Nanomolar Potency Research Articles

Biological evaluation of indolactams for in vitro bryostatin 1-like activity

Small molecule activators of protein kinase C (PKC) have traditionally been classified as either tumor promoters or suppressors. Although bryostatin 1 has well established anti-cancer activity, most natural products that target the PKC regulator domain exhibit tumor promotion properties. In this study, we examine a focused library of indolactam analogues in cell-based assays to establish the structural features of the scaffold that enhance bryostatin 1-like activity. These systematic biological assessments identified specific indole substitution patterns that impart diminished tumor promotion behavior in vitro for indolactam analogues, while still maintaining nanomolar potency for PKC.

The Potent Dihydroorotate Dehydrogenase Inhibitor, Hosu-53, Exhibits Compelling Monotherapy Efficacy in Multiple Myeloma and Augments CD47 Targeted Therapy

Worldwide, multiple myeloma (MM) is the second most common hematological malignancy characterized by the expansion of aberrant mature plasma cells in the bone marrow. Despite the FDA approval of several drugs with different mechanisms of action such as immunomodulatory drugs (IMiDs) and proteosome inhibitors (PIs), MM remains a challenging incurable disease where most patients ultimately relapse and become refractory to available . The resistant nature of MM underscores the need for novel therapeutic strategies that can directly affect MM cells while enhancing the outcome with MM current standard of care (SOC). Among the most promising MM SOC agents is monoclonal antibodies (mAb) such as the CD38 antibody daratumumab, which significantly improved the management of newly diagnosed and relapsed/refractory MM. Therefore, we aimed to develop novel therapies that can enhance mAb therapy in MM. Dihydroorotate dehydrogenase (DHODH) is an enzyme which mediates the fourth and rate-limiting step in the de novo pyrimidine synthesis pathway converting dihydroorotate to orotate, the precursor of uridine. Pyrimidine starvation using DHODH inhibitors has been shown to have potent antiproliferative activity in several malignancies including acute myeloid leukemia (AML) and MM. Thus, we developed a novel potent DHODH inhibitor, HOSU-53 for the treatment of hematological malignancies. We previously discovered the ability of HOSU-53 to modulate surface CD38 expression and found that HOSU-53 had potent monotherapy activity in MM and provided impressive synergy in combination with daratumumab using the NCI-H929 MM cell line derived xenograft (CDX) model (2022 AACR annual meeting abstracts). Herein, we further expanded our studies to pre-clinically develop HOSU-53 as a new potential MM therapy and explore additional synergistic immunotherapy combinations that could maintain efficacy in the case of anti-CD38 therapy resistance. We evaluated the in vitro antiproliferative efficacy of HOSU-53 against a panel of MM cell lines and found nanomolar potency validating that HOSU-53 would have monotherapy efficacy. Indeed, we conducted in vivo studies using two additional MM CDX subcutaneous (s.c) models, OPM-2 and RPMI-8226, and found significant tumor delay and survival advantage. In the OPM-2 model, HOSU-53 has a median survival of 54-days compared to vehicle at 28-days, while the median survival for HOSU-53 was 60-days compared to vehicle at 26-days in the RPMI-8226 model. We further verified HOSU-53 efficacy using the disseminated MM1.S luciferase CDX model and found a significant prolonged survival in the HOSU-53 cohort (median survival53-days) compared to vehicle (median survival28-days) that was further enhanced with isatuximab combination resulting in superior survival benefit (median survival 69-days). Given the continuous challenge in maintaining a durable response with anti-CD38 therapies due to resistance, we sought to explore additional combination regimens to maximize HOSU-53 efficacy. Currently there is significant clinical interest in CD47 antibody therapy such as magrolimab for both solid tumors and hematological malignancies. Our previous work suggested a strong synergy between CD47 antibodies and HOSU-53 in AML with curative potential (2022 ASH annual meeting abstracts), and hypothesized that we would observe similar synergy with HOSU-53 in combination with anti-CD47 in MM. Indeed, we found complete tumor regression in all mice treated with HOSU-53 + B6.H12 anti-CD47 therapy in the NCI-H929 s.c CDX model and significant prolonged survival and reduced bioluminescence in the NCI-H929 luciferase disseminated CDX model. Together, these two studies validate a potential clinical benefit to explore HOSU-53 + anti-CD47 regimen in MM patients. Furthermore, we observed that calreticulin (pro-phagocytosis signal) was modulated post HOSU-53 in vitro treatment suggesting its role in the observed synergy between HOSU-53 and CD47 blockade therapy. In summary, we show compelling survival benefit for HOSU-53 as a monotherapy which is further enhanced when combined with anti-CD38 or anti-CD47 therapies. HOSU-53 is expected to enter phase 1 clinical trials in 2024 and our data is supportive for its expansion into MM.

