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In aging and disease, cellular NAD+is depleted by catabolism to nicotinamide (NAM) and NAD+supple-mentation is being pursued to enhance human healthspan and lifespan. Activation of nicoti namide phosphoribosyl -transferase (NAMPT), the rate-limiting step in NAD+biosynthesis, has potential to increase salvage of NAM. Novel NAMPT positive allosteric modulators (N-PAMs) were discovered in addition to demonstration of NAMPT activati on by biogenic phenols. The mechanism of activation was revealed through synthesis of novel chemical probes, new NAMPT co-crystal structures, and enzyme kinetics. Binding to a rear channel in NAMPT regulates NAM binding and turnover, with biochemical observations being replicated by NAD+measurements in human cells. The mechanism of action of N-PAMs identifies, for the first time, the role of the rear channel in regulation of NAMPT turnover coupled to feedback inhibition by NAM. N-PAM inhibition of low affinity, non-productive NAM binding via the rear channel, causes a right-shif t in KI(NAM) that accompanies an increase in enzyme activity. Conversion of an N-PAM to a high-affinity l igand blocks both high and low affinity NAM binding, ablating enzyme activity. In the presence of an N-PAM, NAMPT boosts NAD+biosynthesis at higher NAM concentrations, in addition to relieving inhibition by NAD+. Since cellular stress often leads to enhanced catabolism of NAD+to NAM, this mechanism is relevant to supporting cellular N AD+levels in aging and disease. The tight regulation of cellular NAMPT is differentially regulated by N-PAMs and other activators, indicating that different classes of pharmacological activators may be engineered for cell and tissue selectivity.

Acquired resistance to fulvestrant and palbociclib is a new challenge to treatment of estrogen receptor positive (ER+) breast cancer. ER is expressed in most resistance settings; thus, bromodomain and extra-terminal protein inhibitors (BETi) that target BET-amplified ER-mediated transcription have therapeutic potential. Novel pyrrolopyridone BETi leveraged novel interactions with L92/L94 confirmed by a cocrystal structure of 27 with BRD4. Optimization of BETi using growth inhibition in fulvestrant-resistant (MCF-7:CFR) cells was confirmed in endocrine-resistant, palbociclib-resistant, and ESR1 mutant cell lines. 27 was more potent in MCF-7:CFR cells than six BET inhibitors in clinical trials. Transcriptomic analysis differentiated 27 from the benchmark BETi, JQ-1, showing downregulation of oncogenes and upregulation of tumor suppressors and apoptosis. The therapeutic approach was validated by oral administration of 27 in orthotopic xenografts of endocrine-resistant breast cancer in monotherapy and in combination with fulvestrant. Importantly, at an equivalent dose in rats, thrombocytopenia was mitigated.

Antiviral agents blocking SARS-CoV-2 viral replication are desperately needed to complement vaccination to end the COVID-19 pandemic. Viral replication and assembly are entirely dependent on two viral cysteine proteases: 3C-like protease (3CLpro) and the papain-like protease (PLpro). PLpro also has deubiquitinase (DUB) activity, removing ubiquitin (Ub) and Ub-like modifications from host proteins, disrupting the host immune response. 3CLpro is inhibited by many known cysteine protease inhibitors, whereas PLpro is a relatively unusual cysteine protease, being resistant to blockade by such inhibitors. A high-throughput screen of biased and unbiased libraries gave a low hit rate, identifying only CPI-169 and the positive control, GRL0617, as inhibitors with good potency (IC50 < 10 lower case Greek μM). Analogues of both inhibitors were designed to develop structure-activity relationships; however, without a co-crystal structure of the CPI-169 series, we focused on GRL0617 as a starting point for structure-based drug design, obtaining several co-crystal structures to guide optimization. A series of novel 2-phenylthiophene-based non-covalent SARS-CoV-2 PLpro inhibitors were obtained, culminating in low nanomolar potency. The high potency and slow inhibitor off-rate were rationalized by newly identified ligand interactions with a 'BL2 groove' that is distal from the active site cysteine. Trapping of the conformationally flexible BL2 loop by these inhibitors blocks binding of viral and host protein substrates; however, until now it has not been demonstrated that this mechanism can induce potent and efficacious antiviral activity. In this study, we report that novel PLpro inhibitors have excellent antiviral efficacy and potency against infectious SARS-CoV-2 replication in cell cultures. Together, our data provide structural insights into the design of potent PLpro inhibitors and the first validation that non-covalent inhibitors of SARS-CoV-2 PLpro can block infection of human cells with low micromolar potency.

