Reducing NSDs activity through specific lysine-HMTase inhibitors appears promising to help suppressing cancer growth.

Over the last few months, we have been doing extensive virtually ligand screening of thousands of selected compounds along with a large number of HMTase assays. Here a short video of the top 10 molecules that tightly bind (inhibit) the histone lysine binding pocket of the SET domain of NSD2/MMSET/WHSC1, calculated in an “open-conformation (able to dock the histone lysine H3K36).

Cancer initiation and progression are controlled by both genetic and epigenetic events. Both genetic and epigenetic alterations of transcriptional co-regulators are key features in carcinogenesis onset with aberrant gene functions and changes in gene expression levels.

Histone modifications along with other epigenetic mechanisms such as DNA methylation maintain gene activity states and are key in regulating a wide range of cellular processes. Alterations and deregulations in the function of enzymes that modify histones alter the array and levels of histone marks and ultimately affect the control of chromatin-based processes. It leads to dramatic changes in gene expression profiles, which eventually contribute to oncogenic transformation and the development of cancer.

Histones are the stage of multiple post-translational modifications. Specific residues on histones H2A / H2B, H3 and H4 can be modified by methylation (Lysine / Arginine), acetylation (Lysine), citrullination (Arginine), phosphorylation (Serine / Threonine), ubiquitination (Lysine), sumoylation (Lysine), ADP-ribosylation (Lysine), butyrylation (Lysine), propionylation (Lysine) and glycosylation (Serine / Threonine).

Amongst the array of covalent histone modifications, lysine methylation is one of the prominent signaling pathway in chromatin-regulatory mechanism. Lysine-histone methyltransferases (HMTases) are transcriptional co-regulators that target specific lysines on H3 and H4, and can transfer up to three methyl groups (Kme1, Kme2, and Kme3) on histone tails. Lysine methylation, or any of the other histone modifications, can have both activating and repressive functions on transcription events. All the covalent histone modifications contribute to finely regulating the diverse activities associated with the chromatin and may be referred as a language of covalent histone modifications or histone code that is still obscure.

One fascinating aspect of the regulation of the transcription lies in the ballet between histones modifiers “readers” and “writers”, both being regulated leading to various physiological output based on the cellular context. This appends complexity to our current limited understanding of gene transcription and its implication in human diseases.

Background: The nuclear receptor binding SET domain (NSD) protein is a family of three histone-lysine N-methyltransferase (HMTase), NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1 that are critical in maintaining the chromatin integrity. NSD1 methylates H3K36 and H4K20 and is associated with acute myeloid leukemia, multiple myeloma, and lung cancer. The NSD1-NUP98 translocation plays a significant role in childhood acute myeloid leukemia with NUP98-NSD1 being an active H3K36 methylase. NSD1 is amplified in multiple myeloma, lung cancer, neuroblastomas and glioblastomas. NSD2 methylates H3K36 and is linked to prostate cancer and multiple myeloma. Over expression of NSD2 in myeloma cells leads to aberrantly high levels of H3K36 di-methylation, accompanied by a decrease in H3K27 methylation. NSD2 is found over expressed in fifteen different cancers and is associated with tumor aggressiveness or prognosis in most types of cancers. NSD3 methylates H3K36 and is associated with both lung and breast cancers along with the acute myeloid leukemia. The amplification of either NSD1 or NSD2 triggers the cellular transformation. NSD3 is found amplified in breast cancer cell lines and primary breast carcinomas. Reducing NSDs activity through specific and selective lysine-HMTase inhibitors appears promising to help suppressing cancer growth.

Little is known about the NSD pathways and our understanding of the histone Lysine-HMTase mechanism is partial. The SET domain of NSD1 has specific mechanisms to recognize histone marks unlike other HMTase. The precise catalytic activities of the NSDs are obscure and discrepancies exist hindering progress in understanding their exact biological functions and pathways in cancer pathogenesis. In this study, we explored the in vitro catalytic activities on histone substrates to understand the substrate recognition and to pave the way for the design of selective and specific NSD inhibitors usable in cancer therapies.

Methods: We used both biochemical and computational methods to understand the substrates recognition by the NSDs and to investigate the structural mechanisms happening in the SET domain during the binding of histone tails.

Results: A key regulatory and a recognition mechanism is driven by the flexibility of a loop at the interface of the SET and postSET region who rotates ~45° and translated 7Å opening the SET domain for the binding of the peptide ligand. This regulatory loop acts as a seat belt for the ligand and contributes to the discrimination and the substrate specificity. In vitro, The SET domain of the NSDs favor H3 recognition and are able to methylate a range of substrate. To reconcile with the in vivo activities previously reported on H3K36 and H4K20, we propose a cross-talk mechanism controlling the substrate recognition.

A photo of Lennart’s crystal photos of PRMT5, a 72Kda human argine-methyltransferase. This is one of the hits we obtained and we are currently optimizing the crystallization conditions.

Thought of You from Ryan J Woodward on Vimeo.

