Structure-Function Insights into the Dual Role in Nucleobase and Nicotinamide Metabolism and a Possible Use in Cancer Gene Therapy of the URH1p Riboside Hydrolase.

The URH1p enzyme from the yeast Saccharomyces cerevisiae has gained significant interest due to its role in nitrogenous base metabolism, particularly involving uracil and nicotinamide salvage. Indeed, URH1p was initially classified as a nucleoside hydrolase (NH) with a pronounced preference for uridine substrate but was later shown to also participate in a Preiss-Handler-dependent pathway for recycling of both endogenous and exogenous nicotinamide riboside (NR) towards NAD+ synthesis. Here, we present the detailed enzymatic and structural characterisation of the yeast URH1p enzyme, a member of the group I NH family of enzymes. We show that the URH1p has similar catalytic efficiencies for hydrolysis of NR and uridine, advocating a dual role of the enzyme in both NAD+ synthesis and nucleobase salvage. We demonstrate that URH1p has a monomeric structure that is unprecedented for members of the NH homology group I, showing that oligomerisation is not strictly required for the N-ribosidic activity in this family of enzymes. The size, thermal stability and activity of URH1p towards the synthetic substrate 5-fluoruridine, a riboside precursor of the antitumoral drug 5-fluorouracil, make the enzyme an attractive tool to be employed in gene-directed enzyme-prodrug activation therapy against solid tumours.

Dual-targeted NAMPT inhibitors as a progressive strategy for cancer therapy.

In mammals, nicotinamide phosphoribosyltransferase (NAMPT) is a crucial enzyme in the nicotinamide adenine dinucleotide (NAD+) synthesis pathway catalyzing the condensation of nicotinamide (NAM) with 5-phosphoribosyl-1-pyrophosphate (PRPP) to produce nicotinamide mononucleotide (NMN). Given the pivotal role of NAD+ in a range of cellular functions, including DNA synthesis, redox reactions, cytokine generation, metabolism, and aging, NAMPT has become a promising target for many diseases, notably cancer. Therefore, various NAMPT inhibitors have been reported and classified as first and second-generation based on their chemical structures and design strategies, dual-targeted being one. However, most NAMPT inhibitors suffer from several limitations, such as dose-dependent toxicity and poor pharmacokinetic properties. Consequently, there is no clinically approved NAMPT inhibitor. Hence, research on discovering more effective and less toxic dual-targeted NAMPT inhibitors with desirable pharmacokinetic properties has drawn attention recently. This review summarizes the previously reported dual-targeted NAMPT inhibitors, focusing on their design strategies and advantages over the single-targeted therapies.

Thiophenyl Derivatives of Nicotinamide Are Metabolized by the NAD Salvage Pathway into Unnatural NAD Derivatives That Inhibit IMPDH and Are Toxic to Peripheral Nerve Cancers.

N-Pyridinylthiophene carboxamide (compound 21) displays activity against peripheral nerve sheath cancer cells and mouse xenografts by an unknown mechanism. Through medicinal chemistry, we identified a more active derivative, compound 9, and found that only analogues with structures similar to nicotinamide retained activity. Genetic screens using compound 9 found that both NAMPT and NMNAT1, enzymes in the NAD salvage pathway, are necessary for activity. Compound 9 is metabolized by NAMPT and NMNAT1 into an adenine dinucleotide (AD) derivative in a cell-free system, cultured cells, and mice, and inhibition of this metabolism blocked compound activity. AD analogues derived from compound 9 inhibit IMPDH in vitro and cause cell death by inhibiting IMPDH in cells. These findings nominate these compounds as preclinical candidates for the development of tumor-activated IMPDH inhibitors to treat neuronal cancers.

Toward Next-Generation Molecular Imaging with a Clinical Low-Field (0.064 T) Point-of-Care MRI Scanner.

