Synthesis of Novobiocin Based Anti-cancerous Hsp90 C- terminal Inhibitors

The research described in this thesis focuses on heat shock protein 90 kDa (Hsp90), which is a molecular chaperone that contributes to the correct folding of around 200 proteins. It is an important protein that is associated with cancer survival and has been a focus of anticancer drug development for over two decades. Previous studies have shown that novobiocin (an FDA approved drug) acts as a weak inhibitor of Hsp90 via binding to the C-terminal domain (CTD) of Hsp90 and that modification of novobiocin has been shown to increase its anticancer activity. In this thesis, we designed and synthesized a series of analogues based on 4’ substituted novobiocin that focused on four modifications to improve its anticancer efficacy: In chapter 2 we aimed to: 1) replace the sugar moieties attached on the 4'-hydroxyl group; 2) substitute the 3'-amino group; 3) replace the novoise group with other sugars; in chapter 3 we aim to: 4) Develop of a covalent targeting strategy to identify any active cysteine residues of Hsp90 CTD, which could react with Michael acceptors added to novobiocin 4’. We designed and synthesized a number of glycosidic type novobiocin analogues via an optimized phase transfer catalysis. The resulted compounds showed increased antiproliferative activity. Protected sugar moieties are far less active than deprotected ones, however, difficulties of deprotection limited further development of such types of inhibitors. Nevertheless, we report compound 68, an indole-amide analogue of novobiocin, to be a new lead structure with IC50 = 23.4 μM. Also, 4’-Acrylate Michael acceptor type novobiocin Hsp90 inhibitors mildly improve anti-proliferative activity and 4’ substitution of cinnamic or sulfone fluoride group significantly increase the drug potency. Proteomic analysis of drug-Hsp90 complex confirmed the covalent modification of C597/C598. Overall, our project added new synthetic and biological knowledge into the design of non-covalent Hsp90 CTD inhibitors, more importantly, opened the gate for covalent Hsp90 CTD inhibitors drug design for the first time. Non-covalent candidate 68 and Michael acceptor type covalent candidate 108, 138 are identified as lead structures

Therapeutic targets and potential delivery systems of melatonin in osteoarthritis

Osteoarthritis (OA) is a highly prevalent age-related musculoskeletal disorder that typically results in chronic pain and disability. OA is a multifactorial disease, with increased oxidative stress, dysregulated inflammatory response, and impaired matrix metabolism contributing to its onset and progression. The neurohormone melatonin, primarily synthesized by the pineal gland, has emerged as a promising therapeutic agent for OA due to its potential to alleviate inflammation, oxidative stress, and chondrocyte death with minimal adverse effects. The present review provides a comprehensive summary of the current understanding regarding melatonin as a promising pharmaceutical agent for the treatment of OA, along with an exploration of various delivery systems that can be utilized for melatonin administration. These findings may provide novel therapeutic strategies and targets for inhibiting the advancement of OA

Covalent penicillin-protein conjugates elicit anti-drug antibodies that are clonally and functionally restricted

Many archetypal and emerging classes of small-molecule therapeutics form covalent protein adducts. In vivo, both the resulting conjugates and their off-target side-conjugates have the potential to elicit antibodies, with implications for allergy and drug sequestration. Although β-lactam antibiotics are a drug class long associated with these immunological phenomena, the molecular underpinnings of off-target drug-protein conjugation and consequent drug-specific immune responses remain incomplete. Here, using the classical β-lactam penicillin G (PenG), we probe the B and T cell determinants of drug-specific IgG responses to such conjugates in mice. Deep B cell clonotyping reveals a dominant murine clonal antibody class encompassing phylogenetically-related IGHV1, IGHV5 and IGHV10 subgroup gene segments. Protein NMR and x-ray structural analyses reveal that these drive structurally convergent binding modes in adduct-specific antibody clones. Their common primary recognition mechanisms of the penicillin side-chain moiety (phenylacetamide in PenG)—regardless of CDRH3 length—limits cross-reactivity against other β-lactam antibiotics. This immunogenetics-guided discovery of the limited binding solutions available to antibodies against side products of an archetypal covalent inhibitor now suggests future potential strategies for the ‘germline-guided reverse engineering’ of such drugs away from unwanted immune responses

Differential metabolites of bronchoalveolar lavage fluid from coal worker's pneumoconiosis patients

