Poor Drug-like Properties Research Articles

Prodrug activation in malaria parasites mediated by an imported erythrocyte esterase, acylpeptide hydrolase (APEH).

The continued emergence of antimalarial drug resistance highlights the need to develop new antimalarial therapies. Unfortunately, new drug development is often hampered by poor drug-like properties of lead compounds. Prodrugging temporarily masks undesirable compound features, improving bioavailability and target penetration. We have found that lipophilic diester prodrugs of phosphonic acid antibiotics, such as fosmidomycin, exhibit significantly higher antimalarial potency than their parent compounds (1). However, the activating enzymes for these prodrugs were unknown. Here, we show that an erythrocyte enzyme, acylpeptide hydrolase (APEH) is the major activating enzyme of multiple lipophilic ester prodrugs. Surprisingly, this enzyme is taken up by the malaria parasite, Plasmodium falciparum, where it localizes to the parasite cytoplasm and retains enzymatic activity. Using a novel fluorogenic ester library, we characterize the structure activity relationship of APEH, and compare it to that of P. falciparum esterases. We show that parasite-internalized APEH plays an important role in the activation of substrates with branching at the alpha carbon, in keeping with its exopeptidase activity. Our findings highlight a novel mechanism for antimicrobial prodrug activation, relying on a host-derived enzyme to yield activation at a microbial target. Mutations in prodrug activating enzymes are a common mechanism for antimicrobial drug resistance (2-4). Leveraging an internalized host enzyme would circumvent this, enabling the design of prodrugs with higher barriers to drug resistance.

Discovery of Monovalent Direct Degraders of BRD4 that Act via the Recruitment of DCAF11.

Targeted protein degradation (TPD) using the ubiquitin proteasome system (UPS) is a rapidly growing drug discovery modality to eliminate pathogenic proteins. Strategies for TPD have focused on heterobifunctional degraders that often suffer from poor drug-like properties, and molecular glues that rely on serendipitous discovery. Monovalent "direct" degraders represent an alternative approach, in which small molecules bind to a target protein and induce degradation of that protein through the recruitment of an E3 ligase complex. Using an ultra-high throughput cell-based screening platform, degraders of the bromodomain extraterminal protein BRD4 were identified and optimized to yield a lead compound, PLX-3618. In this paper, we demonstrate that PLX-3618 elicited UPS-mediated selective degradation of BRD4, resulting in potent antitumor activity in vitro and in vivo. Characterization of the degradation mechanism identified DCAF11 as the E3 ligase required for PLX-3618-mediated degradation of BRD4. Protein-protein interaction studies verified a BRD4:PLX-3618:DCAF11 ternary complex, and mutational studies provided further insights into the DCAF11-mediated degradation mechanism. Collectively, these results demonstrate the discovery and characterization of a novel small molecule that selectively degrades BRD4 through the recruitment of the E3 substrate receptor, DCAF11, and promotes potent antitumor activity in vivo.

Ex Vivo Drug Evaluation Using Human Ocular Cells: A Contribution to Drug Discovery

The field of therapeutics is fundamentally and heavily dependent on drug discovery. The record number of new agents being developed probably conceals how ardent the task is [1]. Drug discovery is indeed a long, costly, and high-risk process that takes over 10-15 years with an average cost of over $1-2 billion for a new drug to be approved for clinical use [2]. What is astonishing is the failure rate of candidate drugs. By most accounts over 90% of drug candidates fail to be approved for clinical use. Lack of clinical efficacy and poor drug-like properties along with unmanageable toxicity are among the frequent reasons for their failure. The transition between basic and clinical science, known colloquially and the “valley of death”, is where most promising discoveries meet their demise [3]. Multiple efforts have been made to overcome this difficult situation that adversely affects modern therapeutics [2].

Pharmacological Characterization of SDX-7320/Evexomostat: A Novel Methionine Aminopeptidase Type 2 Inhibitor with Anti-tumor and Anti-metastatic Activity.

