Co-crystal Structure Research Articles

Prioritizing Computational Cocrystal Prediction Methods for Experimental Researchers: A Review to Find Efficient, Cost-Effective, and User-Friendly Approaches

Pharmaceutical cocrystals offer a versatile approach to enhancing the properties of drug compounds, making them an important tool in drug formulation and development by improving the therapeutic performance and patient experience of pharmaceutical products. The prediction of cocrystals involves using computational and theoretical methods to identify potential cocrystal formers and understand the interactions between the active pharmaceutical ingredient and coformers. This process aims to predict whether two or more molecules can form a stable cocrystal structure before performing experimental synthesis, thus saving time and resources. In this review, the commonly used cocrystal prediction methods are first overviewed and then evaluated based on three criteria: efficiency, cost-effectiveness, and user-friendliness. Based on these considerations, we suggest to experimental researchers without strong computational experiences which methods and tools should be tested as a first step in the workflow of rational design of cocrystals. However, the optimal choice depends on specific needs and resources, and combining methods from different categories can be a more powerful approach.

Discovery of Thiazole Carboxamides as Novel Vanin-1 Inhibitors for Inflammatory Bowel Disease Treatment.

Inflammatory bowel disease (IBD) is a clinically heterogeneous disease demanding more therapeutic targets and intervention strategies. Vanin-1, an oxidative stress-regulating protein, has emerged as a promising target for alleviating inflammation and oxidative stress. In this study, a series of thiazole carboxamide derivatives as vanin-1 inhibitors were designed and synthesized. The preferred compound, X17, demonstrated potent inhibition against vanin-1 at the protein, HT-29 cell, and tissue levels, whose binding mode with the target was confirmed via the cocrystal structure. X17 achieved a high bioavailability of 81% in rats, accompanied by concentration-dependent inhibition of serum vanin-1. In a DSS-induced mouse colitis model, X17 exhibited potent anti-inflammatory and antioxidant activities, repressing the inflammatory factor expressions and myeloperoxidase activity, elevating the colonic glutathione reserve, and restoring the intestinal barrier. Collectively, these findings depict the discovery of a potent vanin-1 inhibitor, providing an opportunity for further drug candidate development for treating IBD.

Discovery of CHD1 Antagonists for PTEN-Deficient Prostate Cancer.

CHD1 is a chromodomain-helicase DNA-binding protein that preferentially recognizes di- and trimethylated lysine 4 on histone H3 (H3K4me2/3). Genetic studies have established CHD1 as a synthetic lethal target in phosphatase and tensin homologue (PTEN)-deficient cancers. Despite this attractive therapeutic link, no inhibitors or antagonists of CHD1 have been reported to date. Herein, we report the discovery of UNC10142, a first-in-class small molecule antagonist of the tandem chromodomains of CHD1 that binds with an IC50 of 1.7 ± 0.2 μM. A cocrystal structure revealed a unique binding mode and competition pull-down experiments in cell lysates confirmed endogenous target engagement. Treatment of PTEN-deficient prostate cancer cells with UNC10142 led to a dose-dependent reduction in viability while PTEN-intact prostate cancer cells were unaffected, phenocopying genetic loss of CHD1. Overall, this study demonstrates the ligandability of the CHD1 chromodomains and suggests more potent and selective antagonists could translate to compounds of therapeutic value in PTEN-deficient cancers.

