Alkylamine-tethered molecules recruit FBXO22 for targeted protein degradation.

Targeted protein degradation (TPD) relies on small molecules to recruit proteins to E3 ligases to induce their ubiquitylation and degradation by the proteasome. Only a few of the approximately 600 human E3 ligases are currently amenable to this strategy. This limits the actionable target space and clinical opportunities and thus establishes the necessity to expand to additional ligases. Here we identify and characterize SP3N, a specific degrader of the prolyl isomerase FKBP12. SP3N features a minimal design, where a known FKBP12 ligand is appended with a flexible alkylamine tail that conveys degradation properties. We found that SP3N is a precursor and that the alkylamine is metabolized to an active aldehyde species that recruits the SCFFBXO22 ligase for FKBP12 degradation. Target engagement occurs via covalent adduction of Cys326 in the FBXO22 C-terminal domain, which is critical for ternary complex formation, ubiquitylation and degradation. This mechanism is conserved for two recently reported alkylamine-based degraders of NSD2 and XIAP, thus establishing alkylamine tethering and covalent hijacking of FBXO22 as a generalizable TPD strategy.

Discovery of Molecular Glue Degraders via Isogenic Morphological Profiling.

Molecular glue degraders (MGDs) are small molecules that degrade proteins of interest via the ubiquitin-proteasome system. While MGDs were historically discovered serendipitously, approaches for MGD discovery now include cell-viability-based drug screens or data mining of public transcriptomics and drug response datasets. These approaches, however, have target spaces restricted to the essential proteins. Here we develop a high-throughput workflow for MGD discovery that also reaches the nonessential proteome. This workflow begins with the rapid synthesis of a compound library by sulfur(VI) fluoride exchange chemistry coupled to a morphological profiling assay in isogenic cell lines that vary in levels of the E3 ligase CRBN. By comparing the morphological changes induced by compound treatment across the isogenic cell lines, we were able to identify FL2-14 as a CRBN-dependent MGD targeting the nonessential protein GSPT2. We envision that this workflow would contribute to the discovery and characterization of MGDs that target a wider range of proteins.

Covalent Inhibition of Wild-Type HIV-1 Reverse Transcriptase Using a Fluorosulfate Warhead.

Covalent inhibitors of wild-type HIV-1 reverse transcriptase (CRTIs) are reported. Three compounds derived from catechol diether non-nucleoside inhibitors (NNRTIs) with addition of a fluorosulfate warhead are demonstrated to covalently modify Tyr181 of HIV-RT. X-ray crystal structures for complexes of the CRTIs with the enzyme are provided, which fully demonstrate the covalent attachment, and confirmation is provided by appropriate mass shifts in ESI-TOF mass spectra. The three CRTIs and six noncovalent analogues are found to be potent inhibitors with both IC50 values for in vitro inhibition of WT RT and EC50 values for cytopathic protection of HIV-1-infected human T-cells in the 5-320 nM range.

Structural and pharmacological evaluation of a novel non-nucleoside reverse transcriptase inhibitor as a promising long acting nanoformulation for treating HIV.

Combination antiretroviral therapy (cART) has been proven effective in inhibiting human immunodeficiency virus type 1 (HIV-1) infection and has significantly improved the health outcomes in acquired immune deficiency syndrome (AIDS) patients. The therapeutic benefits of cART have been challenged because of the toxicity and emergence of drug-resistant HIV-1 strains along with lifelong patient compliance resulting in non-adherence. These issues also hinder the clinical benefits of non-nucleoside reverse transcriptase inhibitors (NNRTIs), which are one of the vital components of cART for the treatment of HIV-1 infection. In this study, using a computational and structural based drug design approach, we have discovered an effective HIV -1 NNRTI, compound I (Cmpd I) that is very potent in biochemical assays and which targets key residues in the allosteric binding pocket of wild-type (WT)-RT as revealed by structural studies. Furthermore, Cmpd I exhibited very potent antiviral activity in HIV-1 infected T cells, lacked cytotoxicity (therapeutic index >100,000), and no significant off-target effects were noted in pharmacological assays. To address the issue of non-adherence, we developed a long-acting nanoformulation of Cmpd I (Cmpd I-NP) using poly (lactide-coglycolide) (PLGA) particles. The pharmacokinetic studies of free and nanoformulated Cmpd I were carried out in BALB/c mice. Intraperitoneal administration of Cmpd I and Cmpd I-NP in BALB/c mice revealed prolonged serum residence time of 48 h and 30 days, respectively. The observed serum concentrations of Cmpd I in both cases were sufficient to provide >97% inhibition in HIV-1 infected T-cells. The significant antiviral activity along with favorable pharmacological and pharmacokinetic profile of Cmpd I, provide compelling and critical support for its further development as an anti-HIV therapeutic agent.

