Co-crystal Complex Research Articles

Two new flavonoids from the leaves of Garcinia smeathmannii, in vitro and in silico anti-inflammatory potentials

Garcinia smeathmannii is a well-known plant for its uses in the effective treatment of intestinal parasites, skin eruptions and skin burns. The dichloromethane-methanol (2:3) crude extract of the leaves of G. smeathmannii led to the isolation and characterization of twenty compounds (1−20) using chromatographic and spectroscopic techniques. Extracts and compounds were screened in vitro for their anti-inflammatory (ROS), antiglycation and antileishmanial (L. tropica) activities. Compounds were also screened for their in silico anti-inflammatory activities using Maestro 4.2.1 software with the co-crystal complex structures of the ovine oCOX-1: meloxicam (PDB Id: 4O1Z) and murine mCOX-2: meloxicam (PDB Id: 4M11) proteins. An unprecedented flavonol (1) and a flavone dimer (2) together with eighteen known compounds (3−20) were characterized. All the tested samples in vitro revealed no antiglycation and antileishmanial activities. Beside, extracts revealed moderate anti-inflammatory activities (IC50 ranging from 24.1 ± 2.0 to 34.7 ± 0.8 μg/mL). Only compound (13) revealed an anti-inflammatory activity which was 9.33 times more active than the reference (Ibuprofen, IC50 = 11.2 ± 1.9 μg/mL) with IC50 of 1.2 ± 0.0 μg/mL. Compounds (2–9, 11–13 and 19–20) were docked and the docking scores were ranging from −10.178 to −6.119 (kcal/mol) which were in agreement with the experimental anti-inflammatory activity. These results are in agreement with the traditional uses of the leave of G. smeathmannii as cataplasm for skin eruption and as analgesic agent.

Discovery of N-Benzylpiperidinol derivatives as USP7 inhibitors against Hematology

USP7 has been recognized as a potential target for the treatment of hematologic malignancies by stabilizing multiple cancer-relevant proteins. Nevertheless, drug-like USP7 inhibitors are still lacking. Herein, compound J21 (USP7 IC50: 41.35 ± 2.16 nM) was discovered based on the structure of L55 and its co-crystal complex with USP7. Additionally, J21 exhibited greater metabolic stability (T1/2: 1.25 h, Cmax: 394.1 ± 48.3 ng/mL, and AUC0-t: 597.8 ± 44.8 ng/mL∙h) compared to L55. These findings may further pave the way for the development of USP7 inhibitors for the treatment of hematologic malignancies.

Structure-based design and synthesis of BML284 derivatives: A novel class of colchicine-site noncovalent tubulin degradation agents

Our previous studies have demonstrated that BML284 is a colchicine-site tubulin degradation agent. To improve its antiproliferative properties, 45 derivatives or analogs of BML284 were designed and synthesized based on the cocrystal structure of BML284 and tubulin. Among them, 5i was the most potent derivative, with IC50 values ranging from 0.02 to 0.05 μM against the five tested tumor cell lines. Structure–activity relationship studies verified that the N1 atom of the pyrimidine ring was the key functional group for its tubulin degradation ability. The 5i–tubulin cocrystal complex revealed that the binding pattern of 5i to tubulin is similar to that of BML284. However, replacing the benzodioxole ring with an indole ring strengthened the hydrogen bond formed by the 2-amino group with E198, which improved the antiproliferative activity of 5i. Compound 5i effectively suppressed tumor growth at an intravenous dose of 40 mg/kg (every 2 days) in paclitaxel sensitive A2780S and paclitaxel resistant A2780T ovarian xenograft models, with tumor growth inhibition values of 79.4% and 82.0%, respectively, without apparent side effects, showing its potential to overcome multidrug resistance. This study provided a successful example of crystal structure–guided discovery of 5i as a colchicine-targeted tubulin degradation agent, expanding the scope of targeted protein degradation.