Distribution and diversity of classical deacylases in bacteria

Classical Zn2+-dependent deac(et)ylases play fundamental regulatory roles in life and are well characterized in eukaryotes regarding their structures, substrates and physiological roles. In bacteria, however, classical deacylases are less well understood. We construct a Generalized Profile (GP) and identify thousands of uncharacterized classical deacylases in bacteria, which are grouped into five clusters. Systematic structural and functional characterization of representative enzymes from each cluster reveal high functional diversity, including polyamine deacylases and protein deacylases with various acyl-chain type preferences. These data are supported by multiple crystal structures of enzymes from different clusters. Through this extensive analysis, we define the structural requirements of substrate selectivity, and discovered bacterial de-d-/l-lactylases and long-chain deacylases. Importantly, bacterial deacylases are inhibited by archetypal HDAC inhibitors, as supported by co-crystal structures with the inhibitors SAHA and TSA, and setting the ground for drug repurposing strategies to fight bacterial infections. Thus, we provide a systematic structure-function analysis of classical deacylases in bacteria and reveal the basis of substrate specificity, acyl-chain preference and inhibition.

A novel cocrystal of indole-3-propionic acid and nicotinamide: structure design, preparation, characterization and preliminary physiochemical properties evaluation

Indole-3-propionic acid (IPA), an intestinal microbiota produced metabolite, has been supposed as a promising clinical drug candidate to ameliorate progressive neurological and neuropsychiatric disorders associated with gut microbiota dysbiosis due to age-related infirmity. In this study, in order to enhance the aqueous solubility of IPA, nicotinamide (NIC) was selected as a coformer to synthesize a novel cocrystal (IPA-NIC) which was characterized by single crystal X-ray diffraction (SCXRD), powder X-ray diffraction (PXRD), thermal analysis, and Fourier transform infrared spectroscopy (FT-IR). The cocrystal structure was found to be in a 2:1 stoichiometric ratio consisting of four IPA molecules and two NIC molecules linked through heteromolecular hydrogen bonds within an asymmetric unit. The physiochemical properties of solubility, dissolution and permeability were demonstrated to be simultaneously improved. Preliminary forced studies under thermal (60 °C) and humid (92.5% RH) conditions indicated that the cocrystal remained stable which would be beneficial to the further development as a potential drug candidate.

Cladophorol-A is an inhibitor of cyclic GMP-AMP synthase

Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) is an enzyme sensor of double-stranded DNA (dsDNA) that serves to trigger activation of the cGAS-stimulator of interferon genes (STING) pathway. Excessive activation of this pathway has been demonstrated to contribute to various forms of inflammatory disease. As such, cGAS has arisen as a potential therapeutic target with broad potential applications. Using a pathway-targeted cell-based screening approach, we identified the natural product Cladophorol A as a new class of non-cytotoxic cGAS inhibitor (cell-based IC50 = 370 nM). An x-ray co-crystal structure at 2.75 Å resolution revealed that Cladophorol A inhibits cGAS by binding to its active site within the conserved adenosine nucleobase binding site.

Characterization of CL-20/HMX cocrystallization and its effects on GAP-based propellants during thermal aging process

The incorporation of HMX and CL-20 as component particles can greatly improve the energy efficiency of composite propellants. Nevertheless, the occurrence of cocrystallization arises when HMX and CL-20 exists together. Further investigation is needed to determine the effects of the CL-20/HMX cocrystallization on the aging features of the propellants. The present study examines the evolutionary trends and effects of the CL-20/HMX cocrystal on the process of solid propellant aging. Experimental thermal aging studies were carried out on GAP-based composite propellants at temperatures of 60 °C/180-day and 70 °C/90-day. An investigation of density bottle, differential scanning calorimetry, X-ray diffraction, Fourier transform infrared spectroscopy, and uniaxial tensile testing were performed on the samples. The results indicate that the cocrystallization behaviours of CL-20/HMX undergo three distinct stages during propellant aging. The reaction rate accelerates most during the middle aging stage. As the GAP-based propellant ages, the recently developed cocrystal structure deteriorates the interfacial characteristics between the propellant particles and the matrix, leading to the formation of cavities inside the material. This will further enhance the decrease in elastic modulus, ultimate strength, and maximum elongation of the propellants. Among them, the most notable distinction from the aging behaviours of propellants without the addition of particles is the increase of the elastic modulus. Furthermore, a strong linear correlation was observed between variation of the elastic modulus and the cocrystal content of CL-20/HMX. This correlation offers a reliable indicator for monitoring the extent of cocrystallization phenomena.

