaller fragments. In this way, two new MurA inhibitors with antibacterial activity were obtained and are available for further optimization.

Encoding BRAF inhibitor functions in protein degraders.

Various BRAF kinase inhibitors were developed to treat cancers carrying the BRAFV600E mutation. First-generation BRAF inhibitors could lead to paradoxical activation of the MAPK pathway, limiting their clinical usefulness. Here, we show the development of two series of BRAFV600E-targeting PROTACs and demonstrate that the exchange of the inhibitor scaffold from vemurafenib to paradox-breaker ligands resulted in BRAFV600E degraders that did not cause paradoxical ERK activation.

Targeting the deubiquitinase USP7 for degradation with PROTACs.

Targeting deubiquitinating enzymes (DUBs) has emerged as a promising therapeutic approach in several human cancers and other diseases. DUB inhibitors are exciting pharmacological tools but often exhibit limited cellular potency. Here we report PROTACs based on a ubiquitin-specific protease 7 (USP7) inhibitor scaffold to degrade USP7. By investigating several linker and E3 ligand types, including novel cereblon recruiters, we discovered a highly selective USP7 degrader tool compound that induced apoptosis of USP7-dependent cancer cells. This work represents one of the first DUB degraders and unlocks a new drug target class for protein degradation.

Towards discovery of inhibitors of the undecaprenyl-pyrophosphate phosphatase BacA by virtual high-throughput screening.

Increasing resistance to common antibiotics is becoming a major challenge that requires the development of new antibacterial agents. Peptidoglycan is an essential heteropolymer of the bacterial envelope that ensures the integrity and shape of all bacteria and is also an important target for antibiotics. The biosynthesis of peptidoglycan depends on a lipid carrier, undecaprenyl phosphate. As a byproduct of peptidoglycan polymerization, the lipid carrier is released as undecaprenyl pyrophosphate, which must be recycled to allow new polymerization cycles. To this end, it undergoes a dephosphorylation process catalyzed by the membrane phosphatase BacA, which is specific and highly conserved in bacteria. In the present study, we identified small molecules displaying inhibitory potency towards BacA. We began by preparing a commercial compound library, followed by high-throughput virtual screening by ensemble docking using the 3D structure of BacA and molecular dynamics snapshots to account for the flexibility of the protein. Of 83 compounds computationally selected and tested in a biochemical assay, one sulfamoylthiophene molecule showed significant inhibition of the undecaprenyl pyrophosphate dephosphorylation activity catalyzed by BacA. Subsequently, an additional 33 scaffold analogs were selected and acquired, of which 6 compounds exhibited BacA inhibition. The IC50 values of these compounds ranged from 42 to 366 µM. In addition, significant antibacterial activity against Escherichia coli was observed in TolC/PAP2-depleted strains. We believe that the overall strategy followed in this study and the identified class of inhibitors provide a solid foundation for the further development of potent BacA-targeted inhibitors and the discovery of novel antibacterial compounds.

A Set of Experimentally Validated Decoys for the Human CC Chemokine Receptor 7 (CCR7) Obtained by Virtual Screening.

We present a state-of-the-art virtual screening workflow aiming at the identification of novel CC chemokine receptor 7 (CCR7) antagonists. Although CCR7 is associated with a variety of human diseases, such as immunological disorders, inflammatory diseases, and cancer, this target is underexplored in drug discovery and there are no potent and selective CCR7 small molecule antagonists available today. Therefore, computer-aided ligand-based, structure-based, and joint virtual screening campaigns were performed. Hits from these virtual screenings were tested in a CCL19-induced calcium signaling assay. After careful evaluation, none of the in silico hits were confirmed to have an antagonistic effect on CCR7. Hence, we report here a valuable set of 287 inactive compounds that can be used as experimentally validated decoys.

ProBiS-Dock: A Hybrid Multitemplate Homology Flexible Docking Algorithm Enabled by Protein Binding Site Comparison.

The protein data bank (PDB) is a rich source of protein ligand structures, but ligands are not explicitly used in current docking algorithms. We have developed ProBiS-Dock, a docking algorithm complementary to the ProBiS-Dock Database (J. Chem. Inf. Model. 2021, 61, 4097-4107) that treats small molecules and proteins as fully flexible entities and allows conformational changes in both after ligand binding. A new scoring function is described that consists of a binding site-specific scoring function (ProBiS-Score) and a general statistical scoring function. ProBiS-Dock enables rapid docking of small molecules to proteins and has been successfully validated in silico against standard benchmarks. It enables rapid search for new active ligands by leveraging existing knowledge in the PDB. The potential of the software for drug development has been confirmed in vitro by the discovery of new inhibitors of human indoleamine 2,3-dioxygenase 1, an enzyme that is an attractive target for cancer therapy and catalyzes the first rate-determining step of l-tryptophan metabolism via the kynurenine pathway. The software is freely available to academic users at http://insilab.org/probisdock.

Redox active or thiol reactive? Optimization of rapid screens to identify less evident nuisance compounds.

Compounds that exhibit assay interference or undesirable mechanisms of bioactivity are routinely encountered in assays at various stages of drug discovery. We observed that assays for the investigation of thiol-reactive and redox-active compounds have not been collected in a comprehensive review. Here, we review these assays and subject them to experimental optimization to improve their reliability. We demonstrate the usefulness of our assay cascade by assaying a library of bioactive compounds, chemical probes, and a set of approved drugs. These high-throughput assays should complement the array of wet-lab and in silico assays during the initial stages of hit discovery campaigns to pursue only hit compounds with tractable mechanisms of action.