My image is the hexameric assembly of the Quinolinate phosphoribosyl transferase enzyme (BNA6) from S. cerevisiae. Quinolinic acid phosphoribosyl transferase (QAPRTase, EC 2.4.2.19) is a 32 kDa enzyme encoded by the BNA6 gene in yeast and catalyzes the  formation of nicotinate mononucleotide from quinolinate and 5-phosphoribosyl-1-pyrophosphate (PRPP). QAPRTase plays a key role in the tryptophan degradation pathway via kynurenine, leading to the de novo biosynthesis of NAD and clearing the neurotoxin quinolinate. The image shows the hexameric surface with electrostatic properties. The structure was solved by APBS and visualized and rendered with VMD 1.8.7.  The electrostatic field lines are displayed and are “emerging” out of the 3 active sites.

From my 2008 paper “Comprehensive x-ray structural studies of the quinolinate phosphoribosyl transferase (BNA6) from Saccharomyces cerevisiae”; E. di Luccio, and D.K. Wilson, (2008) Biochemistry, 47(13); 4039-50. Epub Mar 6, 2008.

Link to Biophysical Society website.

Unfortunately, the heated debate between “experimentalists” and bioinformaticians is far from being over. In a nutshell, the biology world can be roughly divided into 3 worlds. The experimentalists doing only wet biology occupy the first category. They trust only what is inside an Eppendorf tube. At the opposite, we found the second category: the bioinformatics world. The bioinformaticians put into equations what the experimentalists found. Then the bioinformaticians try to understand, and model with an ever-increasing accuracy any biological elements. Their work can be a genomic analysis or predicting the 3D structure of a protein among others. The third category of scientist sits in the middle. They believe that using both wet and dry biology is the right combo to better understand everything.

A fringe of hardcore experimentalist does not believe in all the bioinformatics tools and their power to answer some fundamental questions. Of course, that leads to endless debates amongst scientists. *Yawn* The main argument of experimentalists is “A computer program can’t explain or predict a biological phenomena because there are too many unknowns. If you don’t do wet biology/biochemistry in a tube, it isn’t science. ” Well…sure…Last time I checked, there is a descent number of BS analysis coming from experimentalists. Right? Also, there are numbers of wrong analysis using only bioinformatics too. *Facepalm right here*

As for myself, I am a strong advocate of using the best of both worlds. For instance in structural biology, an X-ray structure gives an accurate snapshot of a 3D structure…but that’s it. What about the flexibility during functions? What about the domain motions? How to accurately understand the mechanism of an enzyme? How does the substrate enter the active site? How the product leaves? How does a protein interact with a partner?

There is a huge gap between the world of known structures and the universe of known protein sequences. Structural genomic projects are unable to keep up with newly discovered genes. How to accurately gain access to nearly all of the 3D protein structure of the whole proteome? Those are just few questions among many more.

Bioinformatics offer many great tools to answers all those questions as long as they are properly used and validated. Unfortunately, some scientists don’t agree with that.  Well! This is 2010, people. It is time to embrace the 30+ years of steady developments in both computer science and bioinformatics and to apply them to biology, broadly define. In science, most of the “easy” stuff has been already unravel. What is left? More difficult and complex problems. In order to make significant contributions in Science, one has to cross boundaries with mixed methods approach. Inter-disciplinary is the way to go!

Here some of the figures of my grant.

Zinc fingers is typically a domain of about 60 amino acids that fold around one or more zinc ions and is found in over 400 eukaryotic proteins, many of which are involved in the regulation of gene expression and in the maintenance of chromatin structure. Zinc fingers typically show a C4HC3 signature (four cysteines, one histidine, three cysteines) with characteristic cysteine spacing and with additional conserved residues, most notably a tryptophan or other aromatic amino acid preceding the final cysteine pair. Studies have suggested a role for zinc fingers as nucleosome interaction determinants. However their functions are still elusive and controversial, as a variety of functions have been suggested, including phosphoinositide binding and E3 ubiquitin ligase activity. In addition to their role as a DNA-binding module,  zinc finger have been shown to mediate protein-protein and protein-lipid interactions as well.

What about the electrostatic surface properties of zinc-finger domains? Here an example with the models of the 4 zinc-fingers of one of the histone methyl-transferase I’m working on. On the figures, blue is positively charged, and red is negative. The large positive (blue) area will bind to the DNA. But, on the other face, there is room for binding to some positively charged partners. Fascinating!

The climb to cruising altitude was really smooth, thanks to the temperature inversion. However the downside of a temperature inversion is bad to poor visibility of such air masses. After few minutes I was overflying Winters and decided to head north for a little while. No other aircrafts were around me and the radio was quiet. Perfect!

After some time, I headed back to Davis and started my descent while turning over Winters. I overflew KEDU at 1600ft then descended a bit to 1400ft to stay underneath SMF airspace. After few moments of flying south Davis, I overflew the city to take some photos of downtown Davis. Not easy to fly with one hand and take pictures with the other.

As usual I had a great time. I can’t get enough of that!