Deracemization of Atropisomeric Biaryls Enabled by Copper Catalysis.

Atropisomeric biaryls have found crucial applications in versatile chiral catalysts as well as in ligands for transition metals. Herein, we have developed an efficient crystallization-induced deracemization (CID) method to access chiral biaryls from their racemates with a chiral ammonium salt under copper catalysis including BINOL, NOBIN, and BINAM derivatives. After being significantly accelerated by its bidentate diamine ligand, the copper catalyst exhibits high efficiency and selectivity in racemizing biaryl skeletons, and the cocrystal complex would be enantioselectively formed together with chiral ammonium salt, which on acid-quenching would directly deliver chiral biaryl without further chromatographic purification. This CID process is easily scalable, and the chiral ammonium salt was nicely recoverable. Ligand effect studies showed that bulky alkyl substitution was an indispensable element to ensure efficient racemization, which probably proceeds via a radical-cation intermediate and further allows axial rotation by forming a delocalized radical.

Facile synthesis of a 2,5-dihydroxyterephthalic acid-based charge transfer cocrystal for photoelectric applications

The assembly of 2,5-dihydroxyterephthalic acid donor and 1,10-phenanthroline acceptor afforded a charge transfer cocrystal complex, which was applied in photoelectric detector and phosphor-converted light-emitting diodes.

Discovery of a potent and highly selective inhibitor of SIRT6 against pancreatic cancer metastasis in vivo

Pancreatic cancer, one of the most aggressive malignancies, has no effective treatment due to the lack of targets and drugs related to tumour metastasis. SIRT6 can promote the migration of pancreatic cancer and could be a potential target for antimetastasis of pancreatic cancer. However, highly selective and potency SIRT6 inhibitor that can be used in vivo is yet to be discovered. Here, we developed a novel SIRT6 allosteric inhibitor, compound 11e, with maximal inhibitory potency and an IC50 value of 0.98 ± 0.13 μmol/L. Moreover, compound 11e exhibited significant selectivity against other histone deacetylases (HADC1‒11 and SIRT1‒3) at concentrations up to 100 μmol/L. The allosteric site and the molecular mechanism of inhibition were extensively elucidated by cocrystal complex structure and dynamic structural analyses. Importantly, we confirmed the antimetastatic function of such inhibitors in four pancreatic cancer cell lines as well as in two mouse models of pancreatic cancer liver metastasis. To our knowledge, this is the first study to reveal the in vivo effects of SIRT6 inhibitors on liver metastatic pancreatic cancer. It not only provides a promising lead compound for subsequent inhibitor development targeting SIRT6 but also provides a potential approach to address the challenge of metastasis in pancreatic cancer.

Product contamination during mechanochemical synthesis of praziquantel co-crystal, polymeric dispersion and cyclodextrin complex

This paper aims to evaluate the product contamination by elemental impurities during the mechanochemical synthesis of praziquantel (PZQ) co-crystal, polymeric dispersion and cyclodextrin complex by grinding. To assess that, PZQ was co-ground with malic acid (MA), Poloxamer F-127 (F-127) and hydroxypropyl-β-cyclodextrin (HPβCD) in high-energy vibrational mills using stainless steel and agate grinding tools, applying different processing time (30 and 90 min). Differential scanning calorimetry and X-ray powder diffraction confirmed the formation of the targeted products, regardless of applied processing time and grinding tool type. After digestion of the solid powder products, the levels of selected elemental impurities were analysed by inductively coupled plasma mass spectrometry (ICP-MS). The analysis revealed that the content of Mg, Ca, and V are below the limit of quantification in all samples analysed. The contents of P and Na are not related to the type of ball mill and reaction time, but to the starting materials themselves, considering that Na is found in HPβCD and MA, while P was found in F-127. The detected Si impurities in the co-ground products can be related to the use of the agate balls and jars, while the presence of Cr and Fe can be related to the use of the stainless steel grinding tools. The risk assessment showed that the oral administration of the prepared co-ground products in quantities corresponding to regular PZQ oral doses resulted in only insignificant exposure to Cr. Finally, the use of agate grinding tools should be preferred, as administration of such products results in lower Cr exposure. The presented elemental impurities did not lead to any significant drug degradation as PZQ content at the end of the six-month testing period was still in the range of 95–105 % of the initial content. Regardless, ICP-MS analysis of the elemental impurities should be considered in regular quality control procedures in the development and production of novel pharmaceutical products prepared by grinding.