Andrographolide Derivatives Target the KEAP1/NRF2 Axis and Possess Potent Anti-SARS-CoV-2 Activity.

Naturally occurring compounds represent a vast pool of pharmacologically active entities. One of such compounds is andrographolide, which is endowed with many beneficial properties, including the activity against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). To initiate a drug repurposing or hit optimization campaign, it is imperative to unravel the primary mechanism(s) of the antiviral action of andrographolide. Here, we showed by means of a reporter gene assay that andrographolide exerts its anti-SARS-CoV-2 effects by inhibiting the interaction between Kelch-like ECH-associated protein 1 (KEAP1) and nuclear factor erythroid 2-related factor 2 (NRF2) causing NRF2 upregulation. Moreover, we demonstrated that subtle structural modifications of andrographolide could lead to derivatives with stronger on-target activities and improved physicochemical properties. Our results indicate that further optimization of this structural class is warranted to develop novel COVID-19 therapies.

Fragment-Sized and Bidentate (Immuno)Proteasome Inhibitors Derived from Cysteine and Threonine Targeting Warheads.

Constitutive- and immunoproteasomes are part of the ubiquitin-proteasome system (UPS), which is responsible for the protein homeostasis. Selective inhibition of the immunoproteasome offers opportunities for the treatment of numerous diseases, including inflammation, autoimmune diseases, and hematologic malignancies. Although several inhibitors have been reported, selective nonpeptidic inhibitors are sparse. Here, we describe two series of compounds that target both proteasomes. First, benzoxazole-2-carbonitriles as fragment-sized covalent immunoproteasome inhibitors are reported. Systematic substituent scans around the fragment core of benzoxazole-2-carbonitrile led to compounds with single digit micromolar inhibition of the ß5i subunit. Experimental and computational reactivity studies revealed that the substituents do not affect the covalent reactivity of the carbonitrile warhead, but mainly influence the non-covalent recognition. Considering the small size of the inhibitors, this finding emphasizes the importance of the non-covalent recognition step in the covalent mechanism of action. As a follow-up series, bidentate inhibitors are disclosed, in which electrophilic heterocyclic fragments, i.e., 2-vinylthiazole, benzoxazole-2-carbonitrile, and benzimidazole-2-carbonitrile were linked to threonine-targeting (R)-boroleucine moieties. These compounds were designed to bind both the Thr1 and ß5i-subunit-specific residue Cys48. However, inhibitory activities against (immuno)proteasome subunits showed that bidentate compounds inhibit the ß5, ß5i, ß1, and ß1i subunits with submicromolar to low-micromolar IC50 values. Inhibitory assays against unrelated enzymes showed that compounds from both series are selective for proteasomes. The presented nonpeptidic and covalent derivatives are suitable hit compounds for the development of either ß5i-selective immunoproteasome inhibitors or compounds targeting multiple subunits of both proteasomes.

Discovery of selective fragment-sized immunoproteasome inhibitors.

Proteasomes contribute to maintaining protein homeostasis and their inhibition is beneficial in certain types of cancer and in autoimmune diseases. However, the inhibition of the proteasomes in healthy cells leads to unwanted side-effects and significant effort has been made to identify inhibitors specific for the immunoproteasome, especially to treat diseases which manifest increased levels and activity of this proteasome isoform. Here, we report our efforts to discover fragment-sized inhibitors of the human immunoproteasome. The screening of an in-house library of structurally diverse fragments resulted in the identification of benzo[d]oxazole-2(3H)-thiones, benzo[d]thiazole-2(3H)-thiones, benzo[d]imidazole-2(3H)-thiones, and 1-methylbenzo[d]imidazole-2(3H)-thiones (with a general term benzoXazole-2(3H)-thiones) as inhibitors of the chymotrypsin-like (ß5i) subunit of the immunoproteasome. A subsequent structure-activity relationship study provided us with an insight regarding growing vectors. Binding to the ß5i subunit was shown and selectivity against the ß5 subunit of the constitutive proteasome was determined. Thorough characterization of these compounds suggested that they inhibit the immunoproteasome by forming a disulfide bond with the Cys48 available specifically in the ß5i active site. To obtain fragments with biologically more tractable covalent interactions, we performed a warhead scan, which yielded benzoXazole-2-carbonitriles as promising starting points for the development of selective immunoproteasome inhibitors with non-peptidic scaffolds.

Synthesis and Biochemical Evaluation of Warhead-Decorated Psoralens as (Immuno)Proteasome Inhibitors.

The immunoproteasome is a multicatalytic protease that is predominantly expressed in cells of hematopoietic origin. Its elevated expression has been associated with autoimmune diseases, various types of cancer, and inflammatory diseases. Selective inhibition of its catalytic activities is therefore a viable approach for the treatment of these diseases. However, the development of immunoproteasome-selective inhibitors with non-peptidic scaffolds remains a challenging task. We previously reported 7H-furo[3,2-g]chromen-7-one (psoralen)-based compounds with an oxathiazolone warhead as selective inhibitors of the chymotrypsin-like (ß5i) subunit of immunoproteasome. Here, we describe the influence of the electrophilic warhead variations at position 3 of the psoralen core on the inhibitory potencies. Despite mapping the chemical space with different warheads, all compounds showed decreased inhibition of the ß5i subunit of immunoproteasome in comparison to the parent oxathiazolone-based compound. Although suboptimal, these results provide crucial information about structure-activity relationships that will serve as guidance for the further design of (immuno)proteasome inhibitors.