Molecular Recognition of FDA-Approved Small Molecule Protein Kinase Drugs in Protein Kinases

Protein kinases are key enzymes that catalyze the covalent phosphorylation of substrates via the transfer of the γ-phosphate of ATP, playing a crucial role in cellular proliferation, differentiation, and various cell regulatory processes. Due to their pivotal cellular role, the aberrant function of kinases has been associated with cancers and many other diseases. Consequently, competitive inhibition of the ATP binding site of protein kinases has emerged as an effective means of curing these diseases. Decades of intense development of protein kinase inhibitors (PKIs) resulted in 71 FDA-approved PKI drugs that target dozens of protein kinases for the treatment of various diseases. How do FDA-approved protein kinase inhibitor PKI drugs compete with ATP in their own binding pocket? This is the central question we attempt to address in this work. Based on modes of non-bonded interactions and their calculated interaction strengths by means of the advanced double hybrid DFT method B2PLYP, the molecular recognition of PKI drugs in the ATP-binding pockets was systematically analyzed. It was found that (1) all the FDA-approved PKI drugs studied here form one or more hydrogen bond(s) with the backbone amide N, O atoms in the hinge region of the ATP binding site, mimicking the adenine base; (2) all the FDA-approved PKI drugs feature two or more aromatic rings. The latter reach far and deep into the hydrophobic regions I and II, forming multiple CH-π interactions with aliphatic residues L(3), V(11), A(15), V(36), G(51), L(77) and π-π stacking interactions with aromatic residues F(47) and F(82), but ATP itself does not utilize these regions extensively; (3) all FDA-approved PKI drugs studied here have one thing in common, i.e., they frequently formed non-bonded interactions with a total of 12 residues L(3),V(11), A(15), K(17), E(24),V(36),T(45), F(47), G(51), L(77), D(81) and F(82) in the ATP binding. Many of those 12 commonly involved residues are highly conserved residues with important structural and catalytic functional roles. K(17) and E(24) are the two highly conserved residues crucial for the catalytic function of kinases. D(81) and F(82) belong to the DFG motif; T(45) was dubbed the gate keeper residue. F(47) is located on the hinge region and G(51) sits on the linker that connects the hinge to the αD-helix. It is this targeting of highly conserved residues in protein kinases that led to promiscuous PKI drugs that lack selectivity. Although the formation of hydrogen bond(s) with the backbone of the hinge gives PKI drugs the added binding affinity and the much-needed directionality, selectivity is sacrificed. That is why so many FDA-approved PKI drugs are known to have multiple targets. Moreover, off-target-mediated toxicity caused by a lack of selectivity was one of the major challenges facing the PKI drug discovery community. This work suggests a road map for future PKI drug design, i.e., targeting non-conserved residues in the ATP binding pocket to gain better selectivity so as to avoid off-target-mediated toxicity.     

Umbelliferone Phosphate as a Substrate for Acid and Alkaline Phosphatase

Abstract

The results of a complete study of 8 substrates for acid and alkaline phosphatase indicated 7-hydroxycoumarin (umbelliferone) phosphate to be the best substrate for the analysis of these enzymes. Using this ester, from 10^?6 to 2 × 10^?2 units per ml. of alkaline phosphatase and 10^?5 to 0.06 units per ml. of acid phosphatase can be determined with an accuracy and precision of about 1.5%. Samples of serum as small as 1 μ;1. can be assayed. Analysis is performed by a direct initial reaction rate method in 2–3 minutes.     

Identification of Small Molecule Inhibitors against Mycobacteria in Activated Macrophages

Mycobacterial pathogens are intrinsically resistant to many available antibiotics, making treatment extremely challenging, especially in immunocompromised individuals and patients with underlying and chronic lung conditions. Even with lengthy therapy and the use of a combination of antibiotics, clinical success for non-tuberculous mycobacteria (NTM) is achieved in fewer than half of the cases. The need for novel antibiotics that are effective against NTM is urgent. To identify such new compounds, a whole cell high-throughput screen (HTS) was performed in this study. Compounds from the Chembridge DIVERSet library were tested for their ability to inhibit intracellular survival of M. avium subsp. hominissuis (MAH) expressing dtTomato protein, using fluorescence as a readout. Fifty-eight compounds were identified to significantly inhibit fluorescent readings of MAH. In subsequent assays, it was found that treatment of MAH-infected THP-1 macrophages with 27 of 58 hit compounds led to a significant reduction in intracellular viable bacteria, while 19 compounds decreased M. abscessus subsp. abscessus (Mab) survival rates within phagocytic cells. In addition, the hit compounds were tested in M. tuberculosis H37Ra (Mtb) and 14 compounds were found to exhibit activity in activated THP-1 cells. While the majority of compounds displayed inhibitory activity against both replicating (extracellular) and non-replicating (intracellular) forms of bacteria, a set of compounds appeared to be effective exclusively against intracellular bacteria. The efficacy of these compounds was examined in combination with current antibiotics and survival of both NTM and Mtb were evaluated within phagocytic cells. In time-kill dynamic studies, it was found that co-treatment promoted increased bacterial clearance when compared with the antibiotic or compound group alone. This study describes promising anti-NTM and anti-Mtb compounds with potential novel mechanisms of action that target intracellular bacteria in activated macrophages.     