Highly stable supercapacitive performance on a copper-based co-crystal complex

The article discusses the synthesis and characterization of a new Cu-based complex, [Cu(Hpz)4SO4]]•[Cu(Hpz)4(H2O)SO4]•H2O (1), obtained via solvothermal method with the ligand 1H-pyrazole (Hpz). The complex exhibits a co-crystal structure composed of two independent units. The crystal structure was further confirmed by PXRD Rietveld refinement. In three-electrode system tests, the electrode made from this co-crystal complex displayed a high specific capacitance and a good cycling stability. At a scan rate of 3 mV s−1, the maximum specific capacitance was 242.4 F g−1, and after 2000 cycles at a current density of 2 A g−1 in a 1 M KOH solution, a capacitance of 95.5% was retained.

Unravelling the allosteric binding mode of αD-VxXXB at nicotinic acetylcholine receptors.

αD-conotoxins are 11kDa homodimers that potently inhibit nicotinic acetylcholine receptors (nAChRs) through a non-competitive (allosteric) mechanism. In this study, we describe the allosteric binding mode of the granulin-like C-terminal (CTD) of VxXXB bound to Lymnea stagnalis acetylcholine binding protein (Ls-AChBP), a soluble homologue of the extracellular ligand-binding domain of nAChRs. This co-crystal complex revealed a novel allosteric binding site for nAChR antagonists outside the C-loop that caps the orthosteric site defined by the nAChR agonist nicotine and the antagonist epibatidine. Mutational and docking studies on Ls-AChBP supported a two-site binding mode for full-length VxXXB, with the first CTD binding site located outside the C-loop as seen in the co-crystal complex, with a second CTD binding site located near the N-terminal end of the adjacent subunit of AChBP. These results provide new structural insight into a novel allosteric mechanism of nAChR inhibition and define the cooperative binding mode of the N-terminal domain linked granulin core domains of αD-conotoxins.

Virtual Screening and Biological Evaluation of Novel Low Molecular Weight Protein Tyrosine Phosphatase Inhibitor for the Treatment of Insulin Resistance.

Protein tyrosine phosphatases (PTPs) play an essential way in diseases including cancer, obesity, diabetes and autoimmune disorders. As a member of PTPs, low molecular weight PTP (LMPTP) has been a well-recognized anti-insulin resistance target in obesity. However, the number of reported LMPTP inhibitors is limited. Our research aims to discover a novel LMPTP inhibitor and evaluate its biological activity against insulin resistance. A virtual screening pipeline based on the X-ray co-crystal complex of LMPTP was constructed. Enzyme inhibition assay and cellular bioassay were used to evaluate the activity of screened compounds. The screening pipeline rendered 15 potential hits from Specs chemical library. Enzyme inhibition assay identified compound F9 (AN-465/41163730) as a potential LMPTP inhibitor with a K i value of 21.5 ± 7.3 μM. Cellular bioassay showed F9 could effectively increase the glucose consumption of HepG2 cells as a result of releasing insulin resistance by regulating PI3K-Akt pathway. In summary, this study presents a versatile virtual screening pipeline for potential LMPTP inhibitor discovery and provides a novel-scaffold lead compound that is worthy of further modification to get more potent LMPTP inhibitors.