Low-field (LF) MRI promises soft-tissue imaging without the expensive, immobile magnets of clinical scanners but generally suffers from limited detection sensitivity and contrast. The sensitivity boost provided by hyperpolarization can thus be highly synergistic with LF MRI. Initial efforts to integrate a continuous-bubbling SABRE (signal amplification by reversible exchange) hyperpolarization setup with a portable, point-of-care 64 mT clinical MRI scanner are reported. Results from 1H SABRE MRI of pyrazine and nicotinamide are compared with those of benchtop NMR spectroscopy. Comparison with MRI signals from samples with known H2O/D2O ratios allowed quantification of the SABRE enhancements of imaged samples with various substrate concentrations (down to 3 mM). Respective limits of detection and quantification of 3.3 and 10.1 mM were determined with pyrazine 1H polarization (PH) enhancements of ∼1900 (PH ∼0.04%), supporting ongoing and envisioned efforts to realize SABRE-enabled MRI-based molecular imaging.

An Investigation into the Transdermal Behavior of Active Ingredients by Combination of Experiments and Multiscale Simulations.

Transdermal behavior is a critical aspect of studying delivery systems and evaluating the efficacy of cosmetics. However, existing methods face challenges such as lengthy experiments, high cost, and limited model accuracy. Therefore, developing accurate transdermal models is essential for formulation development and effectiveness assessment. In this study, we developed a multiscale model to describe the transdermal behavior of active ingredients in the stratum corneum. Molecular dynamics simulations were used to construct lipid bilayers and determine the diffusion coefficients of active ingredients in different regions of these bilayers. These diffusion coefficients were integrated into a multilayer lipid pathway model using finite element simulations. The simulation results were in close agreement with our experimental results for three active ingredients (mandelic acid (MAN), nicotinamide (NIC), and pyruvic acid (PYR)), demonstrating the effectiveness of our multiscale model. This research provides valuable insights for advancing transdermal delivery methods.

Multidimensional Optimization of R-LE001 for New Leads with Enhanced Antifungal Profiles.

Scaffold hopping and structural fine-tuning are important strategies for agrochemical innovation. Multidimensional optimization of the prevalidated antifungal lead R-LE001 was conducted via the design, synthesis, and bioevaluation of 53 new compounds differing in either scaffold or substituent. The antifungal structure-activity relationship (SAR) revealed that a number of amides containing 2-(2-oxazolinyl) aniline (NHPhOx) or 2-(2-thiazolinyl) aniline (NHPhthiOx) demonstrated a more promising antifungal effect than both R-LE001 and the positive control boscalid. Specifically, compound 10 (encoded LEX-K01) shows an excellent antifungal effect against Botrytis cinerea with an EC50 value lower than 0.11 µM. This small change leads to a significant improvement (over 1 order of magnitude) in bioactivity compared to that of either R-LE001 (EC50 = 1.41 µM) or boscalid (EC50 = 2.01 µM) and fluxapyroxad (EC50 = 4.35 µM). With much lower resistance factors, LEX-K01 (10) was more efficacious against the two boscalid-resistant strains of B. cinerea TZ01 and NJBH2017. A combination of LEX-K01 (10) and boscalid in a ratio of 1:3 showed synergistic effects against resistant B. cinerea TZ01 and NJBH2017, with SR values of 3.01 and 2.55, respectively. LEX-K01 (10) has a curative efficacy (70.3%) more prominent than that of boscalid (51.2%) in controlling disease caused by B. cinerea. The molecular docking simulation of LEX-K01 (10) with the SDH protein of B. cinerea displayed four hydrogen bonds with amino acid residues TYR144, ARG88, TRP81, and SER84, rationalizing a stronger affinity than boscalid. The scanning electron microscopy (SEM) characteristic revealed that it could cause an obvious collapse of B. cinerea mycelium. This work indicates that LEX-K01 (10) has the potential to be further explored as a new antifungal agent.

Synthesis and biological evaluation of novel pyrazole scaffold.

synthesis of a pyrazole containing compound was achieved by reacting phenyl hydrazine with (E)-2-((4-bromophenyl) diazinyl)-1-phenylbutane-1,3-dione to produce 4-((4-bromophenyl) diazinyl)-5-methyl-1,3-diphenyl-pyrazole and characterization using mass spectrometer, 1H NMR and 13C NMR. The pharmacological evaluation of the synthesized compound, denoted as (KA5), against Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 29213 and Clostridiums sporogeneses ATCC 19404, indicate that there is no promising antibacterial activity. However, KA5 shows a competitive anticancer activity (IC50: 8.5µM) upon its evaluation against hepatocellular carcinoma cell line (HepG 2) compared to sorafenib (IC50: 4.51µM). Moreover, human skin fibroblast (HSF) was used to investigate the effect of KA5 on normal cell lines, (IC50: 5.53µM). The presented biological evaluations resulted in better understanding of structure-activity relationship for 1, 3, 4-trisubstituted pyrazoles and revealed a great opportunity for more investigations for novel pyrazole-containing anticancer agents.