Biochemical analysis of EGFR exon20 insertion variants insASV and insSVD and their inhibitor sensitivity

Somatic mutations in the epidermal growth factor receptor (EGFR) are a major cause of non-small cell lung cancer. Among these structurally diverse alterations, exon 20 insertions represent a unique subset that rarely respond to EGFR tyrosine kinase inhibitors (TKIs). Therefore, there is a significant need to develop inhibitors that are active against this class of activating mutations. Here, we conducted biochemical analysis of the two most frequent exon 20 insertion variants, V769_D770insASV (insASV) and D770_N771insSVD (insSVD) to better understand their drug sensitivity and resistance. From kinetic studies, we found that EGFR insASV and insSVD are similarly active, but have lower Km, ATP values compared to the L858R variant, which contributes to their lack of sensitivity to 1st-3rd generation EGFR TKIs. Biochemical, structural, and cellular studies of a diverse panel of EGFR inhibitors revealed that the more recently developed compounds BAY-568, TAS6417, and TAK-788 inhibit EGFR insASV and insSVD in a mutant-selective manner, with BAY-568 being the most potent and selective versus wild-type (WT) EGFR. Cocrystal structures with WT EGFR reveal the binding modes of each of these inhibitors and of poziotinib, a potent but not mutantselective inhibitor, and together they define interactions shared by the mutant-selective agents. Collectively, our results show that these exon20 insertion variants are not inherently inhibitor resistant, rather they differ in their drug sensitivity from WT EGFR. However, they are similar to each other, indicating that a single inhibitor should be effective for several of the diverse exon 20 insertion variants.

Bioinspired Photothermal Metal-Organic Framework Cocrystal with Ultra-Fast Water Transporting Channels for Solar-Driven Interfacial Water Evaporation.

Herein, a bioinspired metal-organic framework (MOF) cocrystal produced from the co-assembly of a MOF [Ni3(hexaiminobenzene)2, Ni3(HIB)2] and p-chloranils (CHLs) is reported. Because of the 2D conjugation nature and the formation of persistent anion radicals, this cocrystal shows an excellent photothermal property, and is further used as an absorber in solar-driven interfacial water evaporation. The solar-driven interfacial water evaporation rate (4.04 kg m-2 h-1) is among the best compared with those of previously reported photothermal materials. Molecular dynamics simulation results suggested that the rotating of the CHL molecules relative to the MOF planes tuned the pore size to enable the ultra-fast water transporting, and thus ultra-high water transporting rates (1.11 × 1011 and 3.21 × 1011 H2O s-1 channel-1 at 298.2 and 323.0 K, respectively) for layered cocrystal structures, that are much higher than that of aquaporins (≈1.1 × 1010 H2O s-1 channel-1 at 298.2 K), are observed. The superior solar-driven water evaporation performance is thus attributed to the synergistic effect of the ultra-fast water transporting pores together with the excellent photothermal property of the cocrystal. This research provided a biomimetic strategy of rational design and production of charge transfer cocrystals to modulate their pores and photothermal properties for solar-driven interfacial water evaporation.

Discovery and Characterization of BAY-184: A New Potent and Selective Acylsulfonamide-Benzofuran In Vivo-Active KAT6AB Inhibitor.

KAT6A and KAT6B genes are two closely related lysine acetyltransferases that transfer an acetyl group from acetyl coenzyme A (AcCoA) to lysine residues of target histone substrates, hence playing a key role in chromatin regulation. KAT6A and KAT6B genes are frequently amplified in various cancer types. In breast cancer, the 8p11-p12 amplicon occurs in 12-15% of cases, resulting in elevated copy numbers and expression levels of chromatin modifiers like KAT6A. Here, we report the discovery of a new acylsulfonamide-benzofuran series as a novel structural class for KAT6A/B inhibition. These compounds were identified through high-throughput screening and subsequently optimized using molecular modeling and cocrystal structure determination. The final tool compound, BAY-184 (29), was successfully validated in an in vivo proof-of-concept study.