Charge transfer of cocrystal complex featuring efficient aggregation-induced emission and self-powered photo-electrochemical photodetector

A novel cocrystal complex CZIPA-AD assembled by twisting D-A molecule 5-(9H-carbazol-9-yl)isophthalic (CZIPA) and acridine (AD) through a facial solution volatilization process is reported. The combination of photophysical investigation and DFT analysis verify a reasonable cooperation of hybridized local and charge transfer (HLCT) and aggregation-induced emission (AIE) character. The orbital overlap on CZIPA (for local excited (LE) state) and orbital spatial separation between CZIPA and AD (for charge transfer (CT) state) satisfy the formation of HLCT character. The presence of AD molecules preclude the formation of π-stacking between the carbazole halves, preventing the quenching of PL emission in the aggregate state. It shows intense orange emission in aggregated solid state with high PLQY of 80% and prolonged PL lifetime of 38.57 ns. A self-powered photo-electrochemical photodetector fabricated from the cocrystal thin film exhibits a remarkable detectivity (4.78 × 1010 Jones) in neutral aqueous solution, which is at lest one order of magnitude higher than other reported inorganic materials measured in alkaline solution with additional bias potential.

Application of Angiotensin Receptor-Neprilysin Inhibitor in Chronic Kidney Disease Patients: Chinese Expert Consensus.

Chronic kidney disease (CKD) is a global public health problem, and cardiovascular disease is the most common cause of death in patients with CKD. The incidence and prevalence of cardiovascular events during the early stages of CKD increases significantly with a decline in renal function. More than 50% of dialysis patients die from cardiovascular disease, including coronary heart disease, heart failure, arrhythmia, and sudden cardiac death. Therefore, developing effective methods to control risk factors and improve prognosis is the primary focus during the diagnosis and treatment of CKD. For example, the SPRINT study demonstrated that CKD drugs are effective in reducing cardiovascular and cerebrovascular events by controlling blood pressure. Uncontrolled blood pressure not only increases the risk of these events but also accelerates the progression of CKD. A co-crystal complex of sacubitril, which is a neprilysin inhibitor, and valsartan, which is an angiotensin receptor blockade, has the potential to be widely used against CKD. Sacubitril inhibits neprilysin, which further reduces the degradation of natriuretic peptides and enhances the beneficial effects of the natriuretic peptide system. In contrast, valsartan alone can block the angiotensin II-1 (AT1) receptor and therefore inhibit the renin–angiotensin–aldosterone system. These two components can act synergistically to relax blood vessels, prevent and reverse cardiovascular remodeling, and promote natriuresis. Recent studies have repeatedly confirmed that the first and so far the only angiotensin receptor–neprilysin inhibitor (ARNI) sacubitril/valsartan can reduce blood pressure more effectively than renin–angiotensin system inhibitors and improve the prognosis of heart failure in patients with CKD. Here, we propose clinical recommendations based on an expert consensus to guide ARNI-based therapeutics and reduce the occurrence of cardiovascular events in patients with CKD.

Advances in computer-aided drug design for type 2 diabetes

ABSTRACT Introduction The number of diabetic patients is increasing, posing a heavy social and economic burden worldwide. Traditional drug development technology is time-consuming and costly, and the emergence of computer-aided drug design (CADD) has changed this situation. This study reviews the applications of CADD in diabetic drug designing. Areas covered In this article, the authors focus on the advance in CADD in diabetic drug design by elaborating the discovery, including peroxisome proliferator-activated receptor (PPAR), G protein-coupled receptor 40 (GPR40), dipeptidyl peptidase-IV (DDP-IV), protein tyrosine phosphatase 1B (PTP1B), sodium-dependent glucose transporter 2 (SGLT-2), and glucokinase (GK). Some drug discovery of these targets is related to CADD strategies. Expert opinion There is no doubt that CADD has contributed to the discovery of novel anti-diabetic agents. However, there are still many limitations and challenges, such as lack of co-crystal complex, dynamic simulations, water, and metal ion treatment. In the near future, artificial intelligence (AI) may be a promising strategy to accelerate drug discovery and reduce costs by identifying candidates. Moreover, AlphaFold, a deep learning model that predicts the 3D structure of proteins, represents a considerable advancement in the structural prediction of proteins, especially in the absence of homologous templates for protein structures.