Effects of the G-quadruplex-binding drugs quarfloxin and CX-5461 on the malaria parasite Plasmodium falciparum

Plasmodium falciparum is the deadliest causative agent of human malaria. This parasite has historically developed resistance to most drugs, including the current frontline treatments, so new therapeutic targets are needed. Our previous work on guanine quadruplexes (G4s) in the parasite's DNA and RNA has highlighted their influence on parasite biology, and revealed G4 stabilising compounds as promising candidates for repositioning. In particular, quarfloxin, a former anticancer agent, kills blood-stage parasites at all developmental stages, with fast rates of kill and nanomolar potency. Here we explored the molecular mechanism of quarfloxin and its related derivative CX-5461. In vitro, both compounds bound to P. falciparum-encoded G4 sequences. In cellulo, quarfloxin was more potent than CX-5461, and could prevent establishment of blood-stage malaria in vivo in a murine model. CX-5461 showed clear DNA damaging activity, as reported in human cells, while quarfloxin caused weaker signatures of DNA damage. Both compounds caused transcriptional dysregulation in the parasite, but the affected genes were largely different, again suggesting different modes of action. Therefore, CX-5461 may act primarily as a DNA damaging agent in both Plasmodium parasites and mammalian cells, whereas the complete antimalarial mode of action of quarfloxin may be parasite-specific and remains somewhat elusive.

Potent MOR Agonists from 2′-Hydroxy-5,9-dimethyl-N-phenethyl Substituted-6,7-benzomorphans and from C8-Hydroxy, Methylene and Methyl Derivatives of N-Phenethylnormetazocine

(−)-5,9-Dimethyl-6,7-benzomorphan (normetazocine) derivatives with a para-OH or ortho-F substituent in the aromatic ring of the N-phenethyl moiety were synthesized and found to have subnanomolar potency at MOR, and both were fully efficacious in vitro. These new compounds, (1R,5R,9R)-6,11-dimethyl-3-(2-fluorophenethyl)-1,2,3,4,5,6-hexahydro-2,6-methanobenzo[d]azocin-8-ol and (1R,5R,9R)-6,11-dimethyl-3-(4-hydroxyphenethyl)-1,2,3,4,5,6-hexahydro-2,6-methanobenzo[d]azocin-8-ol, were more potent than the unsubstituted compound N-phenethylnormetazocine and about 30 or 40 times more potent than morphine, respectively. A variety of substituents in the ortho, meta, or para position in the aromatic ring of the N-phenethyl moiety were synthesized, 25 of these compounds, and found to have varying effects on potency and efficacy as determined by the forskolin-induced cAMP accumulation assay. The N-phenethyl moiety was also modified by increasing chain length to form a N-phenylpropyl side chain with and without a para-nitro moiety, and by an N-cinnamyl side chain. Also, an indole ethylamine normetazocine was synthesized to replace the N-phenethylamine side chain in normetazocine. The phenylpropylamine, propenylamine (cinnamyl) and the para-nitropropylamine had little or no MOR potency. The indole-ethylamine on the normetazocine nucleus, however, had moderate potency (MOR EC50 = 12 nM), and was fully efficacious (%Emax = 102%) in the cAMP assay. Retention of the N-phenethyl moiety and the addition of alkyl and alkenyl moieties on C8 in (−)-N-phenethylnormetazocine gave a C8-methylene derivative that had subnanomolar potency at MOR and a C8-methyl analog that had nanomolar potency. Five C8-substituted compounds were synthesized.

Structure-based design of potent FABP4 inhibitors with high selectivity against FABP3

Fatty-acid binding protein 4 (FABP4) presents an attractive target for therapeutic intervention in metabolic and inflammatory diseases in recent years. However, highly similar three-dimensional structures and fatty acid binding ability of multiple FABP family members pose a significant challenge in design of FABP4-selective inhibitors. Particularly, inhibition of FABP3 raises safety concerns such as cardiac dysfunction and exercise intolerance. Here, we reported the discovery of new FABP4 inhibitors with high selectivity over FABP3 by exploiting the little structural difference in the ligand binding pockets of FABP4 and FABP3. On the basis of our previously reported FABP4 inhibitors with nanomolar potency, different substituents were further introduced to perfectly occupy two sub-pockets of FABP4 that are distinct from those of FABP3. Remarkably, a single methyl group introduction leads to the discovery of compound C3 that impressively exhibits a 601-fold selectivity over FABP3 when maintained nanomolar binding affinity for FABP4. Moreover, C3 also shows good metabolic stability and potent cellular anti-inflammatory activity, making it a promising inhibitor for further development. Therefore, the present study highlights the utility of the structure-based rational design strategy for seeking highly selective and potent inhibitors of FABP4 and the importance of identifying the appropriate subsite as well as substituent for gaining the desired selectivity.