Methionine aminopeptidase type 2 (METAP2) is a ubiquitous, evolutionarily conserved metalloprotease fundamental to protein biosynthesis which catalyzes removal of the N-terminal methionine residue from nascent polypeptides. METAP2 is an attractive target for cancer therapeutics based upon its over-expression in multiple human cancers, the importance of METAP2-specific substrates whose biological activity may be altered following METAP2 inhibition, and additionally, that METAP2 was identified as the target for the anti-angiogenic natural product, fumagillin. Irreversible inhibition of METAP2 using fumagillin analogues has established the anti-angiogenic and anti-tumor characteristics of these derivatives; however, their full clinical potential has not been realized due to a combination of poor drug-like properties and dose-limiting central nervous system (CNS) toxicity. This report describes the physicochemical and pharmacological characterization of SDX-7320 (evexomostat), a polymer-drug conjugate of the novel METAP2 inhibitor (METAP2i) SDX-7539. In vitro binding, enzyme, and cell-based assays demonstrated that SDX-7539 is a potent and selective METAP2 inhibitor. In utilizing a high molecular weight, water-soluble polymer to conjugate the novel fumagillol-derived, cathepsin-released, METAP2i SDX-7539, limitations observed with prior generation, small molecule fumagillol derivatives were ameliorated including reduced CNS exposure of the METAP2i, and prolonged half-life enabling convenient administration. Multiple xenograft and syngeneic cancer models were utilized to demonstrate the anti-tumor and anti-metastatic profile of SDX-7320. Unlike polymer-drug conjugates in general, reductions in small molecule-equivalent efficacious doses following polymer conjugation were observed. SDX-7320 has completed a phase I clinical safety study in patients with late-stage cancer and is currently being evaluated in multiple phase Ib/II clinical studies in patients with advanced solid tumors.

Resveratrol natural product inspired compound as a potent neuroprotectant against acute oxidative stress.

Resveratrol as well as natural products biosynthetically derived from it, have been shown to have protective effects against oxidative stress. However, these compounds possess poor druglike properties. At sub-nanomolar concentrations, a novel compound (RVM-6) inspired by a resveratrol natural product prolongs synaptic viability in neuromuscular junctions in D. melanogaster exposed to acute oxidative stress.

Exploring the Antibacterial Potential of Semisynthetic Phytocannabinoid: Tetrahydrocannabidiol (THCBD) as a Potential Antibacterial Agent against Sensitive and Resistant Strains of Staphylococcus aureus.

Antimicrobial resistance (AMR) is one of the most challenging problems and is responsible for millions of deaths every year. We therefore urgently require new chemical entities with novel mechanisms of action. Phytocannabinoids have been adequately reported for the antimicrobial effect but not seriously pursued because of either stringent regulatory issues or poor drug-like properties. In this regard, the current work demonstrated the antibacterial potential of tetrahydrocannabidiol (THCBD, 4), a semisynthetic phytocannabinoid, against Staphylococcus aureus, the second-most widespread bug recognized by the WHO. THCBD (4) was generated from cannabidiol and subjected to extensive antibacterial screening. In in vitro studies, THCBD (4) demonstrated a potent MIC of 0.25 μg/mL against Gram-positive bacteria, S. aureus ATCC-29213. It is interesting to note that THCBD (4) has demonstrated strong effectiveness against efflux pump-overexpressing (SA-1199B, SA-K2191, SA-K2192, and Mupr-1) and multidrug-resistant (MRSA-15187) S. aureus strains. THCBD (4) has also shown a good effect in kill kinetic assays against ATCC-29213 and MRSA-15187. In the checkerboard assay, THCBD (4) has shown additive/indifference effects with several well-known clinically used antibiotics, tetracycline, mupirocin, penicillin G, and ciprofloxacin. THCBD (4) also exhibited good permeability in the artificial skin model. Most importantly, THCBD (4) has significantly reduced CFU in mice's in vivo skin infection models and also demonstrated decent plasma exposure with 16-17% oral bioavailability. Acute dermal toxicity of THCBD (4) suggests no marked treatment-related impact on gross pathophysiology. This attractive in vitro and in vivo profile of plant-based compounds opens a new direction for new-generation antibiotics and warrants further detailed investigation.