Synthesis, Spectroscopic, Characterization and X-ray Structures of Lanthanide(III) Complexes Derived from 1,5-bis(phenyl(pyridin-2-yl)methylene)carbonohydrazide

The use of 1,5-bis (phenyl (pyridin-2-yl) methylene) carbonohydrazide (H₂L) in the coordination chemistry of lanthanides (III) yielded complexes in which two ligand molecules are present. The synthesis was carried out using a Ln/H₂L ratio of 1/2 to lead mononuclear complexes of [Ln(H₂L)₂(<i>η</i>²-NO₃)_3-x](NO₃)_x (Ln=La (1), Sm (4), Gd (6) and Yb (7)), [Ln(H₂L)₂(NO₃)₃] (Ln=Pr(2), Eu(5) and a co-crystal {[Nd(H₂L)₂(<i>η</i>²-NO₃)₂(<i>η¹</i>-NO₃)], [Nd(H₂L)₂(<i>η²-</i>NO₃)(<i>h¹-</i>NO₃)(H₂O)]}.(NO₃).2CH₃OH} (3). The structures of the complexes (2) and (3)were solved by X-ray crystallography on a single crystal. In the mononuclear complex of Pr<SUP>III</SUP>, two neutral ligand molecules act in tridentate fashion. In the co-crystal complex, one of the Nd<SUP>III</SUP> atom is coordinated by two ligand molecules acting tridentately in neutral form and three nitrate anions acting in bidentate fashion while the second Nd<SUP>III</SUP> atom is coordinated by two ligand molecules acting tridentately in neutral form, one monodentate nitrate anion, one bidentate nitrate anion and one water molecule. The neutrality of the complex is ensured by one free nitrate anion. Two free methanol molecules are present. Complex (2) crystallizes in the triclinic space group P-1 with the following parameters: a=10.128 (2) Å, b=12.2285 (19) Å, c=20.816 (5) Å, α=85.634 (4), β=81.870 (4)°, γ=87.210 (5)°, V=2542.9 (9) ų, Z=2, R₁=0.0640, wR₂=0.1377. Complex (3) crystallizes in the monoclinic space group P2₁/c with the following parameters: a=10.4554 (12) Å, b=44.089 (6) Å, c=23.212 (3) Å, β=90.851 (2)°, V=10699 (2) ų, Z=4, R₁=0.0630, wR₂=0.1690. The coordination sphere of the twelve-coordinated Pr<SUP>III</SUP> atom is best described as distorted icosahedron. In the co-crystal one of the Nd<SUP>III</SUP> atom is eleven-coordinated while the second Nd<SUP>III</SUP> atom is ten-coordinated. The environments around the Nd<SUP>III</SUP> atoms are respectively best described as distorted pentacapped trigonal prism and a distorted bicapped square antiprism, respectively. Supramolecular structures are consolidated by numerous hydrogen bonds.

Room-Temperature Phosphorescent Co-Crystal Showing Direct White Light and Photo-Electric Conversion.

The development of molecular crystalline materials with efficient room-temperature phosphorescence has been obtained much attention due to their fascinating photophysical properties and potential applications in the fields of data storage, bioimaging and photodynamic therapy. Herein, a new co-crystal complex [(DCPA) (AD)2] (DCPA = 9,10-di (4-carboxyphenyl)anthracene; AD = acridine) has been synthesized by a facile solvothermal process. Crystal structure analysis reveals that the co-crystal possesses orderly and alternant arrangement of DCPA donors and AD acceptors at molecular level. Fixed by strong hydrogen bonds, the DCPA molecule displays seriously twisty spatial conformation. Density functional theory (DFT) calculations show well separation of HOMO and LUMO for this co-crystal system, suggesting the efficient triplet excitons generation. Photoluminescence measurements show intensive cyan fluorescence (58.20 ns) and direct white phosphorescence (325 µs) emission at room-temperature. The transient current density–time curve reveals a typical switching electric response under the irradiation of simulated light, reveal that the [(DCPA) (AD)2] co-crystal has a high photoelectric response performance.