Discovery of C-3 isoxazole substituted thiochromone S,S-dioxide derivatives as potent and selective inhibitors for monoamine oxidase B (MAO-B)

Developing new scaffolds for highly potent and selective inhibitors of human Monoamine Oxidase B (hMAO-B) is a crucial objective in enhancing the efficacy and safety in the clinical treatment of neurodegenerative diseases. In this study, we have identified a series of C-3 isoxazole-substituted thiochromone S,S-dioxide derivatives that exhibit strong inhibitory activity against hMAO-B. The strategy of oxidizing thiochromone to thiochromone S,S-dioxide solves the key defect of extreme insolubility observed for thiochromone analogues. In addition, the sulfone group contributes extra hydrogen(H)-bonding interactions with Tyr435, which significantly increases the activity of thiochromone S,S-dioxide derivatives against hMAO-B. Furthermore, the presence of isoxazole group provides potential H-bonding interaction and electrostatic interaction with the residue of Tyr326, while the rigid aryl ring introduces a potential steric conflict with Phe208 of hMAO-A to improve both potency and selectivity. In our investigations, several compounds (9c, 10c, 10e, 10g, 10l and 10m) demonstrate remarkable single-digit nanomolar potency. These compounds exhibit favorable cytotoxicity profiles in both differentiated SH-SY5Y and HVSMC cells, without apparent cardiotoxic effects. Moreover, compounds 10e and 10h do not lead to an increase in ROS levels in differentiated SH-SY5Y cells, further demonstrating their potential as safe and effective hMAO-B inhibitors. These findings indicate that the C-3 isoxazole substituted thiochromone S,S-dioxide analogues are potential leading compounds for the development of selective inhibitors with high potency.

DDDR-13. ST-401, A BRAIN PENETRATING MICROTUBULE TARGETING AGENT THAT KILLS GLIOBLASTOMA IN INTERPHASE AND BY DISRUPTING MITOCHONDRIA FUNCTION

Abstract The development of small molecular entities that can effectively penetrate the blood-brain barrier and kill glioblastoma (GBM) is crucial in Neuro-Oncology. ST-401, a unique microtubule depolymerizer, has shown remarkable potential as a therapeutic agent for GBM in PD-GBM isolates (nanomolar potencies) and in vivo preclinical GBM models (potencies DNA-damaging agents). This study aims to investigate the novel mechanism of action of ST-401 and its impact on GBM cell death. We recently showed that increasing concentrations of ST-401 disrupts mitochondrial function, leading to cell death initially in interphase and then in mitosis. Through single-cell RNA sequencing analysis, significant changes in mRNA related to mitochondrial functions were observed, including ribosomal proteins and OXPHOS genes. The study focuses on the role of mitochondria in GBM tumorigenesis and explores how ST-401 affects mitochondrial OXPHOS function. We identified three GBM cell lines (U251, SNB-19, and SF-539) with increasing vulnerability to ST-401, respectively, as measured by using WST-1, an index of mitochondria OXPHOS. Further investigation using the Seahorse platform demonstrated that SF-539 cells treated with ST-401 exhibited a loss of spare capacitance and significantly reduced mitochondrial function, including basal and maximal OXPHOS response, as well as ATP production. In conclusion, our results emphasize the existence of specific subtypes of GBM that show vulnerability or resistance to the antitumor effects of ST-401. Identifying these subtypes is crucial for advancing precision Neuro-Oncology and facilitating human trials. Our results also suggest that ST-401's inhibition of microtubule function and induction of cell death in interphase are associated with the loss of mitochondrial respiratory function. Given the limited treatment options for brain tumors and the challenges in delivering drugs across the blood-brain barrier, the development of brain-penetrant drugs like ST-401 holds great promise. Funded by NIH (CA244213 to NS and NS106924 to NS).

High-Throughput Screen of Microbial Metabolites Identifies F1FO ATP Synthase Inhibitors as New Leads for Naegleria fowleri Infection.