Structure-based design of a phosphotyrosine-masked covalent ligand targeting the E3 ligase SOCS2

The Src homology 2 (SH2) domain recognizes phosphotyrosine (pY) post translational modifications in partner proteins to trigger downstream signaling. Drug discovery efforts targeting the SH2 domains have long been stymied by the poor drug-like properties of phosphate and its mimetics. Here, we use structure-based design to target the SH2 domain of the E3 ligase suppressor of cytokine signaling 2 (SOCS2). Starting from the highly ligand-efficient pY amino acid, a fragment growing approach reveals covalent modification of Cys111 in a co-crystal structure, which we leverage to rationally design a cysteine-directed electrophilic covalent inhibitor MN551. We report the prodrug MN714 containing a pivaloyloxymethyl (POM) protecting group and evidence its cell permeability and capping group unmasking using cellular target engagement and in-cell 19F NMR spectroscopy. Covalent engagement at Cys111 competitively blocks recruitment of cellular SOCS2 protein to its native substrate. The qualified inhibitors of SOCS2 could find attractive applications as chemical probes to understand the biology of SOCS2 and its CRL5 complex, and as E3 ligase handles in proteolysis targeting chimera (PROTACs) to induce targeted protein degradation.

PBRICS: A Novel Fragmentation Method for Explainable Property Prediction of Drug-Like Small Molecules.

Generative artificial intelligence algorithms have shown to be successful in exploring large chemical spaces and designing novel and diverse molecules. There has been considerable interest in developing predictive models using artificial intelligence for drug-like properties, which can potentially reduce the late-stage attrition of drug candidates or predict the properties of novel AI-designed molecules. Concurrently, it is important to understand the contribution of functional groups toward these properties and modify them to obtain property-optimized lead compounds. As a result, there is an increasing interest in the development of explainable property prediction models. However, current explainable approaches are mostly atom-based, where, often, only a fraction of a fragment is shown to be significant. To address the above challenges, we have developed a novel domain-aware molecular fragmentation approach termed post-processing of BRICS (pBRICS), which can fragment small molecules into their functional groups. Multitask models were developed to predict various properties, including the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties. The fragment importance was explained using the gradient-weighted class activation mapping (Grad-CAM) approach. The method was validated on data sets of experimentally available matched molecular pairs (MMPs). The explanations from the model can be useful for medicinal chemists to identify the fragments responsible for poor drug-like properties and optimize the molecule. The explainability approach was also used to identify the reason behind false positive and false negative MMP predictions. Based on evidence from the existing literature and our analysis, some of these mispredictions were justified. We propose that the quantity, quality, and diversity of the training data will improve the accuracy of property prediction algorithms for novel molecules.

Metal-Based Nanoparticles for Cancer Metalloimmunotherapy.

Although the promise of cancer immunotherapy has been partially fulfilled with the unprecedented clinical success of several immunotherapeutic interventions, some issues, such as limited response rate and immunotoxicity, still remain. Metalloimmunotherapy offers a new form of cancer immunotherapy that utilizes the inherent immunomodulatory features of metal ions to enhance anticancer immune responses. Their versatile functionalities for a multitude of direct and indirect anticancer activities together with their inherent biocompatibility suggest that metal ions can help overcome the current issues associated with cancer immunotherapy. However, metal ions exhibit poor drug-like properties due to their intrinsic physicochemical profiles that impede in vivo pharmacological performance, thus necessitating an effective pharmaceutical formulation strategy to improve their in vivo behavior. Metal-based nanoparticles provide a promising platform technology for reshaping metal ions into more drug-like formulations with nano-enabled engineering approaches. This review provides a general overview of cancer immunotherapy, the immune system and how it works against cancer cells, and the role of metal ions in the host response and immune modulation, as well as the impact of metal ions on the process via the regulation of immune cells. The preclinical studies that have demonstrated the potential of metal-based nanoparticles for cancer metalloimmunotherapy are presented for the representative nanoparticles constructed with manganese, zinc, iron, copper, calcium, and sodium ions. Lastly, the perspectives and future directions of metal-based nanoparticles are discussed, particularly with respect to their clinical applications.