Optimization of Pyrazoles as Phenol Surrogates to Yield Potent Inhibitors of Macrophage Migration Inhibitory Factor.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that is implicated in the regulation of inflammation, cell proliferation, and neurological disorders. MIF is also an enzyme that functions as a keto-enol tautomerase. Most potent MIF tautomerase inhibitors incorporate a phenol, which hydrogen bonds to Asn97 in the active site. Starting from a 113-µm docking hit, we report results of structure-based and computer-aided design that have provided substituted pyrazoles as phenol alternatives with potencies of 60-70 nm. Crystal structures of complexes of MIF with the pyrazoles highlight the contributions of hydrogen bonding with Lys32 and Asn97, and aryl-aryl interactions with Tyr36, Tyr95, and Phe113 to the binding.

From in silico hit to long-acting late-stage preclinical candidate to combat HIV-1 infection.

The HIV-1 pandemic affecting over 37 million people worldwide continues, with nearly one-half of the infected population on highly active antiretroviral therapy (HAART). Major therapeutic challenges remain because of the emergence of drug-resistant HIV-1 strains, limitations because of safety and toxicity with current HIV-1 drugs, and patient compliance for lifelong, daily treatment regimens. Nonnucleoside reverse transcriptase inhibitors (NNRTIs) that target the viral polymerase have been a key component of the current HIV-1 combination drug regimens; however, these issues hamper them. Thus, the development of novel more effective NNRTIs as anti-HIV-1 agents with fewer long-term liabilities, efficacy on new drug-resistant HIV-1 strains, and less frequent dosing is crucial. Using a computational and structure-based design strategy to guide lead optimization, a 5 µM virtual screening hit was transformed to a series of very potent nanomolar to picomolar catechol diethers. One representative, compound I, was shown to have nanomolar activity in HIV-1-infected T cells, potency on clinically relevant HIV-1 drug-resistant strains, lack of cytotoxicity and off-target effects, and excellent in vivo pharmacokinetic behavior. In this report, we show the feasibility of compound I as a late-stage preclinical candidate by establishing synergistic antiviral activity with existing HIV-1 drugs and clinical candidates and efficacy in HIV-1-infected humanized [human peripheral blood lymphocyte (Hu-PBL)] mice by completely suppressing viral loads and preventing human CD4+ T-cell loss. Moreover, a long-acting nanoformulation of compound I [compound I nanoparticle (compound I-NP)] in poly(lactide-coglycolide) (PLGA) was developed that shows sustained maintenance of plasma drug concentrations and drug efficacy for almost 3 weeks after a single dose.

Adding a Hydrogen Bond May Not Help: Naphthyridinone vs Quinoline Inhibitors of Macrophage Migration Inhibitory Factor.

Coordination of the ammonium group of Lys32 in the active site of human macrophage migration inhibitory factor (MIF) using a 1,7-naphthyridin-8-one instead of a quinoline is investigated. Both gas- and aqueous-phase DFT calculations for model systems indicate potential benefits for the added hydrogen bond with the lactam carbonyl group, while FEP results are neutral. Three crystal structures are reported for complexes of MIF with 3a, 4a, and 4b, which show that the desired hydrogen bond is formed with O-N distances of 2.8-3.0 Å. Compound 4b is the most potent new MIF inhibitor with Ki and Kd values of 90 and 94 nM; it also has excellent aqueous solubility, 288 µg/mL. Consistent with the FEP results, the naphthyridinones are found to have similar potency as related quinolines in spite of the additional protein-ligand hydrogen bond.

Covalent inhibitors for eradication of drug-resistant HIV-1 reverse transcriptase: From design to protein crystallography.

Development of resistance remains a major challenge for drugs to treat HIV-1 infections, including those targeting the essential viral polymerase, HIV-1 reverse transcriptase (RT). Resistance associated with the Tyr181Cys mutation in HIV-1 RT has been a key roadblock in the discovery of nonnucleoside RT inhibitors (NNRTIs). It is the principal point mutation that arises from treatment of HIV-infected patients with nevirapine, the first-in-class drug still widely used, especially in developing countries. We report covalent inhibitors of Tyr181Cys RT (CRTIs) that can completely knock out activity of the resistant mutant and of the particularly challenging Lys103Asn/Tyr181Cys variant. Conclusive evidence for the covalent modification of Cys181 is provided from enzyme inhibition kinetics, mass spectrometry, protein crystallography, and antiviral activity in infected human T-cell assays. The CRTIs are also shown to be selective for Cys181 and have lower cytotoxicity than the approved NNRTI drugs efavirenz and rilpivirine.

JAK2 JH2 Fluorescence Polarization Assay and Crystal Structures for Complexes with Three Small Molecules.