Background It is a research hotspot to study the changes of metabolites and metabolic pathways in the process of coal worker's pneumoconiosis (CWP) by metabonomics and to explore its pathogenesis. ObjectiveTo study the change of metabolites in bronchoalveolar lavage fluid (BALF) of patients with CWP and explore the metabolic regulation mechanism of the disease. MethodsPatients with CWP who met the national diagnostic criteria according to Diagnosis of occupational pneumoconiosis (GBZ 70-2015) and underwent massive whole lung lavage were selected as the case group, and patients with tracheostenosis who underwent bronchoscopy were selected as the control group. BALF samples were collected from the cases and the controls. After filtering out large particles and mucus, the supernatant was stored in a −80 ℃ refrigerator. The samples were detected and analyzed by liquid chromatography-mass spectrometry after adding extraction solution, cold bath ultrasonication, and high-speed centrifugation, and the metabolic profiles and related data of CWP patients were obtained. The differential metabolites related to the occurrence and development of CWP were screened by multiple statistical analysis; furthermore, we searched the Kyoto Encyclopedia of Genes and Genomes (KEGG) database for potential metabolic pathways involved in the progression. ResultsThere was no significant difference in the general conditions of the subjects, such as weight, height, age, and length of service among the stage I group, the stage II group, the stage III group, and the control group (P˃0.05). When comparing the CWP stage I group with the control group, 48 differential metabolites were screened out, among which 14 were up-regulated and 34 were down-regulated. A total of 66 differential metabolites were screened out between the patients with CWP stage II and the controls, 14 up-regulated and 52 down-regulated differential metabolites. Compared with the control group, 63 differential metabolites were screened out in the patients with CWP stage III, including 11 up-regulated and 52 down-regulated differential metabolites. There were 36 differential metabolites that may be related to the occurrence of CWP, among which 11 differential metabolites were up-regulated, and 25 were down-regulated. Four significant differential metabolic pathways were identified through KEGG database query: linoleic acid metabolic pathway, alanine metabolic pathway, sphingolipid metabolic pathway, and glycerophospholipid metabolic pathway. ConclusionThe metabolomic study of BALF show that there are 36 different metabolites in the occurrence and development of CWP, mainly associating with linoleic acid metabolism, alanine metabolism, sphingolipid metabolism, and glycerophospholipid metabolism pathways

The Effects of an Osteoarthritic Joint Environment on ACL Damage and Degeneration: A Yucatan Miniature Pig Model

Posttraumatic osteoarthritis (PTOA) arises secondary to joint injuries and is characteristically driven by inflammatory mediators. PTOA is often studied in the setting of ACL tears. However, a wide range of other injuries also lead to PTOA pathogenesis. The purpose of this study was to characterize the morphological changes in the uninjured ACL in a PTOA inflammatory environment. We retrospectively reviewed 14 ACLs from 13 Yucatan minipigs, 7 of which had undergone our modified intra-articular drilling (mIAD) procedure, which induced PTOA through inflammatory mediators. Seven ACLs were harvested from mIAD minipigs (PTOA) and seven ACLs from control minipigs with no cartilage degeneration (non-PTOA). ACL degeneration was evaluated using histological scoring systems. IL-1β, NF-κB, and TNF-α mRNA expression in the synovium was measured using qRT-PCR. PTOA minipigs demonstrated significant ACL degeneration, marked by a disorganized extracellular matrix, increased vascularity, and changes in cellular shape, density, and alignment. Furthermore, IL-1β, NF-κB, and TNF-α expression was elevated in the synovium of PTOA minipigs. These findings demonstrate the potential for ACL degeneration in a PTOA environment and emphasize the need for anti-inflammatory disease-modifying therapies following joint injury

Regioselective glycosylation of novobiocin alters activity

Glycosylation is a promising approach to overcome antimicrobial drug resistance. In this study, we investigated Koenigs-Knorr and phase transfer glycosylation on novobiocin. While the former only gave a 4′-OH product, the later produced mainly a kinetic controlled 5-OH product, but still achieved the 4′-OH modification and novoise-glycosylated products (with stronger base), as well as a diglycosylated compound. Investigation on the antibacterial activity indicate that the presence of galactose moiety helps to improve activity possibly via enhanced cellular uptake

Application of Iron Ore Tailings and Phosphogypsum to Create Artificial Rockfills Used in Rock-Filled Concrete

Rock-filled concrete (RFC) has good performance in terms of energy savings, cost reduction, and CO2 emissions as a novel massive concrete construction technology. There have been studies into replacing natural rocks in RFC with large blocks of solid waste, and this method has been used on several construction sites. However, the granular and powdery solid waste utilized in RFC is limited, as a consequence of the special requirement of self-compacting concrete (SCC) in RFC. The goal of this paper is to increase the amount of granular and powdery solid waste in RFC. Iron ore tailing (IOT) and phosphogypsum (PG) were used separately as granular and powdery solid waste. The modified PG, ground blast-furnace slag (GBFS), steel slag, and cement clinker are combined to form parathion gypsum slag cement in a specific proportion, with the ratio of PG, GBFS, steel slag, and cement being 47:47:2:2. To replace the natural rocks in RFC, artificial rockfills made of IOT and parathion gypsum slag cement are used to increase the dosage of solid waste. The artificial rockfills were formed using three methods: compressing, roller compacting, and normal vibrating. When the compressive strength and material costs of the three types of artificial rockfills are compared, the compressing method is the best for maximizing the IOT. In artificial rockfills, the mass fraction of granular solid waste is 83.3%, and the mass fraction of total solid waste is 99.3%