Discovery of pyrimidine-5-carboxamide derivatives as novel salt-inducible kinases (SIKs) inhibitors for inflammatory bowel disease (IBD) treatment

Salt-inducible kinases (SIKs) play a crucial role in inflammation process, acting as molecular switches that regulate the transformation of M1/M2 macrophages. HG-9-91–01 is a SIKs inhibitor with potent inhibitory activity against SIKs in the nanomolar range. However, its poor drug-like properties, including a rapid elimination rate, low in vivo exposure and high plasma protein binding rate, have hindered further research and clinical application. To improve the drug-like properties of HG-9-91–01, a series of pyrimidine-5-carboxamide derivatives were designed and synthesized through a molecular hybridization strategy. The most promising compound 8h was obtained with favorable activity and selectivity on SIK1/2, excellent metabolic stability in human liver microsome, enhanced in vivo exposure and suitable plasma protein binding rate. Mechanism research showed that compound 8h significantly up-regulated the expression of anti-inflammatory cytokine IL-10 and reduced the expression of pro-inflammatory cytokine IL-12 in bone marrow-derived macrophages. Furthermore, it significantly elevated expression of cAMP response element-binding protein (CREB) target genes IL-10, c-FOS and Nurr77. Compound 8h also induced the translocation of CREB-regulated transcriptional coactivator 3 (CRTC3) and elevated the expression of LIGHT, SPHK1 and Arginase 1. Additionally, compound 8h demonstrated excellent anti-inflammatory effects in a DSS-induced colitis model. Generally, this research indicated that compound 8h has the potential to be developed as an anti-inflammatory drug candidate.

Liposomal Delivery of MIW815 (ADU-S100) for Potentiated STING Activation

Stimulator of interferon genes (STING) agonists can improve the anticancer efficacy of immune checkpoint blockade by amplifying tumor immunogenicity. However, the clinical translation of cyclic dinucleotides (CDNs) as STING agonists is hindered by their poor drug-like properties. In this study, we investigated the design criteria for DOTAP/cholesterol liposomes for the systemic delivery of ADU-S100 and delineated the impact of key formulation factors on the loading efficiency, serum stability, and STING agonistic activity of ADU-S100. Our findings demonstrate that the cationic liposomal formulation of ADU-S100 can be optimized to greatly potentiate STING activation in antigen-presenting cells.

Structure-based discovery of small molecules that disaggregate amyloid fibrils from post-mortem Alzheimer's and Parkinson's brains.

Alzheimer's disease (AD) and Parkinson's disease (PD) are the consequences of neuronal death associated with the aggregation, respectively of proteins tau and alpha-synuclein into amyloid fibrils. Thus disaggregation of these fibrils could be therapeutic approaches to these conditions. The small molecule EGCG, abundant in green tea, has long been known to disaggregate amyloid fibrils, but EGCG has poor drug-like properties, failing to fully penetrate the brain. To understand how EGCG can disaggregate stable fibrils, we have cryogenically trapped an intermediate of brain-extracted tau fibrils on the kinetic pathway to EGCG-induced disaggregation and have determined its cryoEM structure. The structure reveals that EGCG molecules stack in polar clefts between the paired helical protofilaments that pathologically define AD. Treating the EGCG binding position as a pharmacophore, we computationally screened thousands of drug-like compounds for compatibility for the pharmacophore, discovering several that experimentally disaggregate brain-derived tau fibrils in vitro and some that limit alpha-synuclein aggregation in vitro and in C. elegans. This work suggests the potential of structure-based, small-molecule drug discovery for amyloid diseases.

Divergent reactivity of usnic acid and evaluation of its derivatives for antiproliferative activity against cancer cells

Natural products continue to be an inspiration for new drugs to treat debilitating diseases such as cancer. Usnic acid is a secondary metabolite isolated predominately from lichen species and has been shown to exhibit antiproliferative properties, however its application is limited by poor drug-like properties and low specificity. We report our work on investigating the reactivity of usnic acid for incorporating heterocyclic rings and the divergent reactivity that can be obtained by simply altering the reaction solvent and temperature. The synthesised derivatives were then tested against HeLa cancer cells for their antiproliferative properties. A number of promising compounds were obtained including 4, 5 and 9 that showed an IC50 of 878, 311 and 116 nM, respectively, against HeLa cancer cells after 48 h of treatment.