CovalentInDB 2.0: an updated comprehensive database for structure-based and ligand-based covalent inhibitor design and screening.

The rational design of targeted covalent inhibitors (TCIs) has emerged as a powerful strategy in drug discovery, known for its ability to achieve strong binding affinity and prolonged target engagement. However, the development of covalent drugs is often challenged by the need to optimize both covalent warhead and non-covalent interactions, alongside the limitations of existing compound libraries. To address these challenges, we present CovalentInDB 2.0, an updated online database designed to support covalent drug discovery. This updated version includes 8303 inhibitors and 368 targets, supplemented by 3445 newly added cocrystal structures, providing detailed analyses of non-covalent interactions. Furthermore, we have employed an AI-based model to profile the ligandability of 144 864 cysteines across the human proteome. CovalentInDB 2.0 also features the largest covalent virtual screening library with 2 030 192 commercially available compounds and a natural product library with 105 901 molecules, crucial for covalent drug screening and discovery. To enhance the utility of these compounds, we performed structural similarity analysis and drug-likeness predictions. Additionally, a new user data upload feature enables efficient data contribution and continuous updates. CovalentInDB 2.0 is freely accessible at http://cadd.zju.edu.cn/cidb/.

Targeting PfCLK3 with Covalent Inhibitors: A Novel Strategy for Malaria Treatment.

Malaria still causes over 600,000 deaths annually, with rising resistance to frontline drugs by Plasmodium falciparum increasing this number each year. New medicines with novel mechanisms of action are, therefore, urgently needed. In this work, we solved the cocrystal structure of the essential malarial kinase PfCLK3 with the reversible inhibitor TCMDC-135051 (1), enabling the design of covalent inhibitors targeting a unique cysteine residue (Cys368) poorly conserved in the human kinome. Chloroacetamide 4 shows nanomolar potency and covalent inhibition in both recombinant protein and P. falciparum assays. Efficacy in parasites persisted after a 6 h washout, indicating an extended duration of action. Additionally, 4 showed improved kinase selectivity and a high selectivity index against HepG2 cells, with a low propensity for resistance (log MIR > 8.1). To our knowledge, compound 4 is the first covalent inhibitor of a malarial kinase, offering promising potential as a lead for a single-dose malaria cure.

Small molecules from antibody pharmacophores (SMAbPs) as a hit identification workflow for immune checkpoints.

Small-molecule modulators of immune checkpoints are poised to revolutionize cancer immunotherapy. However, efficient strategies for hit identification are lacking. We introduce small molecules from antibody pharmacophores (SMAbPs), a workflow leveraging cocrystal structures of checkpoints with antibodies to create pharmacophore maps for virtual screening. Applying SMAbPs to five immune checkpoints yielded hits with submicromolar potency in both cell-free and cellular assays. Notably, SMAbPs identified the most potent T cell immunoglobulin and mucin-domain containing-3 and V-domain immunoglobulin suppressor of T cell activation (VISTA) inhibitors reported to date and first-in-class modulators of B and T lymphocyte attenuator, 4-IBB, and CD27. Targeting inhibitory and costimulatory checkpoints with hits identified through SMAbPs demonstrated remarkable in vivo antitumor activity, exemplified by MG-V-53 (VISTA inhibitor) and MG-C-30 (CD27 agonist), which significantly reduced tumor volumes in MC38 and EG7-OVA mouse models, respectively.

Structure-Guided Conformational Restriction Leading to High-Affinity, Selective, and Cell-Active Tetrahydroisoquinoline-Based Noncovalent Keap1-Nrf2 Inhibitors.