Primary amebic meningoencephalitis (PAM), a brain infection caused by a free-living ameba Naegleria fowleri, leads to an extensive inflammation of the brain and death within 1-18 (median 5) days after symptoms begin. Although natural products have played a significant role in the development of drugs for over a century, research focusing on identifying new natural product-based anti-N. fowleri agents is limited. We undertook a large-scale ATP bioluminescence-based screen of about 10,000 unique marine microbial metabolite mixtures against the trophozoites of N. fowleri. Our screen identified about 100 test materials with >90% inhibition at 50 μg/mL and a dose-response study found 20 of these active test materials exhibiting an EC50 ranging from 0.2 to 2 μg/mL. Examination of four of these potent metabolite mixtures, derived from our actinomycete strains CNT671, CNT756, and CNH301, resulted in the isolation of a pure metabolite identified as oligomycin D. Oligomycin D exhibited nanomolar potency on multiple genotypes of N. fowleri, and it was five- or 850-times more potent than the recommended drugs amphotericin B or miltefosine. Oligomycin D is fast-acting and reached its EC50 in 10 h, and it was also able to inhibit the invasiveness of N. fowleri significantly when tested on a matrigel invasion assay. Since oligomycin is known to manifest inhibitory activity against F1FO ATP synthase, we tested different F1FO ATP synthase inhibitors and identified a natural peptide leucinostatin as a fast-acting amebicidal compound with nanomolar potency on multiple strains.

Structural Optimization of Antimycobacterial Azaaurones Towards Improved Solubility and Metabolic Stability.

While N-acetyl azaaurones have already been disclosed for their potential against tuberculosis (TB), their low metabolic stability remains an unaddressed liability. We now report a study designed to improve the metabolic stability and solubility of the azaaurone scaffold and to identify the structural requirements for antimycobacterial activity. Replacing the N-acetyl moiety for a N-carbamoyl group led to analogues with sub- and nanomolar potencies against M. tuberculosis H37Rv, as well as equipotent against drug-susceptible and drug-resistant M. tuberculosis isolates. The new N-carbamoyl azaaurones exhibited improved microsomal stability, compared to their N-acetylated counterparts, with several compounds displaying moderate to high kinetic solubility. The frequency of spontaneous resistance to azaaurones was observed to be in the range of 10-8 , a value that is comparable to current TB drugs in the market. Overall, these results reveal that azaaurones are amenable to structural modifications to improve metabolic and solubility liabilities, and highlight their potential as antimycobacterial agents.

Bacterial efflux pump modulators prevent bacterial growth in macrophages and under broth conditions that mimic the host environment.

Bacterial efflux pumps are critical for resistance to antibiotics and for virulence. We previously identified small molecules that inhibit efflux pumps (efflux pump modulators, EPMs) and prevent pathogen replication in host cells. Here, we used medicinal chemistry to increase the activity of the EPMs against pathogens in cells into the nanomolar range. We show by cryo-electron microscopy that these EPMs bind an efflux pump subunit. In broth culture, the EPMs increase the potency (activity), but not the efficacy (maximum effect), of antibiotics. We also found that bacterial exposure to the EPMs appear to enable the accumulation of a toxic metabolite that would otherwise be exported by efflux pumps. Thus, inhibitors of bacterial efflux pumps could interfere with infection not only by potentiating antibiotics, but also by allowing toxic waste products to accumulate within bacteria, providing an explanation for why efflux pumps are needed for virulence in the absence of antibiotics.

Efficacy of Belvarafenib with and without Cobimetinib in Preclinical Models of Ras Pathway-Mutant AML