Photocatalytic regeneration of nicotinamide cofactor biomimetics drives biocatalytic reduction by Old Yellow enzymes.

A key approach in developing green chemistry involves converting solar energy into chemical energy of biomolecules through photocatalysis. Photocatalysis can facilitate the regeneration of nicotinamide cofactors during redox processes. Nicotinamide cofactor biomimetics (NCBs) are economical substitutes for natural cofactors. Here, photocatalytic regeneration of NADH and reduced NCBs (NCBsred) using graphitic carbon nitride (g-C3N4) was developed. The process involves g-C3N4 as the photocatalyst, Cp*Rh(bpy)H2O2+ as the electron mediator, and Triethanolamine as the electron donor, facilitating the reduction of NAD+ and various oxidative NCBs (NCBsox) under light irradiation. Notably, the highest reduction yield of 48.32 % was achieved with BANA+, outperforming the natural cofactor NAD+. Electrochemical analysis reveals that the reduction efficiency and capacity of cofactors relies on their redox potentials. Additionally, a coupled photo-enzymatic catalysis system was explored for the reduction of 4-Ketoisophorone by Old Yellow Enzyme XenA. Among all the NCBsox and NAD+, the highest conversion ratio of over 99 % was obtained with BANA+. After recycled for 8 times, g-C3N4 maintained over 93.6 % catalytic efficiency. The photocatalytic cofactor regeneration showcases its outstanding performance with NAD+ as well as NCBsox. This work significantly advances the development of photocatalytic cofactor regeneration for artificial cofactors and its potential application.

Stability and release mechanism of double emulsification (W1/O/W2) for biodegradable pH-responsive polyhydroxybutyrate/cellulose acetate phthalate microbeads loaded with the water-soluble bioactive compound niacinamide.

Microbeads of biodegradable polyhydroxybutyrate (PHB) offer environmental benefits and economic competitiveness. The aim of this study was to encapsulate a water-soluble bioactive compound, niacinamide (NIA), in a pH-responsive natural matrix composed of PHB and cellulose acetate phthalate (CAP) by double emulsification (W1/O/W2) to improve the encapsulation efficiency (%EE) and loading capacity (%LC). PHB was produced in-house by Escherichia coli JM109 pUC19-23119phaCABA-04 without the inducing agent isopropyl ß-D-1-thiogalactopyranoside (IPTG). The influences of PHB and polyvinyl alcohol (PVA) concentrations, stirring rate, PHB/CAP ratio and initial NIA concentration on the properties of NIA-loaded pH-responsive microbeads were studied. The NIA-loaded pH-responsive PHB/CAP microbeads exhibited a spherical core-shell structure. The average size of the NIA-loaded pH-responsive microbeads was 1243.3 ± 11.5 µm. The EE and LC were 33.3 ± 0.5 % and 28.5 ± 0.4 %, respectively. The release profiles of NIA showed pH-responsive properties, as 94.2 ± 3.5 % of NIA was released at pH 5.5, whereas 99.3 ± 2.4 % of NIA was released at pH 7.0. The NIA-loaded pH-responsive PHB/CAP microbeads were stable for >90 days at 4 °C under darkness, with NIA remaining at 73.65 ± 1.86 %. A cytotoxicity assay in PSVK1 cells confirmed that the NIA-loaded pH-responsive PHB/CAP microbeads were nontoxic at concentrations lower than 31.3 µg/mL, in accordance with ISO 10993-5.

Amelioration of tableting properties and dissolution rate of naproxen co-grinded with nicotinamide: preparation and characterization of co-grinded mixture.