Inhibition of the protein-protein interaction between Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2) has been recognized as an attractive approach for treating oxidative stress-related diseases. Here, we present a new series of noncovalent Keap1-Nrf2 inhibitors developed by a conformational restriction strategy of our fluorenone-based compounds previously identified by fragment-based drug discovery. The design was guided by X-ray cocrystal structures, and the subsequent optimization process aimed at improving affinity, cellular activity, and metabolic stability. From the noncyclic compound 7 (Ki = 2.9 μM), a new series of tetrahydroisoquinoline-based Keap1 inhibitors with up to 223-fold improvement in binding affinity (57, Ki = 13 nM), better metabolic stability, and enhanced cellular activity was obtained. In addition, the compounds showed selectivity for the Keap1 Kelch domain across a panel of 15 homologous proteins. We thereby demonstrate the utility of cyclic rigidification in the design of potent and more drug-like Keap1-Nrf2 inhibitors.

Structure-based pose prediction: Non-cognate docking extended to macrocyclic ligands

So-called “cross-docking” is the prediction of the bound configuration of small-molecule ligands that differ from the cognate ligand of a protein co-crystal structure. This is a much more challenging problem than re-docking the cognate ligand, particularly when the new ligand is structurally dissimilar from prior known ones. We have updated the previously introduced PINC (“PINC Is Not Cognate”) benchmark which introduced the idea of temporal segregation to measure cross-docking performance. The temporal set encompasses 846 future ligands for ten targets based on information from the earliest 25% of X-ray co-crystal structures known for each target. Here, we extend the benchmark to include thirteen targets where the bound poses of 128 macrocyclic ligands are to be predicted based on knowledge from structures of bound non-macrocyclic ligands. Performance was roughly equivalent for both the temporally-split non-macrocyclic ligand set and the macrocycle prediction set. Using standard and fully automatic protocols for the Surflex-Dock and ForceGen methods, across the combined 974 non-macrocyclic and macrocyclic ligands, the top-scoring pose family was correct 68% of the time, with the top-two pose families achieving a 79% success rate. Correct poses among all those predicted were identified 92% of the time. These success rates far exceeded those observed for the alternative methods AutoDock Vina and Gnina on both sets.

Elucidating the Unconventional Binding Mode of a DNA-Encoded Library Hit Provides a Blueprint for Sirtuin 6 Inhibitor Development.

Sirtuin 6 (Sirt6), an NAD+-dependent deacylase, has emerged as a promising target for aging-related diseases and cancer. Advancing the medicinal chemistry of Sirt6 modulators is crucial for the development of chemical probes aimed at unraveling the intricate biological functions of Sirt6 and unlocking its therapeutic potential. A proprietary DNA-encoded library yielded Sirt6 inhibitor 2-Pr, displaying remarkable inhibitory activity and isoform-selectivity, and featuring a chemical structure distinct from reported Sirt6 modulators. In this study, we explore the inhibitory mechanism of 2-Pr, evaluating the impact of chemical modifications and presenting a crystal structure of the Sirt6/ADP-ribose/2-Pr complex. Notably, co-crystal structure analysis reveals an unexpected and unprecedented binding mode of Sirt6, with 2-Pr spanning the acyl channel of the enzyme, extending into the acetyl-lysine binding pocket, and reaching toward the C-site. This unique binding mode guides potential avenues for developing potent and selective Sirt6 inhibitors.

Design of a Cereblon construct for crystallographic and biophysical studies of protein degraders

The ubiquitin E3 ligase cereblon (CRBN) is the target of therapeutic drugs thalidomide and lenalidomide and is recruited by most targeted protein degraders (PROTACs and molecular glues) in clinical development. Biophysical and structural investigation of CRBN has been limited by current constructs that either require co-expression with the adaptor DDB1 or inadequately represent full-length protein, with high-resolution structures of degrader ternary complexes remaining rare. We present the design of CRBNmidi, a construct that readily expresses from E. coli with high yields as soluble, stable protein without DDB1. We benchmark CRBNmidi for wild-type functionality through a suite of biophysical techniques and solve high-resolution co-crystal structures of its binary and ternary complexes with degraders. We qualify CRBNmidi as an enabling tool to accelerate structure-based discovery of the next generation of CRBN based therapeutics.