Understanding Hygroscopicity of Theophylline via a Novel Cocrystal Polymorph: A Charge Density Study.

The charge density distribution in a novel cocrystal (1) complex of 1,3-dimethylxanthine (theophylline) and propanedioic acid (malonic acid) has been determined. The molecules crystallize in the triclinic, centrosymmetric space group P1̅, with four independent molecules (Z = 4) in the asymmetric unit (two molecules each of theophylline and malonic acid). Theophylline has a notably high hygroscopic nature, and numerous cocrystals have shown a significant improvement in stability to humidity. A charge density study of the novel polymorph has identified interesting theoretical results correlating the stability enhancement of theophylline via cocrystallization. Topological analysis of the electron density highlighted key differences (up to 17.8) in Laplacian (∇2ρ) between the experimental (EXP) and single-point (SP) models, mainly around intermolecular-bonded carbonyls. Further investigation via molecular electrostatic potential maps reaffirmed that the charge redistribution enhanced intramolecular hydrogen bonding, predominantly for N(2') and N(2) (61.2 and 61.8 kJ mol-1, respectively). An overall weaker lattice energy of the triclinic form (-126.1 kJ mol-1) compared to that of the monoclinic form (-133.8 kJ mol-1) suggests a lower energy threshold to overcome to initiate dissociation. Future work via physical testing of the novel cocrystal in both dissolution and solubility will further solidify the correlation between theoretical and experimental results.

Synthesis and evaluation of FAK inhibitors with a 5-fluoro-7H-pyrrolo[2,3-d]pyrimidine scaffold as anti-hepatocellular carcinoma agents.

Focal adhesion kinase (FAK) is a ubiquitous intracellular non-receptor tyrosine kinase, which is involved in multiple cellular functions, including cell adhesion, migration, invasion, survival, and angiogenesis. In this study, a series of 7H-pyrrolo[2,3-d]pyrimidines were designed and synthesized according to the E-pharmacophores generated by docking a library of 667 fragments into the ATP pocket of the co-crystal complex of FAK and PF-562271 (PDB ID: 3BZ3). The 5-fluoro-7H-pyrrolo[2,3-d]pyrimidine derivatives demonstrated excellent activity against FAK and the cell lines SMMC7721 and YY8103. 2-((2-((3-(Acetamidomethyl)phenyl)amino)-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-methylbenzamide (16c) was selected for further bioactivity evaluations invivo, including preliminary pharmacokinetic profiling in rats and toxicity assays in mice, and tumor growth inhibition studies in a xenograft tumor model. The results showed that 16c did not affect the body weight gain of the animals up to a dose of 200mg/kg, and significantly inhibited tumor growth with a tumor growth inhibition rate of 78.6% compared with the negative control group. Furthermore, phosphoantibody array analyses of a sample of the tumor suggested that 16c inhibited the malignant proliferation of hepatocellular carcinoma (HCC) cells through decreasing the phosphorylation in the FAK cascade.