OBJECTIVE AND SIGNIFICANCE: Reducing the dimensions, when other additives are present, shows potential as a method to improve the dissolution and solubility of biopharmaceutical classification system class II drugs that have poor solubility. In this investigation, the process involved grinding naproxen with nicotinamide with the aim of improving solubility and the rate of dissolution. METHODS: Naproxen was subjected to co-milling with urea, dimethylurea, and nicotinamide using a planetary ball mill for a duration of 90 min, maintaining a 1:1 molar ratio for the excipients (screening studies). The co-milled combinations, naproxen in its pure milled form, and a physical mixture were subjected to analysis using X-ray powder diffraction (XRPD), scanning electron microscopy (SEM), and solubility assessment. The mixture displaying the highest solubility (naproxen-nicotinamide) was chosen for further investigation, involving testing for intrinsic dissolution rate (IDR) and Fourier-transform infrared spectroscopy (FTIR) after co-milling for both 90 and 480 min. RESULTS AND CONCLUSION: The co-milled combination, denoted as S-3b and consisting of the most substantial ratio of nicotinamide to naproxen at 1:3, subjected to 480 min of milling, exhibited a remarkable 45-fold increase in solubility and a 9-fold increase in IDR. XRPD analysis of the co-milled samples demonstrated no amorphization, while SEM images portrayed the aggregates of naproxen with nicotinamide. FTIR outcomes negate the presence of any chemical interactions between the components. The co-milled sample exhibiting the highest solubility and IDR was used to create a tablet, which was then subjected to comprehensive evaluation for standard attributes. The results revealed improved compressibility and dissolution properties.

Efficient Screening of Pharmaceutical Cocrystals by Microspacing In-Air Sublimation.

Cocrystal screening and single-crystal growth remain the primary obstacles in the development of pharmaceutical cocrystals. Here, we present a new approach for cocrystal screening, microspacing in-air sublimation (MAS), to obtain new cocrystals and grow high-quality single crystals of cocrystals within tens of minutes. The method possesses the advantages of strong designable ability of devices, user-friendly control, and compatibility with materials, especially for the thermolabile molecules. A novel drug-drug cocrystal of favipiravir (FPV) with salicylamide (SAA) was first discovered by this method, which shows improved physiochemical properties. Furthermore, this method proved effective in cultivating single crystals of FPV-isonicotinamide (FPV-INIA), FPV-urea, FPV-nicotinamide (FPV-NIA), and FPV-tromethamine (FPV-Tro) cocrystals, and the structures of these cocrystals were determined for the first time. By adjusting the growth temperature and growth distance precisely, we also achieved single crystals of 10 different paracetamol (PCA) cocrystals and piracetam (PIR) cocrystals, which underscores the versatility and efficiency of this method in pharmaceutical cocrystal screening.

Tofacitinib and peficitinib inhibitors of Janus kinase for autoimmune disease treatment: a quantum biochemistry approach.

Autoimmune inflammatory diseases, such as rheumatoid arthritis (RA) and ulcerative colitis, are associated with an uncontrolled production of cytokines leading to the pronounced inflammatory response of these disorders. Their therapy is currently focused on the inhibition of cytokine receptors, such as the Janus kinase (JAK) protein family. Tofacitinib and peficitinib are JAK inhibitors that have been recently approved to treat rheumatoid arthritis. In this study, an in-depth analysis was carried out through quantum biochemistry to understand the interactions involved in the complexes formed by JAK1 and tofacitinib or peficitinib. Computational analyses provided new insights into the binding mechanisms between tofacitinib or peficitinib and JAK1. The essential amino acid residues that support the complex are also identified and reported. Additionally, we report new interactions, such as van der Waals; hydrogen bonds; and alkyl, pi-alkyl, and pi-sulfur forces, that stabilize the complexes. The computational results revealed that peficitinib presents a similar affinity to JAK1 compared to tofacitinib based on their interaction energies.

A Magnesium Binding Site And The Anomeric Effect Regulate The Abiotic Redox Chemistry Of Nicotinamide Nucleotides.