Molecular mechanism for regulating APOBEC3G DNA editing function by the non-catalytic domain

APOBEC3G, part of the AID/APOBEC cytidine deaminase family, is crucial for antiviral immunity. It has two zinc-coordinated cytidine-deaminase domains. The non-catalytic N-terminal domain strongly binds to nucleic acids, whereas the C-terminal domain catalyzes C-to-U editing in single-stranded DNA. The interplay between the two domains is not fully understood. Here, we show that DNA editing function of rhesus macaque APOBEC3G on linear and hairpin loop DNA is enhanced by AA or GA dinucleotide motifs present downstream in the 3’-direction of the target-C editing sites. The effective distance between AA/GA and the target-C sites is contingent on the local DNA secondary structure. We present two co-crystal structures of rhesus macaque APOBEC3G bound to ssDNA containing AA and GA, revealing the contribution of the non-catalytic domain in capturing AA/GA DNA. Our findings elucidate the molecular mechanism of APOBEC3G’s cooperative function, which is critical for its antiviral role and its contribution to mutations in cancer genomes.

Discovery of glycosidated glycyrrhetinic acid derivatives: Natural product-based soluble epoxide hydrolase inhibitors

There are few reports on soluble epoxide hydrolase (sEH) structure-activity relationship studies using natural product-based scaffolds. In this study, we discovered that C-30 urea derivatives of glycyrrhetinic acid such as 33, rather than C-20/C-3 urea derivatives, possess in vitro sEH inhibitory capabilities. Furthermore, we explored the impact of stereoconfigurations at C-3 and C-18 positions, and glycosidic bonds at the 3-OH on the compound's activity. Consequently, a glycoside of 33, specifically 49Cα containing alpha-oriented mannose, exhibited promising in vivo efficacy in alleviating carrageenan-induced paw edema and acetic acid-induced writhing. Meanwhile, 49Cα demonstrated potential in mitigating acute pancreatitis by modulating the ratios of anti-inflammatory epoxyeicosatrienoic acids (EETs) to pro-inflammatory dihydroxyeicosatrienoic acids (DHETs). The co-crystal structure of sEH in complex with 49Cα revealed that the N-tetrahydropyranylmethylene urea hydrogen bonded with the residues within the sEH tunnel, contrasting with the mannose component that extended beyond the tunnel's confines. Our findings highlight 49Cα (coded LQ-38) as a promising candidate for anti-inflammatory and analgesic effects, and pave the way for the future rational design of triterpenoid-based sEH inhibitors.

Scalable Synthesis of Organic Core/Shell Architectures toward Dual-Wavelength Optical Waveguides.

Organic core/shell heterostructures have undergone rapid progress in materials chemistry owing to the integration of a wide array of unique properties. Nonetheless, the intricate challenge of regulating homogeneous nucleation and phase separation processes in excessively analogous cocrystal structures presents a formidable barrier to expanding the synthesis strategy for organic core/shell heterostructures. Herein, we successfully achieved a phase separation growth process facilitated by the organic alloy interface layer through a dynamic visualization to capture the intricate morphological evolution. By finely regulating the nucleation process, homogeneous self-assembly induced by high chemical and structural compatibility is circumvented, enabling the formation of organic core/shell heterostructures. Notably, this core/shell architecture boasts dual-wavelength emission at 496 and 696 nm, accompanied by an optical loss coefficient of 0.092 dB per micrometer. This methodology shows potential for extending to the scalable design of other conformational cocrystal heterostructure systems, thereby offering valuable insights into the realm of organic photonics.