Structural Insight into the Design on Oleanolic Acid Derivatives as Potent Protein Tyrosine Phosphatase 1B Inhibitors

Oleanolic acid derivatives act as newer protein tyrosine phosphatase 1B(PTP-1B) inhibitors for type 2 diabetes mellitus(T2DM). In order to understand the structural requirement of PTP-1B inhibitors, 52 oleanolic acid derivatives were divided into a training set(34 compounds) and a test set(18 compounds). The highly reliable and predictive 3D-QSAR models were constructed by CoM FA, CoM SIA and topomer Co MFA methods, respectively. The results showed that the cross validated coefficient(q2) and non-cross-validated coefficient(R~2) were 0.554 and 0.999 in the CoM FA model, 0.675 and 0.971 in the CoM SIA model, and 0.628 and 0.939 in the topomer Co MFA model, which suggests that three models are robust and have good exterior predictive capabilities. Furthermore, ten novel inhibitors with much higher inhibitory potency were designed. Our design strategy was that(i) the electronegative substituents(Cl,-CH_2OH, OH and-CH_2Cl) were introduced into the double bond of ring C,(ii) the hydrogen bond acceptor groups(C≡N and N atom), electronegative groups(C≡N, N atom,-COOH and-COOCH_3) and bulky substituents(C_6H_5N) were connected to the C-3 position, which would result in generating potent and selective PTP-1B inhibitors. We expect that the results in this paper have the potential to facilitate the process of design and to develop new potent PTP-1B inhibitors.     

Acid Phosphatase Assay with a Wireless Magnetoelastic Biosensor

Abstract

A wireless remote‐query disposable magnetoelastic (ME) biosensor was developed for the assay of acid phosphatase (ACP). The sensor was fabricated by applying a magnetoelastic ribbon with a layer of pH‐sensitive polymer and upon it a sensing film containing bovine serum albumin (BSA) and adenosine‐5′‐monophosphate (5′‐AMP). The ACP‐catalyzed hydrolysis of 5′‐AMP decreases the solution pH, resulting in the polymer shrinking and consequently the resonance frequency of the magnetoelastic sensor increasing. The kinetic parameters were measured to be 1.64×10^?3 M (Michaelis constant) and 130 Hz/min (maximum initial rate). The proposed sensor can determine 0.2 to 1.2 U/ml of ACP.     

Acid Phosphatase Assay with a Wireless Magnetoelastic Biosensor

Abstract

A wireless remote‐query disposable magnetoelastic biosensor is developed for the assay of acid phosphatase (ACP). The sensor was fabricated by applying a layer of pH‐sensitive polymer to a magnetoelastic ribbon and, on top of it, a sensing film containing bovine serum albumin and adenosine‐5′‐monophosphate (5′‐AMP), the substrate of ACP. In response to an externally applied time‐varying magnetic field, the magnetoelastic sensor mechanically vibrates at a characteristic frequency that is inversely dependent on the mass of the attached film. Because the magnetoelastic sensor is magnetostrictive, the mechanical vibrations of the sensor launch magnetic flux that can be detected remotely from the sensor using a pick‐up coil. The ACP‐catalyzed hydrolysis of 5′‐AMP decreases the solution pH, resulting in the polymer shrinking and, consequently, the resonance frequency of the magnetoelastic sensor increasing. The experimental condition was optimized to be 37°C, in 2.5 mM sodium citrate buffer solution (pH 6.5). Using citrate as buffer can enhance the ACP activity, partly offsetting the effect of the buffer capacity on the sensitivity. The kinetic parameter were measured to be: 1.64×10^–3 M (Michaelis constant) and 130 Hz/min (maximum initial rate). This work provides a remote enzymatic assay of ACP. The relative standard deviation in the measurements of six sensors in parallel is 3.4%. The proposed sensor can determine 0.2～1.2 U/ml of ACP.     