Nicotinamide adenine dinucleotide (NAD+) is a redox active molecule that is universally found in biology. Despite the importance and simplicity of this molecule, few reports exist that investigate which molecular features are important for the activity of this ribodinucleotide. By exploiting the nonenzymatic reduction and oxidation of NAD+ by pyruvate and methylene blue, respectively, we were able to identify key molecular features necessary for the intrinsic activity of NAD+ through kinetic analysis. Such features may explain how NAD+ could have been selected early during the emergence of life. Simpler molecules, such as nicotinamide, that lack an anomeric carbon are incapable of accepting electrons from pyruvate. The phosphate moiety inhibits activity in the absence of metal ions but facilitates activity at physiological pH and model prebiotic conditions by recruiting catalytic Mg2+. Reduction proceeds through consecutive single electron transfer events. Of the derivatives tested, including nicotinamide mononucleotide, nicotinamide riboside, 3-(aminocarbonyl)-1-(2,3-dihydroxypropyl)pyridinium, 1-methylnicotinamide, and nicotinamide, only NAD+ and nicotinamide mononucleotide would be capable of efficiently accepting and donating electrons within a nonenzymatic electron transport chain. The data are consistent with early metabolic chemistry exploiting NAD+ or nicotinamide mononucleotide and not simpler molecules.

Vitamin B3 Containing Polymers for Nanodelivery.

Polymeric nanoparticles (NPs) with an integrated dual delivery system enable the controlled release of bioactive molecules and drugs, providing therapeutic advantages. Key design targets include high biocompatibility, cellular uptake, and encapsulating efficiency. In this study, a polymer library derived from niacin, also known as vitamin B3 is synthesized. The library comprises poly(2-(acryloyloxy)ethyl nicotinate) (PAEN), poly(2-acrylamidoethyl nicotinate) (PAAEN), and poly(N-(2-acrylamidoethyl)nicotinamide) (PAAENA), with varying hydrophilicity in the backbone and pendant group linker. All polymers are formulated, and those with increased hydrophobicity yield NPs with homogeneous spherical distribution and diameters below 150 nm, as confirmed by scanning electron microscopy and dynamic light scattering. Encapsulation studies utilizing a model drug, neutral lipid orange (NLO), reveal the influence of polymer backbone on encapsulation efficiency. Specifically, efficiencies of 46% and 96% are observed with acrylate and acrylamide backbones, respectively. Biological investigations showed that P(AEN) and P(AAEN) NPs are non-toxic up to 300 µg mL-1, exhibit superior cellular uptake, and boost cell metabolic activity. The latter is attributed to the cellular release of niacin, a precursor to nicotinamide adenine dinucleotide (NAD), a central coenzyme in metabolism. The results underline the potential of nutrient-derived polymers as pro-nutrient and drug-delivery materials.

Effect of tannic acid binding on the thermal degradation behavior and product toxicity of boscalid.

The effect of tannic acid (TA) binding on the thermal degradation of boscalid was studied in this work. The results revealed that TA binding has a significant impact on boscalid degradation. The degradation rate constant of bound boscalid was reduced, and its corresponding half-life was significantly prolonged compared to the free state. Four identical degradation products were detected in both states through UHPLC-Q-TOF-MS, indicating that degradation products were not affected by TA binding. Based on DFT and MS analysis, the degradation pathways of boscalid included hydroxyl substitution of chlorine atoms and cleavage of CN and CC bonds. The toxicity of B2 and B3 exceeded that of boscalid. In summary, the binding of TA and boscalid significantly affected the thermal degradation rate of boscalid while preserving the types of degradation products. This study contributed to a fundamental understanding of the degradation process of bound pesticide residues in complex food matrices.

Synthesis, activity, and their relationships of 2,4-diaminonicotinamide derivatives as EGFR inhibitors targeting C797S mutation.

The C797S mutation is one of the major factors behind resistance to the third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors. Herein, we describe the discovery of the 2,4-diaminonicotinamide derivative 5j, which shows potent inhibitory activity against EGFR del19/T790M/C797S and L858R/T790M/C797S. We also report the structure-activity relationship of the 2,4-diaminonicotinamide derivatives and the co-crystal structure of 5j and EGFR del19/T790M/C797S.

5-((3-Amidobenzyl)oxy)nicotinamides as SIRT2 Inhibitors: A Study of Constrained Analogs.

SIRT2 is a member of NAD+-dependent sirtuins and its inhibition has been proposed as a promising therapeutic approach for treating human diseases, including neurodegenerative diseases, cancer, and infections. Expanding SIRT2 inhibitors based on the 3-aminobenzyloxy nicotinamide core structure, we have synthesized and evaluated constrained analogs and selected stereoisomers. Our structure-activity relationship (SAR) study has revealed that 2,3-constrained (S)-isomers possess enhanced in vitro enzymatic inhibitory activity against SIRT2 and retain excellent selectivity over SIRT1 and SIRT3, provided that a suitable ring A is used. This current study further explores SIRT2 inhibitors based on the 3-aminobenzyloxy nicotinamide scaffold and contributes to the discovery of potent, selective SIRT2 inhibitors that have been actively pursued for their potential therapeutic applications.

