The elucidation of species-specific receptor pharmacology: a case study using subtype selective para- and meta-carborane estrogen receptor agonists.

Estrogen receptors are essential pharmacological targets for treating hormonal disorders and estrogen-dependent malignancies. Selective activation of estrogen receptor (ER) ß is hypothesized to provide therapeutic benefit with reduced risk of unwanted estrogenic side-effects associated with ERα activity. However, activating ERß without activating α is challenging due to the high sequence and structural homology between the receptor subtypes. We assessed the impact of structural modifications to the parent compound OSU-ERß-12 on receptor subtype binding selectivity using cell-free binding assays. Functional selectivity was evaluated by transactivation in HEK-293 cells overexpressing human or murine estrogen receptors. In vivo selectivity was examined through the uterotrophic effects of the analogs after oral administration in estrogen-naïve female mice. Furthermore, we evaluated the in vivo pharmacokinetics of the analogs following single dose IV and oral administration. Regarding selectivity, a single compound exhibited greater functional selectivity than OSU-ERß-12 for human ERß. However, like others in the meta-carborane series, its poor in vivo pharmacokinetics limit its suitability for further development. Surprisingly, and at odds with their pharmacokinetic and in vitro human activity data, most analogs potently induced uterotrophic effects in estrogen-naïve female mice. Further investigation of activity in HEK293 cells expressing murine estrogen receptors revealed species-specific differences in the ER-subtype selectivity of these analogs. Our findings highlight species-specific receptor pharmacology and the challenges it poses to characterizing developmental therapeutics in preclinical species. Significance Statement This study investigates para- and meta-substituted carborane analogs targeting estrogen receptors, revealing the greater selectivity of carborane analogs for human ERß compared to the mouse homolog. These findings shed light on the intricacies of using preclinical species in drug development to predict human pharmacology. The report also provides insights for the refinement and optimization of carborane analogs as potential therapeutic agents for estrogen-related disease states.

Discovery of Nanomolar Inhibitors for Human Dihydroorotate Dehydrogenase Using Structure-Based Drug Discovery Methods.

We used a structure-based drug discovery approach to identify novel inhibitors of human dihydroorotate dehydrogenase (DHODH), which is a therapeutic target for treating cancer and autoimmune and inflammatory diseases. In the case of acute myeloid leukemia, no previously discovered DHODH inhibitors have yet succeeded in this clinical application. Thus, there remains a strong need for new inhibitors that could be used as alternatives to the current standard-of-care. Our goal was to identify novel inhibitors of DHODH. We implemented prefiltering steps to omit PAINS and Lipinski violators at the earliest stages of this project. This enriched compounds in the data set that had a higher potential of favorable oral druggability. Guided by Glide SP docking scores, we found 20 structurally unique compounds from the ChemBridge EXPRESS-pick library that inhibited DHODH with IC50, DHODH values between 91 nM and 2.7 µM. Ten of these compounds reduced MOLM-13 cell viability with IC50, MOLM-13 values between 2.3 and 50.6 µM. Compound 16 (IC50, DHODH = 91 nM) inhibited DHODH more potently than the known DHODH inhibitor, teriflunomide (IC50, DHODH = 130 nM), during biochemical characterizations and presented a promising scaffold for future hit-to-lead optimization efforts. Compound 17 (IC50, MOLM-13 = 2.3 µM) was most successful at reducing survival in MOLM-13 cell lines compared with our other hits. The discovered compounds represent excellent starting points for the development and optimization of novel DHODH inhibitors.

Papaverine and its derivatives radiosensitize solid tumors by inhibiting mitochondrial metabolism.

Tumor hypoxia reduces the effectiveness of radiation therapy by limiting the biologically effective dose. An acute increase in tumor oxygenation before radiation treatment should therefore significantly improve the tumor cell kill after radiation. Efforts to increase oxygen delivery to the tumor have not shown positive clinical results. Here we show that targeting mitochondrial respiration results in a significant reduction of the tumor cells' demand for oxygen, leading to increased tumor oxygenation and radiation response. We identified an activity of the FDA-approved drug papaverine as an inhibitor of mitochondrial complex I. We also provide genetic evidence that papaverine's complex I inhibition is directly responsible for increased oxygenation and enhanced radiation response. Furthermore, we describe derivatives of papaverine that have the potential to become clinical radiosensitizers with potentially fewer side effects. Importantly, this radiosensitizing strategy will not sensitize well-oxygenated normal tissue, thereby increasing the therapeutic index of radiotherapy.

Coordination Properties of GdDO3A-Based Model Compounds of Bioresponsive MRI Contrast Agents.