A conserved epitope III on hepatitis C virus E2 protein has alternate conformations facilitating cell binding or virus neutralization

Epitope III, a highly conserved amino acid motif of 524APTYSW529 on the hepatitis C virus (HCV) E2 glycoprotein, resides in the critical loop that binds to the host receptor CD81, thus making it one of the most important antibody targets for blocking HCV infections. Here, we have determined the X-ray crystal structure of epitope III at a 2.0-Å resolution when it was captured by a site-specific neutralizing antibody, monoclonal antibody 1H8 (mAb1H8). The snapshot of this complex revealed that epitope III has a relatively rigid structure when confined in the binding grooves of mAb1H8, which confers the residue specificity at both ends of the epitope. Such a high shape complementarity is reminiscent of the "lock and key" mode of action, which is reinforced by the incompatibility of an antibody binding with an epitope bearing specific mutations. By subtly positioning the side chains on the three residues of Tyr527, Ser528, and Trp529 while preserving the spatial rigidity of the rest, epitope III in this cocrystal complex adopts a unique conformation that is different from previously described E2 structures. With further analyses of molecular docking and phage display-based peptide interactions, we recognized that it is the arrangements of two separate sets of residues within epitope III that create these discrete conformations for the epitope to interact selectively with either mAb1H8 or CD81. These observations thus raise the possibility that local epitope III conformational dynamics, in conjunction with sequence variations, may act as a regulatory mechanism to coordinate "mAb1H8-like" antibody-mediated immune defenses with CD81-initiated HCV infections.

Kinetics-Driven Drug Design Strategy for Next-Generation Acetylcholinesterase Inhibitors to Clinical Candidate.

The acetylcholinesterase (AChE) inhibitors remain key therapeutic drugs for the treatment of Alzheimer's disease (AD). However, the low-safety window limits their maximum therapeutic benefits. Here, a novel kinetics-driven drug design strategy was employed to discover new-generation AChE inhibitors that possess a longer drug-target residence time and exhibit a larger safety window. After detailed investigations, compound 12 was identified as a highly potent, highly selective, orally bioavailable, and brain preferentially distributed AChE inhibitor. Moreover, it significantly ameliorated cognitive impairments in different mouse models with a lower effective dose than donepezil. The X-ray structure of the cocrystal complex provided a precise binding mode between 12 and AChE. Besides, the data from the phase I trials demonstrated that 12 had good safety, tolerance, and pharmacokinetic profiles at all preset doses in healthy volunteers, providing a solid basis for its further investigation in phase II trials for the treatment of AD.

Structural insights into CYP107G1 from rapamycin-producing Streptomyces rapamycinicus

Rapamycin is a clinically important macrolide agent with immunosuppressant and antiproliferative properties, produced by the actinobacterium, Streptomyces rapamycinicus. Two cytochrome P450 enzymes are involved in the biosynthesis of rapamycin. CYP107G1 and CYP122A2 catalyze the oxidation reactions of C27 and C9 of pre-rapamycin, respectively. To understand the structural and biochemical features of P450 enzymes in rapamycin biosynthesis, the CYP107G1 and CYP122A2 genes were cloned, their recombinant proteins were expressed in Escherichia coli, and the purified enzymes were characterized. Both enzymes displayed low spin states in the absolute spectra of ferric forms, and the titrations with rapamycin induced type I spectral changes with Kd values of 4.4 ± 0.4 and 3.0 ± 0.3 μM for CYP107G1 and CYP122A2, respectively. The X-ray crystal structures of CYP107G1 and its co-crystal complex with everolimus, a clinical rapamycin derivative, were determined at resolutions of 2.9 and 3.0 Å, respectively. The overall structure of CYP107G1 adopts the canonical scaffold of cytochrome P450 and possesses large substrate pocket. The distal face of the heme group is exposed to solvents to accommodate macrolide access. When the structure of the everolimus-bound CYP107G1 complex (CYP107G1-Eve) was compared to that of the ligand-free CYP107G1 form, no significant conformational change was observed. Hence, CYP107G1 has a relatively rigid structure with versatile loops to accommodate a bulky substrate. The everolimus molecule is bound to the substrate-binding pocket in the shape of a squeezed donut, and its elongated structure is bound perpendicular to a planar heme plane and I-helix.