225 (PB105) - Discovery of novel small molecules that recruit DCAF11 for selective degradation of BRD4

Background: Targeted protein degradation (TPD) using the endogenous ubiquitin (Ub) proteasome system (UPS) is a rapidly growing drug discovery approach to eliminate pathogenic proteins. Strategies for TPD have focused on designing heterobifunctional PROTACs that utilize ligand binding to select E3 ligases (e.g., cereblon and VHL), often resulting in compounds with poor drug-like properties. Monovalent degraders represent an alternative approach, in which small molecules are designed to bind the target protein and induce its degradation through the recruitment of an E3 ligase complex. Monovalent degraders generally have better physico-chemical properties than PROTACs; however, designing compounds to induce degradation is not well precedented and relies on serendipitous discovery. Methods: An E3 ligase agnostic chemical library was screened using our ultra-high throughput cell-based screening (uHTS) platform. Hits were identified by measuring protein degradation of BRD4 using immunofluorescence. A Ub pathway focused CRISPR screen was used to identify the E3 ligase responsible for compound-induced degradation of BRD4. The degradation mechanism was validated using western blot, luciferase-based protein-protein interaction (PPI) and mutational analyses. In vitro antitumor activity was assessed by proliferation and apoptosis assays. NOD/ SCID mice were implanted sub-Q with acute myeloid leukemia (AML) xenograft model MV-4-11 and treated with compounds administered QD. Results: uHTS resulted in identification of selective monovalent degraders that target the bromodomain extra-terminal (BET) protein, BRD4. Degrader series optimization produced PLX-3618, which demonstrated potent degradation of BRD4, without depleting BET family members BRD2 and BRD3. Proteasome and neddylation inhibitors confirmed degradation was mediated via the UPS, and a Ub-focused CRISPR screen identified CUL4DCAF11 as the E3 complex responsible for PLX-3618 induced degradation of BRD4. PPI studies verified a BRD4/PLX-3618/DCAF11 ternary complex; BRD4 point mutational analyses provided further insights into the DCAF11-mediated degradation mechanism. Degradation of BRD4 by PLX-3618 resulted in downregulation of the MYC oncogene and potent anti-proliferative activity against a panel of tumor cell lines, with high sensitivity observed in AML. in vivo, PLX-3618 resulted in complete tumor regression, whereas a pan-BET inhibitor only resulted in tumor growth inhibition without regression. PK/PD analyses provided insight into the unique exposure/response profile of targeted protein degraders. Conclusion: These results demonstrate the efficient discovery of novel monovalent degraders using our proprietary platform. Characterization of the degradation mechanism highlights the discovery of DCAF11 as a novel E3 ligase substrate receptor amenable to recruitment and degradation of BRD4. No conflict of interest.

Structure-based discovery of small molecules that disaggregate Alzheimer’s disease tissue derived tau fibrils in vitro

Alzheimer’s disease (AD) is the consequence of neuronal death and brain atrophy associated with the aggregation of protein tau into fibrils. Thus disaggregation of tau fibrils could be a therapeutic approach to AD. The small molecule EGCG, abundant in green tea, has long been known to disaggregate tau and other amyloid fibrils, but EGCG has poor drug-like properties, failing to fully penetrate the brain. Here we have cryogenically trapped an intermediate of brain-extracted tau fibrils on the kinetic pathway to EGCG-induced disaggregation and have determined its cryoEM structure. The structure reveals that EGCG molecules stack in polar clefts between the paired helical protofilaments that pathologically define AD. Treating the EGCG binding position as a pharmacophore, we computationally screened thousands of drug-like compounds for compatibility for the pharmacophore, discovering several that experimentally disaggregate brain-derived tau fibrils in vitro. This work suggests the potential of structure-based, small-molecule drug discovery for amyloid diseases.