We report a detailed characterization of the thermodynamic stability and dissociation kinetics of Gd3+ complexes with DO3A derivatives containing a (methylethylcarbamoylmethylamino)acetic acid (L1), (methylpropylcarbamoylmethylamino)acetic acid (L2), 2-dimethylamino- N-ethylacetamide (L3), or 2-dimethylamino- N-propylacetamide (L4) group attached to the fourth nitrogen atom of the macrocyclic unit. These ligands are model systems of Ca2+- and Zn2+-responsive contrast agents (CA) for application in magnetic resonance imaging (MRI). The results of the potentiometric studies ( I = 0.15 M NaCl) provide stability constants with log KGdL values in the range 13.9-14.8. The complex speciation in solution was found to be quite complicated due to the formation of protonated species at low pH, hydroxido complexes at high pH, and stable dinuclear complexes in the case of L1,2. At neutral pH significant fractions of the complexes are protonated at the amine group of the amide side chain (log KGdL×H = 7.2-8.1). These ligands form rather weak complexes with Mg2+ and Ca2+ but very stable complexes with Cu2+ (log KCuL = 20.4-22.3) and Zn2+ (log KZnL = 15.5-17.6). Structural studies using a combination of 1H NMR and luminescence spectroscopy show that the amide group of the ligand is coordinated to the metal ion at pH ∼8.5, while protonation of the amine group provokes the decoordination of the amide O atom and a concomitant increase in the hydration number and proton relaxivity. The dissociation of the complexes occurs mainly through a rather efficient proton-assisted pathway, which results in kinetic inertness comparable to that of nonmacrocyclic ligands such as DTPA rather than DOTA-like complexes.

Innovative Design of Ca-Sensitive Paramagnetic Liposomes Results in an Unprecedented Increase in Longitudinal Relaxivity.

Bioresponsive MRI contrast agents sensitive to Ca(II) fluctuations may play a critical role in the development of functional molecular imaging methods to study brain physiology or abnormalities in muscle contraction. A great challenge in their chemistry is the preparation of probes capable of inducing a strong signal variation that could be detected in a robust way. To this end, the incorporation of small molecular weight bioresponsive agents into nanocarriers can improve the overall properties in a few ways: (i) the agent can be delivered into the tissue of interest, increasing the local concentration; (ii) its biokinetic properties and retention time will improve; (iii) the high molecular weight and size of the nanocarrier may cause additional changes in the MRI signal and raise the chances for their detection in functional experiments. In this work, we report the preparation of the new class of liposome-based, Ca-sensitive MRI agents. We synthesized a novel amphiphilic ligand which was incorporated into the liposome bilayer. A remarkable increase of ∼420% in longitudinal relaxivity r1, from 7.3 mM(-1) s(-1) to 38.1 mM(-1) s(-1) at 25 °C and 21.5 MHz in the absence and presence of Ca(II), respectively, was achieved by the most active liposomal formulation. To the best of our knowledge, this is the highest change in r1 observed for Ca-sensitive agents at physiological pH and can be explained by simultaneous Ca-triggered increase in hydration and reduction of local motion of Gd(III) complex, which can be followed at low magnetic fields.

Ultrasmall Nanoplatforms as Calcium-Responsive Contrast Agents for Magnetic Resonance Imaging.

The preparation of ultrasmall and rigid platforms (USRPs) that are covalently coupled to macrocycle-based, calcium-responsive/smart contrast agents (SCAs), and the initial in vitro and in vivo validation of the resulting nanosized probes (SCA-USRPs) by means of magnetic resonance imaging (MRI) is reported. The synthetic procedure is robust, allowing preparation of the SCA-USRPs on a multigram scale. The resulting platforms display the desired MRI activity­i.e., longitudinal relaxivity increases almost twice at 7 T magnetic field strength upon saturation with Ca(2+). Cell viability is probed with the MTT assay using HEK-293 cells, which show good tolerance for lower contrast agent concentrations over longer periods of time. On intravenous administration of SCA-USRPs in living mice, MRI studies indicate their rapid accumulation in the renal pelvis and parenchyma. Importantly, the MRI signal increases in both kidney compartments when CaCl2 is also administrated. Laser-induced breakdown spectroscopy experiments confirm accumulation of SCA-USRPs in the renal cortex. To the best of our knowledge, these are the first studies which demonstrate calcium-sensitive MRI signal changes in vivo. Continuing contrast agent and MRI protocol optimizations should lead to wider application of these responsive probes and development of superior functional methods for monitoring calcium-dependent physiological and pathological processes in a dynamic manner.