A competitive fluorescence polarization (FP) assay is reported for determining binding affinities of probe molecules with the pseudokinase JAK2 JH2 allosteric site. The syntheses of the fluorescent 5 and 6 used in the assay are reported as well as Kd results for 10 compounds, including JNJ7706621, NVP-BSK805, and filgotinib (GLPG0634). X-ray crystal structures of JAK2 JH2 in complex with NVP-BSK805, filgotinib, and diaminopyrimidine 8 elucidate the binding poses.

Systematic Study of Effects of Structural Modifications on the Aqueous Solubility of Drug-like Molecules.

Aqueous solubilities and activities have been measured for 17 members of the quinolinyltriazole series of inhibitors of human macrophage migration inhibitory factor (MIF). Systematic variation of a solvent-exposed substituent provided increases in solubility from 2 µg/mL for the parent compound 3a up to 867 µg/mL. The low solubility of 3a results from its near-planar structure and an intermolecular hydrogen bond, as revealed in a small-molecule X-ray structure. Removal of the hydrogen bond yields a 3-fold increase in solubility, but a 7-fold drop in activity. 5b emerges as the most potent MIF inhibitor with a Ki of 14 nM and good solubility, 47 µg/mL, while 4e has both high potency and solubility.

A Fluorescence Polarization Assay for Binding to Macrophage Migration Inhibitory Factor and Crystal Structures for Complexes of Two Potent Inhibitors.

Human macrophage migration inhibitory factor (MIF) is both a keto-enol tautomerase and a cytokine associated with numerous inflammatory diseases and cancer. Consistent with observed correlations between inhibition of the enzymatic and biological activities, discovery of MIF inhibitors has focused on monitoring the tautomerase activity using l-dopachrome methyl ester or 4-hydroxyphenyl pyruvic acid as substrates. The accuracy of these assays is compromised by several issues including substrate instability, spectral interference, and short linear periods for product formation. In this work, we report the syntheses of fluorescently labeled MIF inhibitors and their use in the first fluorescence polarization-based assay to measure the direct binding of inhibitors to the active site. The assay allows the accurate and efficient identification of competitive, noncompetitive, and covalent inhibitors of MIF in a manner that can be scaled for high-throughput screening. The results for 22 compounds show that the most potent MIF inhibitors bind with Kd values of ca. 50 nM; two are from our laboratory, and the other is a compound from the patent literature. X-ray crystal structures for two of the most potent compounds bound to MIF are also reported here. Striking combinations of protein-ligand hydrogen bonding, aryl-aryl, and cation-π interactions are responsible for the high affinities. A new chemical series was then designed using this knowledge to yield two more strong MIF inhibitors/binders.

Irregularities in enzyme assays: The case of macrophage migration inhibitory factor.

Inhibitors of human macrophage migration inhibitory factor (MIF) previously reported in the literature have been reexamined by synthesis, assaying for tautomerase activity, and protein crystallography. Substantial inconsistencies between prior and current assay results are noted. They appear to arise from difficulties with the tautomerase substrates, solubility issues, and especially covalent inhibition. Incubation time variation shows that 3, 4, 6, and 9 are covalent or slow-binding inhibitors. Two protein crystal structures are provided; one confirms that the twice-discovered 3 is a covalent inhibitor.

A nanotherapy strategy significantly enhances anticryptosporidial activity of an inhibitor of bifunctional thymidylate synthase-dihydrofolate reductase from Cryptosporidium.

Cryptosporidiosis, a gastrointestinal disease caused by protozoans of the genus Cryptosporidium, is a common cause of diarrheal diseases and often fatal in immunocompromised individuals. Bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from Cryptosporidium hominis (C. hominis) has been a molecular target for inhibitor design. C. hominis TS-DHFR inhibitors with nM potency at a biochemical level have been developed however drug delivery to achieve comparable antiparasitic activity in Cryptosporidium infected cell culture has been a major hurdle for designing effective therapies. Previous mechanistic and structural studies have identified compound 906 as a nM C. hominis TS-DHFR inhibitor in vitro, having µM antiparasitic activity in cell culture. In this work, proof of concept studies are presented using a nanotherapy approach to improve drug delivery and the antiparasitic activity of 906 in cell culture. We utilized PLGA nanoparticles that were loaded with 906 (NP-906) and conjugated with antibodies to the Cryptosporidium specific protein, CP2, on the nanoparticle surface in order to specifically target the parasite. Our results indicate that CP2 labeled NP-906 (CP2-NP-906) reduces the level of parasites by 200-fold in cell culture, while NP-906 resulted in 4.4-fold decrease. Moreover, the anticryptosporidial potency of 906 improved 15 to 78-fold confirming the utility of the antibody conjugated nanoparticles as an effective drug delivery strategy.

Design, synthesis, and protein crystallography of biaryltriazoles as potent tautomerase inhibitors of macrophage migration inhibitory factor.