Anticholinesterase Inhibition, Drug-Likeness Assessment, and Molecular Docking Evaluation of Milk Protein-Derived Opioid Peptides for the Control of Alzheimer’s Disease

The drug-likeness and pharmacokinetic properties of 23 dairy-protein-derived opioid peptides were studied using SwissADME and ADMETlab in silico tools. All the opioid peptides had poor drug-like properties based on violations of Lipinski’s rule-of-five. Moreover, prediction of their pharmacokinetic properties showed that the peptides had poor intestinal absorption and bioavailability. Following this, two well-known opioid peptides (βb-casomorphin-5, βb-casomorphin-7) from A1 bovine milk and caffeine (positive control) were selected for in silico molecular docking and in vitro inhibition study with two cholinesterase enzyme receptors important for the pathogenesis of Alzheimer’s disease. Both peptides showed higher binding free energies and inhibitory activities to butyrylcholinesterase (BChE) than caffeine, but in vitro binding energy values were lower than those from the docking model. Moreover, the two casomorphins had lower inhibitory properties against acetylcholinesterase (AChE) than caffeine, although the docking model predicted the opposite. At 1 mg/mL concentrations, βb-casomorphin-5 and βb-casomorphin-7 showed promising results in inhibiting both cholinesterases (i.e., respectively 34% and 43% inhibition of AChE, and 67% and 81% inhibition of BChE). These dairy-derived opioid peptides have the potential to treat Alzheimer’s disease via cholinesterase inhibition. However, appropriate derivatization may be required to improve their poor predicted intestinal absorption and bioavailability.

In silico evaluation of the inhibitory property of Holothuria scabra (sea cucumber) with the catalytic domain of matrix metalloproteinase-1 for collagen degradation via interaction of triterpenoid saponins

Matrix metalloproteinase-1 (MMP-1) is a collagenase that cleaves the collagen present in the extracellular matrix (ECM), which results in skin wrinkling. In this in silico study, the triterpenoid saponins present from Holothuria scabra (desholothurin A, holothurinoside C, pervicoside C), were studied to determine its inhibitory properties against MMP-1. The approach was done via determining its dermatologic activity, its binding affinity against MMP-1 via molecular docking, followed by its pharmacokinetic properties. Dermatologic activity assessment of each triterpenoid saponins gave a Pa value of 0.408 for desholothurin A, 0.481 for holothurinoside C, and 0.297 for pervicoside C. In molecular docking, all triterpenoid saponins exhibited strong (such as conventional hydrogen bonding) and weak molecular interactions (such as van der Waals forces and carbon-carbon interactions). These molecular interactions was described by binding affinity giving desholothurin A a binding energy of -6.03 kcal/mol (37.79 uM), following holothurinoside C with -5.46 kcal/mol (99.14 uM), and pervicoside C with -3.05 kcal/mol (5480 uM). Lastly, in silico pharmacokinetic studies were done, which demonstrated poor drug-like properties of the compounds.

Abstract P5-05-04: Inhibition of HER2+ tumor growth with SDX-7320, a novel MetAP2 inhibitor, alone and in combination with capivasertib/AZD-5363: Reduced expression of hypoxia-inducible and innate-immune system genes