Pyrimidine depletion enhances targeted and immune therapy combinations in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a fatal disease characterized by the accumulation of undifferentiated myeloblasts, and agents that promote differentiation have been effective in this disease but are not curative. Dihydroorotate dehydrogenase inhibitors (DHODHi) have the ability to promote AML differentiation and target aberrant malignant myelopoiesis. We introduce HOSU-53, a DHODHi with significant monotherapy activity, which is further enhanced when combined with other standard-of-care therapeutics. We further discovered that DHODHi modulated surface expression of CD38 and CD47, prompting the evaluation of HOSU-53 combined with anti-CD38 and anti-CD47 therapies, where we identified a compelling curative potential in an aggressive AML model with CD47 targeting. Finally, we explored using plasma dihydroorotate (DHO) levels to monitor HOSU-53 safety and found that the level of DHO accumulation could predict HOSU-53 intolerability, suggesting the clinical use of plasma DHO to determine safe DHODHi doses. Collectively, our data support the clinical translation of HOSU-53 in AML, particularly to augment immune therapies. Potent DHODHi to date have been limited by their therapeutic index; however, we introduce pharmacodynamic monitoring to predict tolerability while preserving antitumor activity. We additionally suggest that DHODHi is effective at lower doses with select immune therapies, widening the therapeutic index.

Synthesis and characterization of pH-sensitive, biotinylated MRI contrast agents and their conjugates with avidin.

Responsive or smart contrast agents (SCAs) provide new opportunities in magnetic resonance imaging (MRI) to examine a number of physiological and pathological events. However, their application in vivo remains challenging. Therefore, much research is focused on the optimization of their properties, to enable their use in additional imaging modalities, pre-targeted delivery, or to increase the local concentration of the agent. The key feature in the SCA synthetic modification is the retention of their physicochemical properties related to the specific MR response. Here, we report the preparation and characterization of pH sensitive SCAs appended with a phosphonate pendant arm and either an aliphatic (GdL(1)) or aromatic linker (GdL(2)). The longitudinal relaxivity of GdL(1) and GdL(2) increases by 146% and 31%, respectively, while the pH decreases from 9 to 5. These two SCAs were converted to the biotinylated systems GdL(3) and GdL(4) and their interaction with avidin was investigated. The binding affinity with avidin was assessed with a fluorescence displacement assay and with MRI phantom experiments in a 3T MRI scanner. The fluorometric assay and MRI E-titrations revealed a 3 : 1 binding mode of GdL(3-4) to avidin with the binding affinity as high as that of the parent avidin-biotin complex. The high binding affinity was confirmed with MRI by a competitive assay. The avidin-GdL(3-4) complexes thus obtained exhibit changes in both r(1) and r(2) that are pH dependent. The results reveal a new pathway for the modification and improvement of SCAs to make them more suitable for in vivo application.

Antibacterial activity of novel bacterial topoisomerase inhibitors against key veterinary pathogens.

Multidrug-resistant bacteria infect companion animals and livestock in addition to their devastating impact on human health. Novel Bacterial Topoisomerase Inhibitors (NBTIs) with excellent activity against Gram-positive bacteria have previously been identified as promising new antibacterial agents. Herein, we evaluate the antibacterial activity of these NBTIs against a variety of important veterinary pathogens and demonstrate outstanding in vitro activity, especially against staphylococci.

1,3-Dioxane-Linked Novel Bacterial Topoisomerase Inhibitors: Expanding Structural Diversity and the Antibacterial Spectrum.

Antibacterial resistance continues its devastation of available therapies. Novel bacterial topoisomerase inhibitors (NBTIs) offer one solution to this critical issue. Two series of amine NBTIs bearing tricyclic DNA-binding moieties as well as amide NBTIs with a bicyclic DNA-binding moiety were synthesized and evaluated against methicillin-resistant Staphylococcus aureus (MRSA). Additionally, these compounds and a series of bicyclic amine analogues displayed high activity against susceptible and drug-resistant Neisseria gonorrhoeae, expanding the spectrum of these dioxane-linked NBTIs.

Structure-Activity Relationship of para-Carborane Selective Estrogen Receptor ß Agonists.