Optimization is reported for biaryltriazoles as inhibitors of the tautomerase activity of human macrophage migration inhibitory factor (MIF), a proinflammatory cytokine associated with numerous inflammatory diseases and cancer. A combined approach was taken featuring organic synthesis, enzymatic assaying, crystallography, and modeling including free-energy perturbation (FEP) calculations. X-ray crystal structures for 3a and 3b bound to MIF are reported and provided a basis for the modeling efforts. The accommodation of the inhibitors in the binding site is striking with multiple hydrogen bonds and aryl-aryl interactions. Additional modeling encouraged pursuit of 5-phenoxyquinolinyl analogues, which led to the very potent compound 3s. Activity was further enhanced by addition of a fluorine atom adjacent to the phenolic hydroxyl group as in 3w, 3z, 3aa, and 3bb to strengthen a key hydrogen bond. It is also shown that physical properties of the compounds can be modulated by variation of solvent-exposed substituents. Several of the compounds are likely the most potent known MIF tautomerase inhibitors; the most active ones are more than 1000-fold more active than the well-studied (R)-ISO-1 and more than 200-fold more active than the chromen-4-one Orita-13.

Structural studies provide clues for analog design of specific inhibitors of Cryptosporidium hominis thymidylate synthase-dihydrofolate reductase.

Cryptosporidium is the causative agent of a gastrointestinal disease, cryptosporidiosis, which is often fatal in immunocompromised individuals and children. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer, bacterial infections, and malaria. Cryptosporidium hominis has a bifunctional thymidylate synthase and dihydrofolate reductase enzyme, compared to separate enzymes in the host. We evaluated lead compound 1 from a novel series of antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines as an inhibitor of Cryptosporidium hominis thymidylate synthase with selectivity over the human enzyme. Complementing the enzyme inhibition compound 1 also has anti-cryptosporidial activity in cell culture. A crystal structure with compound 1 bound to the TS active site is discussed in terms of several van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate (TS), cofactor NADPH and inhibitor methotrexate (DHFR). Another crystal structure in complex with compound 1 bound in both the TS and DHFR active sites is also reported here. The crystal structures provide clues for analog design and for the design of ChTS-DHFR specific inhibitors.

Extension into the entrance channel of HIV-1 reverse transcriptase--crystallography and enhanced solubility.

Non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-RT) are reported that feature extension into the entrance channel near Glu138. Complexes of the parent anilinylpyrimidine 1 and the morpholinoethoxy analog 2j with HIV-RT have received crystallographic characterization confirming the designs. Measurement of aqueous solubilities of 2j, 2k, the parent triazene 2a, and other NNRTIs demonstrate profound benefits for addition of the morpholinyl substituent.

Optimization of diarylazines as anti-HIV agents with dramatically enhanced solubility.

Non-nucleoside inhibitors of HIV-1 reverse transcriptase are reported that have ca. 100-fold greater solubility than the structurally related drugs etravirine and rilpivirine, while retaining high anti-viral activity. The solubility enhancements come from strategic placement of a morpholinylalkoxy substituent in the entrance channel of the NNRTI binding site. Compound 4d shows low-nanomolar activity similar to etravirine towards wild-type HIV-1 and key viral variants.

Crystal structures of HIV-1 reverse transcriptase with picomolar inhibitors reveal key interactions for drug design.

X-ray crystal structures at 2.9 Å resolution are reported for two complexes of catechol diethers with HIV-1 reverse transcriptase. The results help elucidate the structural origins of the extreme antiviral activity of the compounds. The possibility of halogen bonding between the inhibitors and Pro95 is addressed. Structural analysis reveals key interactions with conserved residues P95 and W229 of importance for design of inhibitors with high potency and favorable resistance profiles.

Structure-activity relationship of a new series of reversible dual monoacylglycerol lipase/fatty acid amide hydrolase inhibitors.

The two endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), play independent and nonredundant roles in the body. This makes the development of both selective and dual inhibitors of their inactivation an important priority. In this work we report a new series of inhibitors of monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH). Among them, (±)-oxiran-2-ylmethyl 6-(1,1'-biphenyl-4-yl)hexanoate (8) and (2R)-(-)-oxiran-2-ylmethyl(4-benzylphenyl)acetate (30) stand out as potent inhibitors of human recombinant MAGL (IC(50) (8) = 4.1 µM; IC(50) (30) = 2.4 µM), rat brain monoacylglycerol hydrolysis (IC(50) (8) = 1.8 µM; IC(50) (30) = 0.68 µM), and rat brain FAAH (IC(50) (8) = 5.1 µM; IC(50) (30) = 0.29 µM). Importantly, and in contrast to the other previously described MAGL inhibitors, these compounds behave as reversible inhibitors either of competitive (8) or noncompetitive nature (30). Hence, they could be useful to explore the therapeutic potential of reversible MAGL inhibitors.