Abstract MetAP2 inhibitors have shown clinical anti-tumor activity, but CNS side effects and poor drug-like properties have generally limited their development. SDX-7320 is a polymer-drug conjugate of a novel fumagillin-derived MetAP2 inhibitor (SDX-7539) attached via a cleavable linker to a hydroxypropylmethacrylamide (HPMA) polymer backbone. This conjugation approach is intended to improve safety (i.e., limit CNS penetration) while also improving biodistribution and pharmacokinetics relative to small molecule MetAP2 inhibitors. SDX-7320 recently completed a phase I safety trial in late-stage cancer patients (NCT02743637) and was well-tolerated with no apparent CNS side effects. In prior models of breast cancer, SDX-7320 significantly inhibited the growth of syngeneic EO771 triple-negative breast cancers (TNBC) accelerated by obesity/metabolic dysfunction (i.e., “metabo-oncology”) and also synergized with the PI3Kα inhibitor alpelisib/Piqray® in ER+/Her2- MCF-7 xenografts. The objectives of these experiments were to determine the effect of SDX-7320 on the growth of Her2+ xenografts alone or in combination with the Akt/mTOR inhibitor capivasertib/AZD-5363 and to determine if SDX-7320 could attenuate hyperglycemia induced by capivasertib/AZD-5363. Female athymic, nude mice (9 weeks of age) were surgically implanted with slow-release estrogen pellets (90-day, 0.72 mg, inter-scapular) and three days later 2 x 107 BT474 cells (in Matrigel/RPMI-1640, 50/50, v/v) were injected into the fourth mammary gland. When the group mean tumor volume exceeded 100 mm3, treatment with test agents commenced. SDX-7320 (sc/q4d, 6, 12 mg/kg) exerted significant, dose-dependent inhibition of BT-474 tumor growth (TGI of 61 and 88% respectively), whereas capivasertib/AZD-5363 (po, qd, 100, 200 mg/kg) exhibited significant anti-tumor activity only at 200 mg/kg (54% TGI). The combination of SDX-7320 (12 mg/kg) plus capivasertib/AZD-5363 (200 mg/kg) produced additive effects in that a greater number of mice had tumor regression relative to mice treated with either SDX-7320 or capivasertib/AZD-5363 alone. Tumor tissue from a subset of treatment groups (Vehicle, SDX-7320-12 mg/kg, SDX-7320-12 mg/kg + AZD-5363-200 mg/kg, and AZD-5363-200 mg/kg) was snap frozen and stored at -80oC. PolyA+ RNA was isolated, cDNA libraries were constructed and RNASeq was conducted (range of 20-30 reads per million (RPM) base pairs). Analysis of RNASeq data showed that SDX-7320 alone significantly attenuated the expression of hypoxia-induced genes as well as genes related to the innate immune system. Based on these results, we conclude that SDX-7320 affected tumor growth by limiting the ability of tumors to adapt to a hypoxic microenvironment and by altering the innate tumor-immune microenvironment. In normal male, C57Bl/6 mice, capivasertib/AZD-5363 (200 mg/kg, po) elevated blood glucose after a single dose. Pretreatment with either one dose (24 hours before the Akt/mTOR inhibitor) or multiple doses (Q4D beginning 14 days prior to Akt/mTOR inhibitor) of SDX-7320 (8 mg/kg) significantly inhibited hyperglycemia induced by capivasertib/AZD-5363. In summary SDX-7320 significantly inhibited the growth of BT-474 xenografts, alone and in combination with capivasertib/AZD-5363, which was associated with suppression of hypoxia-induced genes, as well as genes of the innate immune system. In addition, SDX-7320 improved the safety profile of capivasertib/AZD-5363 by attenuating Akt/mTOR inhibitor-induced hyperglycemia, providing further support for the clinical exploration of SDX-7320 in combination with Akt/mTOR inhibitors in Her2+ breast cancer. Citation Format: Peter Cornelius, Benjamin Mayes, Pierre Dufour, Mark Kalinich, Jonathan Wang, Sara Little, Bradley Carver, James Shanahan. Inhibition of HER2+ tumor growth with SDX-7320, a novel MetAP2 inhibitor, alone and in combination with capivasertib/AZD-5363: Reduced expression of hypoxia-inducible and innate-immune system genes [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-05-04.

Selective Recognition of a Single HIV-1 G-Quadruplex by Ultrafast Small-Molecule Screening.

G-quadruplexes (G4s) are implicated in pathological processes such as cancer and infective diseases. Their targeting with G4-ligands has shown therapeutic capacity. Most of the current G4-ligands are planar molecules, do not discriminate among G4s, and have poor druglike properties. The available methods to identify compounds selective for one single G4 are often time-consuming. Here, we describe the development, validation, and application of an affinity-selection mass spectrometry method that employs unlabeled G4 oligonucleotides as targets and allows testing of up to 320 unmodified small molecules in a single tube. As a proof of concept, this method was applied to screen a library of 40 000 druglike molecules against two G4s, transcriptional regulators of the HIV-1 LTR promoter. We identified nonplanar pyrazolopyrimidines that selectively recognize and stabilize the major HIV-1 LTR G4 possibly by fitting and binding through H-bonding in its unique binding pocket. The compounds inhibit LTR promoter activity and HIV-1 replication. We propose this method to prompt the fast development of new G4-based therapeutics.