A Small Molecule Inhibits Protein Disulfide Isomerase and Triggers the Chemosensitization of Cancer Cells

Resistance to chemotherapeutic agents represents a major challenge in cancer research. One approach to this problem is combination therapy, the application of a toxic chemotherapeutic drug together with a sensitizing compound that addresses the vulnerability of cancer cells to induce apoptosis. Here we report the discovery of a new compound class ( T8 ) that sensitizes various cancer cells towards etoposide treatment at subtoxic concentrations. Proteomic analysis revealed protein disulfide isomerase (PDI) as the target of the T8 class. In‐depth chemical and biological studies such as the synthesis of optimized compounds, molecular docking analyses, cellular imaging, and apoptosis assays confirmed the unique mode of action through reversible PDI inhibition.     

A Molecular Description of Acid Phosphatase

Acid phosphatase is ubiquitous in distribution in various organisms. Although it catalyzes simple hydrolytic reactions, it is considered as an interesting enzyme in biological systems due to its involvement in different physiological activities. However, earlier reviews on acid phosphatase reveal some fragmentary information and do not give a holistic view on this enzyme. So, the present review summarizes studies on biochemical properties, structure, catalytic mechanism, and applications of acid phosphatase. Recent advancement of acid phosphatase in agricultural and clinical fields is emphasized where it is presented as potent agent for sustainable agricultural practices and diagnostic marker in bone metabolic disorders. Also, its significance in prostate cancer therapies as a therapeutic target has been discussed. At the end, current studies and prospects of immobilized acid phosphatase are included.     

抗肿瘤缺氧诱导因子-1的小分子抑制剂

肿瘤的缺氧微环境与其增殖、分化、血管生成、能量代谢、耐药性的发生以及患者预后状况密切相关。缺氧诱导因子1(Hypoxia-inducible factor 1, HIF-1)是细胞适应缺氧环境的重要转录因子和调控蛋白,通过调控下游靶基因如EPO、VEGF、GLUT等的表达,促进血管新生及有氧糖酵解以适应缺氧的环境,进而影响肿瘤细胞代谢、血管生成和肿瘤转移等。因此,开发以HIF-1为靶标的小分子抑制剂药物有望成为一种有效的肿瘤治疗方法。本文就HIF-1小分子抑制剂在肿瘤学研究中的进展进行综述,旨在为靶向HIF-1抗肿瘤药物的研发提供新思路。     

高结晶性小分子给体材料应用于全小分子有机太阳能电池中的研究进展

随着新型小分子给体材料和非富勒烯小分子受体材料的开发和应用, 非富勒烯全小分子有机太阳能电池(NF-ASM OSCs)的光电转换效率已经突破15%, 并逐渐接近聚合物太阳能电池的效率. 相比于聚合物电子给体材料, 小分子电子给体材料拥有其独特的优势, 例如合成批次性差异小、分子量明确和易于提纯等; 但是, 对小分子给体材料的结晶性难于精确调控, 使获得合适的纳米级结构的混合膜仍然是一个挑战. 本综述以给体小分子中心共轭单元的扩展为主线, 从分子设计的角度汇总了近年来对苯并二噻吩、萘并二噻吩和二噻并苯并二噻吩类小分子给体材料的结晶性研究, 并为进一步改善电池活性层形貌和获得更高的光伏性能提供了未来发展的建议.     

Identification of Potent Natural Resource Small Molecule Inhibitor to Control Vibrio cholera by Targeting Its Outer Membrane Protein U: An In Silico Approach

Vibrio cholerae causes the diarrheal disease cholera which affects millions of people globally. The outer membrane protein U (OmpU) is the outer membrane protein that is most prevalent in V. cholerae and has already been recognized as a critical component of pathogenicity involved in host cell contact and as being necessary for the survival of pathogenic V. cholerae in the host body. Computational approaches were used in this study to screen a total of 37,709 natural compounds from the traditional Chinese medicine (TCM) database against the active site of OmpU. Following a sequential screening of the TCM database, we report three lead compounds—ZINC06494587, ZINC85510056, and ZINC95910434—that bind strongly to OmpU, with binding affinity values of −8.92, −8.12, and −8.78 kcal/mol, which were higher than the control ligand (−7.0 kcal/mol). To optimize the interaction, several 100 ns molecular dynamics simulations were performed, and the resulting complexes were shown to be stable in their vicinity. Additionally, these compounds were predicted to have good drug-like properties based on physicochemical properties and ADMET assessments. This study suggests that further research be conducted on these compounds to determine their potential use as cholera disease treatment.