👤 Gerlach DL

The aim of the UniProt Knowledgebase (UniProtKB; https://www.uniprot.org/) is to provide users with a comprehensive, high-quality and freely accessible set of protein sequences annotated with functional information. In this publication, we describe ongoing changes to our production pipeline to limit the sequences available in UniProtKB to high-quality, non-redundant reference proteomes. We continue to manually curate the scientific literature to add the latest functional data and use machine learning techniques. We also encourage community curation to ensure key publications are not missed. We provide an update on the automatic annotation methods used by UniProtKB to predict information for unreviewed entries describing unstudied proteins. Finally, updates to the UniProt website are described, including a new tab linking protein to genomic information. In recognition of its value to the scientific community, the UniProt database has been awarded Global Core Biodata Resource status. Show less

Efforts to identify anti-cancer therapeutics and understand tumor-immune interactions are built with in vitro models that do not match the microenvironmental characteristics of human tissues. Using in vitro models which mimic the physical properties of healthy or cancerous tissues and a physiologically relevant culture medium, we demonstrate that the chemical and physical properties of the microenvironment regulate the composition and topology of the glycocalyx. Remarkably, we find that cancer and age-related changes in the physical properties of the microenvironment are sufficient to adjust immune surveillance via the topology of the glycocalyx, a previously unknown phenomenon observable only with a physiologically relevant culture medium. Show less

Lysine-specific demethylase 5A (KDM5A) has recently become a promising target for epigenetic therapy. In this study, we designed and synthesized metal complexes bearing ligands with reported demethylase and p27 modulating activities. The Rh(III) complex 1 was identified as a direct, selective and potent inhibitor of KDM5A that directly abrogate KDM5A demethylase activity via antagonizing the KDM5A-tri-/di-methylated histone 3 protein-protein interaction (PPI) in vitro and in cellulo. Complex 1 induced accumulation of H3K4me3 and H3K4me2 levels in cells, causing growth arrest at G1 phase in the triple-negative breast cancer (TNBC) cell lines, MDA-MB-231 and 4T1. Finally, 1 exhibited potent anti-tumor activity against TNBC xenografts in an in vivo mouse model, presumably via targeting of KDM5A and hence upregulating p27. Moreover, complex 1 was less toxic compared with two clinical drugs, cisplatin and doxorubicin. To our knowledge, complex 1 is the first metal-based KDM5A inhibitor reported in the literature. We anticipate that complex 1 may be used as a novel scaffold for the further development of more potent epigenetic agents against cancers, including TNBC. Show less

Metallo prodrugs that take advantage of the inherent acidity surrounding cancer cells have yet to be developed. We report a new class of pH-activated metallo prodrugs (pHAMPs) that are activated by light- and pH-triggered ligand dissociation. These ruthenium complexes take advantage of a key characteristic of cancer cells and hypoxic solid tumors (acidity) that can be exploited to lessen the side effects of chemotherapy. Five ruthenium complexes of the type [(N,N)₂Ru(PL)]²⁺ were synthesized, fully characterized, and tested for cytotoxicity in cell culture (1_A: N,N = 2,2'-bipyridine (bipy) and PL, the photolabile ligand, = 6,6'-dihydroxybipyridine (6,6'-dhbp); 2_A: N,N = 1,10-phenanthroline (phen) and PL = 6,6'-dhbp; 3_A: N,N = 2,3-dihydro-[1,4]dioxino[2,3-f][1,10]phenanthroline (dop) and PL = 6,6'-dhbp; 4_A: N,N = bipy and PL = 4,4'-dimethyl-6,6'-dihydroxybipyridine (dmdhbp); 5_A: N,N = 1,10-phenanthroline (phen) and PL = 4,4'-dihydroxybipyridine (4,4'-dhbp). The thermodynamic acidity of these complexes was measured in terms of two pK_a values for conversion from the acidic form (X_A) to the basic form (X_B) by removal of two protons. Single-crystal X-ray diffraction data is discussed for 2_A, 2_B, 3_A, 4_B, and 5_A. All complexes except 5_A showed measurable photodissociation with blue light (λ = 450 nm). For complexes 1_A-4_A and their deprotonated analogues (1_B-4_B), the protonated form (at pH 5) consistently gave faster rates of photodissociation and larger quantum yields for the photoproduct, [(N,N)₂Ru(H₂O)₂]²⁺. This shows that low pH can lead to greater rates of photodissociation. Cytotoxicity studies with 1_A-5_A showed that complex 3_A is the most cytotoxic complex of this series with IC₅₀ values as low as 4 μM (with blue light) versus two breast cancer cell lines. Complex 3_A is also selectively cytotoxic, with sevenfold higher toxicity toward cancerous versus normal breast cells. Phototoxicity indices with 3_A were as high as 120, which shows that dark toxicity is avoided. The key difference between complex 3_A and the other complexes tested appears to be higher uptake of the complex as measured by inductively coupled plasma mass spectrometry, and a more hydrophobic complex as compared to 1_A, which may enhance uptake. These complexes demonstrate proof of concept for dual activation by both low pH and blue light, thus establishing that a pHAMP approach can be used for selective targeting of cancer cells. Show less