Selective agonism of the estrogen receptor (ER) subtypes, ERα and ERß, has historically been difficult to achieve due to the high degree of ligand-binding domain structural similarity. Multiple efforts have focused on the use of classical organic scaffolds to model 17ß-estradiol geometry in the design of ERß selective agonists, with several proceeding to various stages of clinical development. Carborane scaffolds offer many unique advantages including the potential for novel ligand/receptor interactions but remain relatively unexplored. We synthesized a series of para-carborane estrogen receptor agonists revealing an ERß selective structure-activity relationship. We report ERß agonists with low nanomolar potency, greater than 200-fold selectivity for ERß over ERα, limited off-target activity against other nuclear receptors, and only sparse CYP450 inhibition at very high micromolar concentrations. The pharmacological properties of our para-carborane ERß selective agonists measure favorably against clinically developed ERß agonists and support further evaluation of carborane-based selective estrogen receptor modulators.

Optimization of TopoIV Potency, ADMET Properties, and hERG Inhibition of 5-Amino-1,3-dioxane-Linked Novel Bacterial Topoisomerase Inhibitors: Identification of a Lead with In Vivo Efficacy against MRSA.

Novel bacterial topoisomerase inhibitors (NBTIs) are among the most promising new antibiotics in preclinical/clinical development. We previously reported dioxane-linked NBTIs with potent antistaphylococcal activity and reduced hERG inhibition, a key safety liability. Herein, polarity-focused optimization enabled the delineation of clear structure-property relationships for both microsomal metabolic stability and hERG inhibition, resulting in the identification of lead compound 79. This molecule demonstrates potent antibacterial activity against diverse Gram-positive pathogens, inhibition of both DNA gyrase and topoisomerase IV, a low frequency of resistance, a favorable in vitro cardiovascular safety profile, and in vivo efficacy in a murine model of methicillin-resistant Staphylococcus aureus infection.

Dendrimeric calcium-sensitive MRI probes: the first low-field relaxometric study.

Different classes of small- or nano-sized calcium-sensitive probes for magnetic resonance imaging (MRI) have been proposed in the last two decades. These compounds have been developed mainly for functional MRI purposes and tested in vivo in different animal models. Most of them are paramagnetic systems that change their relaxivity in the presence of the divalent ion calcium, resulting in increased T1 or T2 contrast. In this work, we report the investigation of their relaxometric behavior at low magnetic fields, specifically the comparison of the monomeric Ca-sensitive probe and the corresponding dendrimer conjugates of generations 0, 1 and 2 (G0, G1 and G2, respectively). As a result, a relaxivity hump between 10 and 100 MHz of the Larmor frequency progressively appeared with an increase in the size of the investigated contrast agent, indicative of a restricted rotational motion of the complexes as long as the size of the molecule increases. The same trend with a more pronounced effect was detectable in the presence of calcium. The relaxivity enhancement for the Ca2+ adducts, primarily caused by an increase of the hydration state of Gd3+, went from ca. 130% for the monomeric probe to ca. 310% for the G2 dendrimer conjugate at 0.5 T and 25 °C. T1 weighted magnetic resonance images acquired at 1 T displayed the strong ability of these systems to change their contrast according to the presence of calcium at this field, thus laying the basis for promising future in vivo applications.

Solid-Phase-Supported Approach for the Preparation of Bioresponsive and Multifunctional MRI Probes.

The development of bifunctional imaging probes can often be challenging with difficult and time-consuming solution phase chemistry protocols and purification techniques. A solid phase synthetic protocol was therefore utilized to produce a functionalized derivative of a potent bismacrocyclic calcium-responsive contrast agent for magnetic resonance imaging. Through a convenient building block approach, the applicability of this methodology in the preparation and simple future development of multifunctional imaging probes was demonstrated.

1,3-Dioxane-Linked Bacterial Topoisomerase Inhibitors with Enhanced Antibacterial Activity and Reduced hERG Inhibition.

The development of new therapies to treat methicillin-resistant Staphylococcus aureus (MRSA) is needed to counteract the significant threat that MRSA presents to human health. Novel inhibitors of DNA gyrase and topoisomerase IV (TopoIV) constitute one highly promising approach, but continued optimization is required to realize the full potential of this class of antibiotics. Herein, we report further studies on a series of dioxane-linked derivatives, demonstrating improved antistaphylococcal activity and reduced hERG inhibition. A subseries of analogues also possesses enhanced inhibition of the secondary target, TopoIV.

Dendrimeric calcium-responsive MRI contrast agents with slow in vivo diffusion.

We report a methodology which enables the preparation of dendrimeric contrast agents sensitive to Ca(2+) when starting from the monomeric analogue. The Ca-triggered longitudinal relaxivity response of these agents is not compromised by undertaking synthetic transformations, despite structural changes. The in vivo MRI studies in the rat cerebral cortex indicate that diffusion properties of dendrimeric contrast agents have great advantages as compared to their monomeric equivalents.