Enhancing physical and chemical stability of hygroscopic hydroxytyrosol by cocrystal formation.

Hydroxytyrosol (HT) is a natural phenolic compound with potent antioxidant activity extracted from olive trees. It is generally a slightly hydrated viscous liquid at ambient conditions, and it is highly susceptible to oxygen due to the presence of catechol moiety. Although encapsulation technique provides HT in powder form, it does not improve its chemical stability. Herein, we propose an efficient solution to the high hygroscopicity and poor stability of HT. Four cocrystals were first reported, and their intermolecular interactions were analyzed in detail. After cocrystallization, the melting point is increased and the hygroscopicity is significantly decreased. HT cocrystals are thus solid at room temperature. Moreover, hydroxytyrosol cocrystals with betaine (HT-BET) and nicotinamide (HT-NIC) demonstrate superior chemical stability than pure HT, olive extract, and HT encapsulation material. Therefore, cocrystallization can be considered as a promising approach to overcome the application obstacles of HT.

Binding Affinity Studies of Nicotinamide N-methyltransferase and Ligands by Saturation Transfer Difference NMR.

INTRODUCTION: Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide with S-adenosine-L-methionine (SAM) as the methyl donor. Abnormal expression of NNMT is associated with many diseases (such as multiple cancers and metabolic and liver diseases), making NNMT a potential therapeutic target. Limited studies concerning the enzymesubstrate/ inhibitor interactions could be found to fully understand the detailed reaction mechanism. METHODS: The binding affinity and ligand binding epitopes of nicotinamide or SAH for binding NNMT and its mutants were determined using saturated transfer difference (STD) nuclear magnetic resonance (NMR) techniques combined with site-directed mutagenesis. RESULTS: The average dissociation constant of WT NNMT with nicotinamide and S-adenosine homocysteine (SAH) was 5.5 ± 0.9 mM and 1.2 ± 0.3 mM, respectively, while the mutants Y20F and Y20G with nicotinamide were up to nearly 4 times and 20 times that of WT and with SAH nearly 2 times and 5 times that of WT. The data suggested that WT had the highest binding affinity for nicotinamide or SAH, followed by Y20F and Y20G, which was consistent with its catalytic activity. CONCLUSION: The binding affinity of nicotinamide and SAH to NNMT and its mutants were obtained by STD NMR in this study. It was found that nicotinamide and SAH bind to WT in a particular orientation, and Y20 is critical for their binding orientation and affinity to NNMT.

A biophysical framework for double-drugging kinases.

Selective orthosteric inhibition of kinases has been challenging due to the conserved active site architecture of kinases and emergence of resistance mutants. Simultaneous inhibition of distant orthosteric and allosteric sites, which we refer to as "double-drugging", has recently been shown to be effective in overcoming drug resistance. However, detailed biophysical characterization of the cooperative nature between orthosteric and allosteric modulators has not been undertaken. Here, we provide a quantitative framework for double-drugging of kinases employing isothermal titration calorimetry, Förster resonance energy transfer, coupled-enzyme assays, and X-ray crystallography. We discern positive and negative cooperativity for Aurora A kinase (AurA) and Abelson kinase (Abl) with different combinations of orthosteric and allosteric modulators. We find that a conformational equilibrium shift is the main principle governing cooperativity. Notably, for both kinases, we find a synergistic decrease of the required orthosteric and allosteric drug dosages when used in combination to inhibit kinase activities to clinically relevant inhibition levels. X-ray crystal structures of the double-drugged kinase complexes reveal the molecular principles underlying the cooperative nature of double-drugging AurA and Abl with orthosteric and allosteric inhibitors. Finally, we observe a fully closed conformation of Abl when bound to a pair of positively cooperative orthosteric and allosteric modulators, shedding light on the puzzling abnormality of previously solved closed Abl structures. Collectively, our data provide mechanistic and structural insights into rational design and evaluation of double-drugging strategies.