Introduction Outcomes for children with acute myeloid leukemia (AML) remain poor, with ~40% dying from refractory leukemia or treatment-related toxicity (Gamis et al. 2014). NRAS, KRAS, and NF1 mutations occur in over 40% of pediatric AMLs (Bolouri et al. 2018), but efforts to therapeutically target the RAS/mitogen activated protein kinase (MAPK) pathway have been largely unsuccessful. Belvarafenib, a novel type II pan-RAF kinase inhibitor that inhibits mutant monomeric BRAF proteins and activated RAF homo- and heterodimers, has shown promising safety/efficacy data in adult solid cancers (Yen et al. 2021). Here, we investigate belvarafenib in preclinical models of AML, both alone and in combination with the allosteric MEK inhibitor, cobimetinib. Methods We used a panel of NRAS (OCI-AML3, HL-60, THP-1) or KRAS (NOMO-1, NB4, and SKM-1) mutant human AML cell lines. Viability was determined by CellTiter-Glo. Synergy was assessed by Bliss Independence and Chou Talalay methods. Transcriptome and proteomic profiling were performed as previously described (Pucciarelli et al. 2020). Mouse AMLs were generated using retroviral insertional mutagenesis (Li et al. 2011). Cryopreserved primary AML cells were injected intravenously into sublethally irradiated recipients that were then treated daily with vehicle, belvarafenib, cobimetinib, or the combination until disease progression. Survival curves were generated using Kaplan-Meier analysis. Results Belvarafenib inhibited the growth of AML cells lines with nanomolar potency at IC50 values ranging from 48nM (OCI-AML3) to 310nM (SKM-1). In all cell lines, belvarafenib and cobimetinib were highly synergistic. Western blotting of OCI-AML3 cells treated with either belvarafenib or cobimetinib at their respective IC50 values (50nM belvarafenib, 20nM cobimetinib) for 4 or 24 hours demonstrated discordant effects on downstream MAPK effector proteins (Fig. 1). Whereas cobimetinib potently suppressed phosphorylated ERK (pERK), belvarafenib unexpectedly had no effect. Phosphorylated S6 (pS6) levels were not reduced by either belvarafenib or cobimetinib at their respective IC50 values, but were dramatically reduced by the drug combination. To further investigate these unanticipated biochemical findings, we treated OCI-AML3 cells with belvarafenib, cobimetinib or the combination and performed kinome profiling and transcriptome sequencing (RNAseq) analysis. Exposure to 10nM cobimetinib inhibited several kinases at 4 hours with rebound activation of kinases involved in cell cycle progression at 24 hours. Cells treated with 100nM belvarafenib displayed down-regulation of multiple kinases that persisted at 24 hours. Cells treated with the combination at these doses exhibited the most potent inhibition of many kinases; this effect recapitulated the effect of high dose (500nM) belvarafenib, but not high dose (50nM) cobimetinib. At the transcriptional level, cells treated with the combination showed far greater suppression of negative regulators of MAPK signaling (e.g., DUSPs, SPRY2/4, SPRED1/2) than either agent alone, suggesting potent inhibition of the MAPK pathway by the combination. Intriguingly, OCI-AML3 cells treated with high-dose belvarafenib or low-dose combination unexpectedly exhibited profound down-regulation of mTORC1-regulated genes on GSEA analysis. We extended these in vitro data by performing preclinical trials in mice transplanted with 5 independent primary Nras- or Kras-mutant AMLs that received belvarafenib (15mg/kg), cobimetinib (2mg/kg), the combination or vehicle by oral gavage (Fig. 2). The combination was well tolerated. Belvarafenib prolonged survival in all 5 trials; in 3 of 5, the addition of low-dose cobimetinib further enhanced survival (median survival: vehicle, 9 days; cobimetinib, 13 days; belvarafenib, 22 days; combination, 32 days; p < 0.0001). Conclusions Belvarafenib showed activity in 6 of 6 Ras mutant human AML cell lines and in 5 of 5 primary Ras mutant murine AMLs. Belvarafenib and cobimetinib displayed synergy in all AML cell lines and in 3 of 5 murine AMLs treated in vivo. Mechanistically, we identified distinct biochemical and transcriptional effects of RAF dimer and MEK inhibition in AML cells. We are characterizing these further and pursuing causes of resistance in primary Nras- and Kras-mutant mouse AMLs that relapsed after an initial response to treatment.

Targeting Covalent and Non-Covalent Btki-Resistant CLL Using the Dual Irreversible/Reversible 4 th Generation BTK Inhibitor LP-168