Antiviral agents blocking SARS-CoV-2 viral replication are desperately needed to complement vaccination to end the COVID-19 pandemic. Viral replication and assembly are entirely dependent on two viral cysteine proteases: 3C-like protease (3CLpro) and the papain-like protease (PLpro). PLpro also has deubiquitinase (DUB) activity, removing ubiquitin (Ub) and Ub-like modifications from host proteins, disrupting the host immune response. 3CLpro is inhibited by many known cysteine protease inhibitors, whereas PLpro is a relatively unusual cysteine protease, being resistant to blockade by such inhibitors. A high-throughput screen of biased and unbiased libraries gave a low hit rate, identifying only CPI-169 and the positive control, GRL0617, as inhibitors with good potency (IC50 < 10 µM). Analogues of both inhibitors were designed to develop structure-activity relationships; however, without a co-crystal structure of the CPI-169 series, we focused on GRL0617 as a starting point for structure-based drug design, obtaining several co-crystal structures to guide optimization. A series of novel 2-phenylthiophene-based non-covalent SARS-CoV-2 PLpro inhibitors were obtained, culminating in low nanomolar potency. The high potency and slow inhibitor off-rate were rationalized by newly identified ligand interactions with a “BL2 groove” that is distal from the active site cysteine. Trapping of the conformationally flexible BL2 loop by these inhibitors blocks binding of viral and host protein substrates; however, until now it has not been demonstrated that this mechanism can induce potent and efficacious antiviral activity. In this study, we report that novel PLpro inhibitors have excellent antiviral efficacy and potency against infectious SARS-CoV-2 replication in cell cultures. Together, our data provide structural insights into the design of potent PLpro inhibitors and the first validation that non-covalent inhibitors of SARS-CoV-2 PLpro can block infection of human cells with low micromolar potency.

Carbohydrates, such as oligo- and polysaccharides, are highly abundant biopolymers that are involved in numerous processes. The study of their structure and functions is commonly based on a material that is isolated from complex natural sources. However, a more precise analysis requires pure compounds with well-defined structures that can be obtained from chemical or enzymatic syntheses. Novel synthetic strategies have increased the accessibility of larger monodisperse polysaccharides, posing a challenge to the analytical methods used for their molecular characterization. Here, we present wide mass range ultrahigh-resolution matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) as a powerful platform for the analysis of synthetic oligo- and polysaccharides. Synthetic carbohydrates 16-, 64-, 100-, and 151-mers were mass analyzed and characterized by MALDI in-source decay FT-ICR MS. Detection of fragment ions generated from glycosidic bond cleavage (or cross-ring cleavage) provided information of the monosaccharide content and the linkage type, allowing for the corroboration of the carbohydrate compositions and structures.

Filoviridae, including Ebola (EBOV) and Marburg (MARV) viruses, are emerging pathogens that pose a serious threat to public health. No agents have been approved to treat filovirus infections, representing a major unmet medical need. The selective estrogen receptor modulator (SERM) toremifene was previously identified from a screen of FDA-approved drugs as a potent EBOV viral entry inhibitor, via binding to EBOV glycoprotein (GP). A focused screen of ER ligands identified ridaifen-B as a potent dual inhibitor of EBOV and MARV. Optimization and reverse-engineering to remove ER activity led to a novel compound 30 (XL-147) showing potent inhibition against infectious EBOV Zaire (0.09 μM) and MARV (0.64 μM). Mutagenesis studies confirmed that inhibition of EBOV viral entry is mediated by the direct interaction with GP. Importantly, compound 30 displayed a broad-spectrum antifilovirus activity against Bundibugyo, Tai Forest, Reston, and Měngla viruses and is the first submicromolar antiviral agent reported for some of these strains, therefore warranting further development as a pan-filovirus inhibitor.