Targeting protein-protein interactions (PPIs) offers tantalizing opportunities for therapeutic intervention for the treatment of human diseases. Modulating PPI interfaces with organic small molecules has been found to be exceptionally challenging, and few candidates have been successfully developed into clinical drugs. Meanwhile, the striking array of distinctive properties exhibited by metal compounds renders them attractive scaffolds for the development of bioactive leads. Here, we report the identification of iridium(iii) compounds as inhibitors of the H-Ras/Raf-1 PPI. The lead iridium(iii) compound 1 exhibited potent inhibitory activity against the H-Ras/Raf-1 interaction and its signaling pathway in vitro and in vivo, and also directly engaged both H-Ras and Raf-1-RBD in cell lysates. Moreover, 1 repressed tumor growth in a mouse renal xenograft tumor model. Intriguingly, the Δ-enantiomer of 1 showed superior potency in the biological assays compared to Λ-1 or racemic 1. These compounds could potentially be used as starting scaffolds for the development of more potent Ras/Raf PPI inhibitors for the treatment of kidney cancer or other proliferative diseases. Show less

A novel iridium(III) complex was synthesized and evaluated for its ability to target JMJD2 enzymatic activity. The iridium(III) complex 1 can inhibit JMJD2 activity and was selective for JMJD2 activity over JARID, JMJD3, and HDAC activities. Moreover, 1 suppressed the trimethylation of the p21 promoter on H3K9me3 and interrupted the JMJD2D-H3K9me3 interactions in human cells, suggesting that it could act as an epigenetic modulator. To our knowledge, 1 represents the first metal-based JMJD2 inhibitor reported in the literature. Show less

Group 9 transition metal complexes have been widely explored as therapeutic agents due to their unique geometry, their propensity to undergo ligand exchanges with biomolecules and their diverse steric and electronic properties. These metal complexes can offer distinct modes of action in living organisms compared to carbon-based molecules. In this study, we investigated the antimicrobial and anti-proliferative abilities of a series of cyclometallated iridium(III) complexes. The iridium(III) complex 1 inhibited the growth of S. aureus with MIC and MBC values of 3.60 and 7.19 μM, respectively, indicating its potent bactericidal activity. Moreover, complex 1 also exhibited cytotoxicity against a number of cancer cell lines, with particular potency against ovarian, cervical and melanoma cells. This cyclometallated iridium(III) complex is the first example of a substitutionally-inert, Group 9 organometallic compound utilized as a direct and selective inhibitor of S. aureus. Show less

We have previously showed that platinum drugs up-regulate SSAT and SMO and down-regulate ODC and SAMDC in the polyamine pathway. Several studies including our own established that platinum drugs combined with polyamine analog DENSPM produces synergistic increase in SSAT activity with polyamine depletion. Since polyamine pathway is an important therapeutic target, we investigated whether agents containing both platinum and polyamines have similar effects on the polyamine pathway. Two complexes i) Pt-spermine with two cisplatin molecules linked to a spermine in the center and ii) Pd-spermine with similar structure i, but Pd (II) substituted for Pt (II) were analyzed with respect to their effect on the expression of genes in polyamine pathway, SSAT and SMO protein expression, SSAT activity and polyamine pools. Pt-, Pd-spermine complexes induced significant down-regulation of SMO, arginase 2 and NRF-2, with no change in SSAT, while cisplatin as a single agent or in combination with DENSPM induced significant up-regulation of SSAT and SMO. The SSAT activity was not induced by either Pt- or Pd-spermine in A2780 cells; SMO protein levels were significantly elevated compared to the no-drug control and to a similar extent as cisplatin/DENSPM. The Pd-spm treatment induced a fall in putrescine levels to 33%, spermidine to 62% and spermine to 72% while Pt-spm did not induce such a decline. Comparative cytotoxicity studies in A2780 cells indicated the potency to be cisplatin> Pd-Spm>Pt-Spm. Although both complexes exhibit a lower potency, the degree of resistance itself is much lower for Pt-spermine and Pd-spermine in that order (2.5 and 7.5, respectively) compared to cisplatin ( approximately 12) as tested in cisplatin resistant A2780/CP cells. These studies suggest that Pd (II)-polyamine complexes may constitute a promising group of inorganic compounds for further studies in the development of novel chemotherapy/adjuvant chemotherapy strategies. Show less

[Ru(tBu2bpy)2(2-appt)](PF6)2 [1.(PF6)2, tBu2bpy = 4,4'-di-tert-butyl-2,2'-bipyridine, 2-appt = 2-amino-4-phenylamino-6-(2-pyridyl)-1,3,5-triazine] and [Re(CO)3(2-appt)Cl] (2) were prepared and characterized by X-ray crystal analysis. The binding of 1.(PF6)2 and 2 to calf thymus DNA (ct DNA) led to increases in the DNA melting temperature (Delta Tm = +12 degrees C), modest hypochromism (29% and 5% of the absorption bands at lambda max = 450 and 376 nm, respectively), and insignificant shifts in the absorption maxima. The binding constants of 1.(PF6)2 and 2 with ct DNA, as determined by absorption titration, are (8.9 +/- 0.5) x 104 and (3.6 +/- 0.1) x 104 dm3 mol-1, respectively. UV-vis absorption titration, DNA melting studies, and competition dialysis using synthetic oligonucleotides [poly(dA-dT)2 and poly(dG-dC)2] revealed that 1.(PF6)2 and 2 exhibit a binding preference for AT sequences. A modeling study on the interaction between 1 or 2 and B-DNA revealed that the minor groove is the most favored binding site and an extensive hydrogen-bonding network is formed. As determined by MTT assays, 1.(PF6)2 and 2 exhibited moderate cytotoxicities toward several human cancer cell lines (KB-3-1, HepG2, and HeLa), as well as a multi-drug-resistant cancer cell line (KB-V-1). According to confocal microscopic and flow cytometric studies, 1.(PF6)2 and 2 induced apoptosis (50-60%) in cancer cells with <5% necrosis detected. Show less