Background: Treatment of chronic lymphocytic leukemia (CLL) has been transformed with targeted therapies including inhibitors of Bruton's tyrosine kinase (BTKi). Currently three covalent BTK inhibitors (cBTKi) are approved for CLL, but most patients eventually relapse, commonly through acquisition of the C481S BTK mutation (Woyach et al. 2014). Pirtobrutinib and nemtabrutinib are non-covalent BTK inhibitors (ncBTKi) developed to target and inhibit C481S mutant BTK. However novel secondary site mutations in BTK, namely T474I and L528W, have been found to confer resistance to both cBTKi and ncBTKi (Wang et al. 2022). Regardless of these mutations, BCR signaling remains intact, suggesting that inhibition of BTK maintains its therapeutic importance. LP-168 is a novel ultra-selective 4 th generation BTKi with an active warhead capable of covalent interaction with WT BTK or non-covalent binding when a BTK C481 mutation is present. Methods: To determine the target selectivity of LP-168 we screened the compound against 468 kinases in the scanMAX Kinase Assay. For validation of covalent binding, dialysis against WT BTK was performed. Primary CLL B cells were isolated by negative selection and treated with LP-168 for all experiments. BCR signaling alterations were assessed via immunoblot to observe changes in target protein phosphorylation. Changes in CLL migration towards CXCL12 or CXCL13 were interrogated by transwell migration. Cytotoxicity towards primary CLL cells was measured via Annexin V/PI flow cytometry with and without HS-5 stromal cell support. Experiments using TMD8 cells were performed following CRISPR modification to insert WT, C481S, or T474I BTK. In vivo survival studies were performed using both the Eμ-TCL1 and Eμ-MTCP1 mouse engraftment models. Results: LP-168 demonstrated selectivity towards BTK with roughly 700-fold selectivity for BTK vs its next off-target. In an enzymatic assay, LP-168 displayed nanomolar potency towards both WT BTK (IC 50=0.11 nM) and C481S BTK (IC 50=1.0 nM). Following 2-hour drugging, LP-168 inhibited BCR signaling in primary CLL B cells at increasing concentrations, demonstrated by reduced phosphorylation of BTK (92%, p<0.0001, n=8) and its immediate downstream target PLCγ2 (41%, p=0.0013, n=8). Migration capacity of primary CLL cells towards CXCL12 and CXCL14 was also found to decrease after 2-hours of treatment with LP-168 (52%, p<0.001, n=10 and 51%, p<0.001, n=10, respectively). After 48 hours of exposure, LP-168 was able to induce cytotoxicity of primary CLL cells alone and in co-culture with HS-5 stromal cells in a dose-dependent manner, with similar or improved efficacy to other BTKi. LP-168 decreased CLL cell production of CCL3 and CCL4 chemokines (CCL3: 93%, p<0.001, n=8; CCL4: 93%, p<0.001, n=8). LP-168 also demonstrated modest cytotoxicity towards TMD8 cells harboring either C481S BTK (24%, p=0.0008 , n=3) or T474I BTK (13%, p=0.0690, n=3), as well as decreased production of CCL3 (C481S: 95%, p<0.0001, n=3; T474I: 99%, p<0.0001, n=3) and CCL4 (C481S: 97%, p<0.0001, n=3; T474I: 98%, p<0.0001, n=3) (Figure 1A). Further, we observed inhibition of downstream BCR signaling in primary CLL B cells harboring C481S via immunoblotting and cytokine production. Finally, to evaluate the in vivo efficacy of LP-168 we tested this compound in both the Eµ-TCL1 and Eµ-MTCP1 models. We treated mice daily via oral gavage and found that 50 mg/kg of LP-168 significantly improved survival in the Eµ-TCL1 model when compared to vehicle (median 51 vs 44 days; p=0.0018) or ibrutinib at 50 mg/kg (median 51 vs 45 days; p=0.0098) and the Eµ-MTCP1 model when compared with vehicle (median 122 vs 62 days; p<0.0001) or ibrutinib at 50 mg/kg (median 122 vs 96 days; p=0.0162) (Figure 1B). Conclusions: Collectively, our data demonstrate that LP-168 is a potent and selective inhibitor of BTK with activity against CLL, even in the presence of mutations that mediate resistance to cBTKi and ncBTKi. These data support the continued preclinical and clinical investigation of LP-168, which is currently being studied in the phase 1 setting of CLL (NCT04775745) and NHL (NCT04993690).

Effective Prevention of Arterial Thrombosis with Albumin-Thrombin Inhibitor Conjugates.

Thromboprophylaxis is indicated in patients at an elevated risk of developing thrombotic disorders, typically using direct oral anticoagulants or low-molecular-weight heparins. We postulated that transient thromboprophylaxis (days-weeks) could be provided by a single dose of an anticoagulant engineered for prolonged pharmacokinetics. In the present work, d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone (PPACK) was used as a model anticoagulant to test the hypothesis that conjugation of thrombin inhibitors to the surface of albumin would provide durable protection against thrombotic insults. Covalent conjugates were formed between albumin and PPACK using click chemistry, and they were tested in vitro using a thrombin activity assay and a clot formation assay. Thromboprophylactic efficacy was tested in mouse models of arterial thrombosis, both chemically induced (FeCl3) and following ischemia-reperfusion (transient middle cerebral artery occlusion; tMCAO). Albumin-PPACK conjugates were shown to have nanomolar potency in both in vitro assays, and following intravenous injection had prolonged circulation. Conjugates did not impact hemostasis (tail clipping) or systemic coagulation parameters in normal mice. Intravenous injection of conjugates prior to FeCl3-induced thrombosis provided significant protection against occlusion of the middle cerebral and common carotid arteries, and injection immediately following ischemia-reperfusion reduced stroke volume measured 3 days after injury by ∼40% in the tMCAO model. The data presented here provide support for the use of albumin-linked anticoagulants as an injectable, long-circulating, safe thromboprophylactic agent. In particular, albumin-PPACK provides significant protection against thrombosis induced by multiple mechanisms, without adversely affecting hemostasis.

Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A

The normally antiviral enzyme APOBEC3A is an endogenous mutagen in human cancer. Its single-stranded DNA C-to-U editing activity results in multiple mutagenic outcomes including signature single-base substitution mutations (isolated and clustered), DNA breakage, and larger-scale chromosomal aberrations. APOBEC3A inhibitors may therefore comprise a unique class of anti-cancer agents that work by blocking mutagenesis, slowing tumor evolvability, and preventing detrimental outcomes such as drug resistance and metastasis. Here we reveal the structural basis of competitive inhibition of wildtype APOBEC3A by hairpin DNA bearing 2′-deoxy-5-fluorozebularine in place of the cytidine in the TC substrate motif that is part of a 3-nucleotide loop. In addition, the structural basis of APOBEC3A’s preference for YTCD motifs (Y = T, C; D = A, G, T) is explained. The nuclease-resistant phosphorothioated derivatives of these inhibitors have nanomolar potency in vitro and block APOBEC3A activity in human cells. These inhibitors may be useful probes for studying APOBEC3A activity in cellular systems and leading toward, potentially as conjuvants, next-generation, combinatorial anti-mutator and anti-cancer therapies.

Enhancing Giardicidal Activity and Aqueous Solubility through the Development of "RetroABZ", a Regioisomer of Albendazole: In Vitro, In Vivo, and In Silico Studies.

Parasitic diseases, including giardiasis caused by Giardia lamblia (G. lamblia), present a considerable global health burden. The limited effectiveness and adverse effects of current treatment options underscore the necessity for novel therapeutic compounds. In this study, we employed a rational design strategy to synthesize retroalbendazole (RetroABZ), aiming to address the limitations associated with albendazole, a commonly used drug for giardiasis treatment. RetroABZ exhibited enhanced in vitro activity against G. lamblia trophozoites, demonstrating nanomolar potency (IC50 = 83 nM), outperforming albendazole (189 nM). Moreover, our in vivo murine model of giardiasis displayed a strong correlation, supporting the efficacy of RetroABZ, which exhibited an eleven-fold increase in potency compared to albendazole, with median effective dose (ED50) values of 5 µg/kg and 55 µg/kg, respectively. A notable finding was RetroABZ's significantly improved water solubility (245.74 µg/mL), representing a 23-fold increase compared to albendazole, thereby offering potential opportunities for developing derivatives that effectively target invasive parasites. The molecular docking study revealed that RetroABZ displays an interaction profile with tubulin similar to albendazole, forming hydrogen bonds with Glu198 and Cys236 of the β-tubulin. Additionally, molecular dynamics studies demonstrated that RetroABZ has a greater number of hydrophobic interactions with the binding site in the β-tubulin, due to the orientation of the propylthio substituent. Consequently, RetroABZ exhibited a higher affinity compared to albendazole. Overall, our findings underscore RetroABZ's potential as a promising therapeutic candidate not only for giardiasis but also for other parasitic diseases.

OR26-01 A Novel Small Molecule Thyrotropin Receptor Antagonist

Abstract Disclosure: G.J. Kahaly: None. A. George: None. L. Steiner: None. T. Weirauch: None. J. Henseling: None. L. Frommer: None. J. Wolf: None. Background & aims: Treatment of both Graves’ hyperthyroidism (GH) and Graves’ orbitopathy (GO) is still far from adequate, hence new therapeutic modalities, which specifically target the autoantigens in GH/GO, are warranted. This study determined the preclinical in vitro efficacy of SYD5115, a novel thyrotropin receptor (TSH-R) antagonist. Methods: SYD5115 is a nanomolar potent low molecular weight TSH-R antagonist with in vivo activity in rats after oral dosing. The compound consists of an N-acetylated dihydropyrrole pyrimidine core containing several substituents such as a difluorinated-spirocyclohexyl ring and a chiral carbamate group on the pyrimidine ring. The TSH-R inhibiting capability of SYD5115 was tested through stimulation of wild type and chimeric TSH-R expressed in Chinese hamster ovary (CHO) cells using-two functional (stimulatory and blocking) cell-based TSH-R-Ab bioassays. TSH-R expressing human orbital fibroblasts, collected from GH+GO patients (GOF), were stimulated with the monoclonal antibody M22 or with stimulatory TSH-R-Ab (TSAb)-positive sera with cyclic adenosine monophosphate (cAMP) or hyaluronic acid (HA) release as read-outs. The effect of SYD5115 on the viability of GOF was tested in both 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide and the “scratch” cell growth assays. Results: The new small molecule SYD5115 significantly and dose-dependently inhibited the TSH-R activation through M22 or TSAb-positive sera in all performed bioassays. Inhibition showed similar levels in the TSAb reporter bioassay and in the cAMP assay with GOF. The % inhibition and compound concentration showed a sigmoidal relationship, with all seven TSAb positive sera markedly inhibited by SYD5115. A SYD5115 dose-dependent inhibition of M22 (10ng/ml, 6 hours)-stimulated HA and/or cAMP-release from GOF was observed. Strong SYD5115-induced inhibitions of M22-stimulated cAMP production in GOF were registered with SYD5115 concentrations of one (p=0.0029), 10 (p<0.0001), 100 (p<0.0001), 1,000 (p<0.0001), and 10,000 (p<0.0001) nM/L, respectively. SYD5115-induced inhibition of M22-stimulated HA production was noted with SYD5115 concentrations of 100 (p=0.0392), 1,000 (p=0.0431), and 10,000 (p=0.0245) nM/L, respectively. The inhibitory activity of SYD5115 was confirmed in a human osteosarcoma U2OS cell line stably expressing human TSH-R with cAMP as read-out. SYD5115 induced 100% inhibition of the M22-induced cAMP levels with a potency of 193 nM. Compared to control, SYD5115 affected neither the growth nor the migration of cultivated GOF. In addition, SYD5115 did not alter the viability of GOF. Conclusions: SYD5115 blocked M22- and TSAb-induced TSH-R activity with a nanomolar potency in TSH-R overexpressed CHO cells as well as primary GOF, which demonstrates the ability of this novel small molecule to inhibit TSH-R overactivity. Presentation: Saturday, June 17, 2023