Abstract 1068: SDX-7320, a novel inhibitor of methionine aminopeptidase 2 (MetAP2), inhibits MCF-7 tumor growth in combination with palbociclib (Ibrance®)

Abstract Small molecule MetAP2 inhibitor TNP-470 previously showed clinical anti-tumor activity, however CNS side effects and poor drug-like properties limited its development. SDX-7320 is a polymer-drug conjugate of a novel MetAP2 inhibitor (SDX-7539), intended to improve biodistribution (limit CNS penetration) and pharmacokinetics relative to small molecule MetAP2 inhibitors. SDX-7320 has recently completed a phase I trial in late-stage cancer patients (NCT02743637). SDX-7320 was previously shown to inhibit the growth of syngeneic EO771 triple-negative breast cancers (TNBC) accelerated by obesity/metabolic dysfunction (“metabo-oncology”). In addition SDX-7320 synergized with the PI3Kα inhibitor alpelisib (Piqray®) to block the growth of ER+/Her2- MCF-7 xenografts. Here, we show that SDX-7320 inhibits the growth of MCF-7 xenografts alone and in combination with the CDK4/6 inhibitor Palbociclib (Ibrance®). Nude mice were implanted with slow release estrogen pellets and then injected with MCF-7 cells in the fourth mammary gland. Treatment began (n=10/group) when tumors exceeded 100 mm³: SDX-7320 (sc/q4d, 8 mg/kg), palbociclib (po, qd, 20 or 40 mg/kg), SDX-7320 plus palbociclib (20 mg/kg), SDX-7320 plus palbociclib (40 mg/kg). MCF-7 tumor growth inhibition at day 31 relative to vehicle-treated mice: SDX-7320, 29% (NS); palbociclib, 24% and 54% at 20 and 40 mg/kg respectively (NS); SDX-7320 plus palbociclib (20 mg/kg), 64% (p<0.05); and SDX-7320 plus palbociclib (40 mg/kg), 82%, (p<0.01). Tumor samples were homogenized in RIPA buffer containing protease and phosphatase inhibitors for analysis of proteins (by WES). Cell-cycle proteins (cyclin E1, E2, cdk2, cdk4) were decreased in tumor tissue from SDX-7320-treated mice relative to vehicle, and were typically lower in tumors from mice treated with SDX-7320 plus palbociclib (40 mg/kg) compared to either vehicle-treated or SDX-7320-treated mice. A key growth factor-signaling protein Akt was reduced in SDX-7320-treated groups (alone and in combination) as was estrogen receptor alpha (ERα). pAkt (S473) was lowest in SDX-7320-treated tumors and in tumors treated with SDX-7320 plus palbociclib (40 mg/kg). The autophagy marker LC3B was elevated in response to palbociclib (20 and 40 mg/kg) but in combination with SDX-7320, the increase was significantly attenuated. Intracellular pathways and proteins linked to emergence of resistance to palbociclib include cell cycle (cyclin E1), growth factor and hormone signaling (Akt, ERα) and autophagy (LC3B). The protein changes observed (representative of these pathways) suggest that combining SDX-7320 with palbociclib (Ibrance®) may extend the period of progression-free survival in patients with ER+/Her2- breast cancer relative to treatment with palbociclib (Ibrance®) alone. Citation Format: Peter Cornelius, Benjamin Mayes, Pierre Dufour, Sara Little, Andrew Slee, Raphael Nir, Adam Nir, Bradley J. Carver, James Shanahan. SDX-7320, a novel inhibitor of methionine aminopeptidase 2 (MetAP2), inhibits MCF-7 tumor growth in combination with palbociclib (Ibrance®) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1068.