The clinical steroidal selective estrogen receptor (ER) degrader (SERD), fulvestrant, is effective in metastatic breast cancer, but limited by poor pharmacokinetics, prompting the development of orally bioavailable, nonsteroidal SERDs, currently in clinical trials. These trials address local breast cancer as well as peripheral metastases, but patients with brain metastases are generally excluded because of the lack of blood-brain barrier penetration. A novel family of benzothiophene SERDs with a basic amino side arm (B-SERDs) was synthesized. Proteasomal degradation of ERα was induced by B-SERDs that achieved the objectives of oral and brain bioavailability, while maintaining high affinity binding to ERα and both potency and efficacy comparable to fulvestrant in cell lines resistant to endocrine therapy or bearing ESR1 mutations. A novel 3-oxyazetidine side chain was designed, leading to 37d, a B-SERD that caused endocrine-resistant ER+ tumors to regress in a mouse orthotopic xenograft model.

Background Resistance to endocrine therapy in estrogen receptor positive (ER+) breast cancer drives mortality and despite new targeted therapies, resistance is the obstacle to progression-free survival. Fulvestrant (FUL), a selective estrogen receptor degrader (SERD) was approved in 2017 for first-line therapy of metaststic disease in postmenopausal women; however, acquired FUL-resistance in both first-line setting and in combination therapy with CDK4/6 inhibitor, palbociclib, has been observed clinically. The poor pharmacokinetics of FUL may contribute to acquired resistance. Methods Using structure-based design, we optimized: 1) novel SERDs bearing a basic side chain (B-SERDs) as an orally bioavailable and brain penetrant alternative to FUL; 2) novel pyridinone-based bromodomains and extra-terminal motif (BET) inhibitors to be used in combination with B-SERDs or FUL. Biochemical assays and growth inhibition of breast cancer cell lines, resistant to tamoxifen, and/or FUL, both in 2D and 3D cultures were used to optimize and select development candidates. Drug metabolism and pharmacokinetics(DMPK) demonstrated oral bioavailability and dose selection for validation in mouse xenograft models of endocrine resistant breast cancer. Results B-SERDs showed equivalence to FUL in cell culture models, with respect to ERα degradation and antiproliferative activity; and in contrast to FUL, were demonstrated to have good oral and brain bioavailability. A development candidate with improved DMPK characteristics was effective in endocrine-resistant ER+ xenograft models. Novel BET inhibitors, optimized for potency in binding to BRD4-BD1 and selectivity over the larger family of bromodomain containing proteins, inhibited growth of tamoxifen and FUL-resistant breast cancer cells.Optimized compounds showed superior in vitro activity to eight BET inhibitors in clinical trials. The optimized BET inhibitor was validated alone and in combination with B-SERD in endocrine-resistant xenograft models. Conclusions The combination of B-SERD with BET inhibitor provides a multi-targeted suppression of ER signaling that may extend progression-free survival and cdircumvent acquired resistance in metastatic ER+ breast cancer. Legal entity responsible for the study Gregory R J Thatcher. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Stereoselective β-rhamnopyranosylation remains a challenge, due to the unfavorable anomeric effect and steric hindrance of the C2-substituent; herein, this challenge is addressed with a gold(I)-catalyzed SN2-like glycosylation protocol employing α-rhamnopyranosyl 2-alkynyl-4-nitro-benzoates as donors.

Stereoselective β-rhamnopyranosylation remains a challenge, due to the unfavorable anomeric effect and steric hindrance of the C2-substituent; herein, this challenge is addressed with a gold(I)-catalyzed SN2-like glycosylation protocol employing α-rhamnopyranosyl 2-alkynyl-4-nitro-benzoates as donors.