Further Quinolizidine Derivatives as Antiarrhythmic Agents- 3.

Fourteen quinolizidine derivatives, structurally related to the alkaloids lupinine and cytisine and previously studied for other pharmacological purposes, were presently tested for antiarrhythmic, and other cardiovascular effects on isolated guinea pig heart tissues in comparison to well-established reference drugs. According to their structures, the tested compounds are assembled into three subsets: (a) N-(quinolizidinyl-alkyl)-benzamides; (b) 2-(benzotriazol-2-yl)methyl-1-(quinolizidinyl)alkyl-benzimidazoles; (c) N-substituted cytisines. All compounds but two displayed antiarrhythmic activity that was potent for compounds 4, 1, 6, and 5 (in ascending order). The last compound (N-(3,4,5-trimethoxybenzoyl)aminohomolupinane) was outstanding, exhibiting a nanomolar potency (EC50 = 0.017 µM) for the increase in the threshold of ac-arrhythmia. The tested compounds shared strong negative inotropic activity; however, this does not compromise the value of their antiarrhythmic action. On the other hand, only moderate or modest negative chronotropic and vasorelaxant activities were commonly observed. Compound 5, which has high antiarrhythmic potency, a favorable cardiovascular profile, and is devoid of antihypertensive activity in spontaneously hypertensive rats, represents a lead worthy of further investigation.

175 Nanomolar potency 3-carboxy-2-methylbenzofuran pendrin inhibitors for treatment of cystic fibrosis

175 Nanomolar potency 3-carboxy-2-methylbenzofuran pendrin inhibitors for treatment of cystic fibrosis

Electrochemical Synthesis of Unnatural Amino Acids via Anodic Decarboxylation of N-Acetylamino Malonic Acid Derivatives.

Broad application of α,α-disubstituted cyclic amino acid derivatives in medicinal chemistry urges for analogue design with improved pharmacokinetic properties. Herein, we disclose an electrochemical approach toward unnatural THF- and THP-containing amino acid derivatives that relies on anodic decarboxylation-intramolecular etherification of inexpensive and readily available N-acetylamino malonic acid monoesters under Hofer-Moest reaction conditions. The decarboxylative cyclization proceeds under constant current conditions in an undivided cell in an aqueous medium without any added base. A successful bioisosteric replacement of the 1-aminocyclohexane-1-carboxylic acid subunit by the THP-containing amino acid scaffold in cathepsin K inhibitor balicatib helped to reduce lipophilicity while retaining low nanomolar enzyme inhibitory potency and comparable microsomal stability.

Design and synthesis of 4th generation EGFR inhibitors against human triple (Del19/T790M/C797S) mutation

Epidermal growth factor receptor (EGFR)-targeted therapy is used to treat EGFR mutation-induced non-small cell lung cancer (NSCLC). However, its efficacy does not last beyond a certain period due to the development of primary and secondary resistance. First and second-generation inhibitors (e.g., gefitinib, erlotinib, and afatinib) induce EGFR T790M mutations, while third-generation inhibitors (e.g., osimertinib) induce C797S as a major target resistance mutation. Therefore, the C797S mutation is being actively researched. In this study, we investigated the structure-activity relationship of several synthesized compounds as fourth-generation inhibitors against the C797S mutation. We identified a compound 13k that displayed nanomolar potency and high selectivity. Moreover, we used a triple mutant xenograft mouse model to evaluate the in vivo efficacy of 13k in inhibiting EGFR C797S, which demonstrated exceptional profiles and satisfactory EGFR C797S inhibition efficacy. Based on its excellent in vitro and in vivo profiles, compound 13k can be considered a promising candidate for treating EGFR C797S mutations.