Breaking News
May 3, 2019 - Vaping and Smoking May Signal Greater Motivation to Quit
May 3, 2019 - Dementia looks different in brains of Hispanics
May 3, 2019 - Short-Staffed Nursing Homes See Drop In Medicare Ratings
May 3, 2019 - Study of teens with eating disorders explores how substance users differ from non-substance users
May 3, 2019 - Scientists develop new video game that may help in the study of Alzheimer’s
May 3, 2019 - Arc Bio introduces Galileo Pathogen Solution product line at ASM Clinical Virology Symposium
May 3, 2019 - Cornell University study uncovers relationship between starch digestion gene and gut bacteria
May 3, 2019 - How to Safely Use Glucose Meters and Test Strips for Diabetes
May 3, 2019 - Anti-inflammatory drugs ineffective for prevention of Alzheimer’s disease
May 3, 2019 - Study tracks Pennsylvania’s oil and gas waste-disposal practices
May 3, 2019 - Creating a better radiation diagnostic test for astronauts
May 3, 2019 - Vegans are often deficient in these four nutrients
May 3, 2019 - PPDC announces seed grants to develop medical devices for children
May 3, 2019 - Study maps out the frequency and impact of water polo head injuries
May 3, 2019 - Research on Reddit identifies risks associated with unproven treatments for opioid addiction
May 3, 2019 - Good smells may help ease tobacco cravings
May 3, 2019 - Medical financial hardship found to be very common among people in the United States
May 3, 2019 - Researchers develop multimodal system for personalized post-stroke rehabilitation
May 3, 2019 - Study shows significant mortality benefit with CABG over percutaneous coronary intervention
May 3, 2019 - Will gene-editing of human embryos ever be justifiable?
May 3, 2019 - FDA Approves Dengvaxia (dengue vaccine) for the Prevention of Dengue Disease in Endemic Regions
May 3, 2019 - Why Tonsillitis Keeps Coming Back
May 3, 2019 - Fighting the opioid epidemic with data
May 3, 2019 - Maggot sausages may soon be a reality
May 3, 2019 - Deletion of ATDC gene prevents development of pancreatic cancer in mice
May 2, 2019 - Targeted Therapy Promising for Rare Hematologic Cancer
May 2, 2019 - Alzheimer’s disease is a ‘double-prion disorder,’ study shows
May 2, 2019 - Reservoir bugs: How one bacterial menace makes its home in the human stomach
May 2, 2019 - Clinical, Admin Staff From Cardiology Get Sneak Peek at Epic
May 2, 2019 - Depression increases hospital use and mortality in children
May 2, 2019 - Vicon and NOC support CURE International to create first gait lab in Ethiopia
May 2, 2019 - Researchers use 3D printer to make paper organs
May 2, 2019 - Viral infection in utero associated with behavioral abnormalities in offspring
May 2, 2019 - U.S. Teen Opioid Deaths Soaring
May 2, 2019 - Opioid distribution data should be public
May 2, 2019 - In the Spotlight: “I’m learning every single day”
May 2, 2019 - 2019 Schaefer Scholars Announced
May 2, 2019 - Podcast: KHN’s ‘What The Health?’ Bye-Bye, ACA, And Hello ‘Medicare-For-All’?
May 2, 2019 - Study describes new viral molecular evasion mechanism used by cytomegalovirus
May 2, 2019 - SLU study suggests a more equitable way for Medicare reimbursement
May 2, 2019 - Scientists discover first gene involved in lower urinary tract obstruction
May 2, 2019 - Researchers identify 34 genes associated with increased risk of ovarian cancer
May 2, 2019 - Many low-income infants receive formula in the first few days of life, finds study
May 2, 2019 - Global study finds high success rate for hip and knee replacements
May 2, 2019 - Taking depression seriously: What is it?
May 2, 2019 - With Head Injuries Mounting, Will Cities Put Their Feet Down On E-Scooters?
May 2, 2019 - Scientists develop small fluorophores for tracking metabolites in living cells
May 2, 2019 - Study casts new light into how mothers’ and babies’ genes influence birth weight
May 2, 2019 - Researchers uncover new brain mechanisms regulating body weight
May 2, 2019 - Organ-on-chip systems offered to Asia-Pacific regions by Sydney’s AXT
May 2, 2019 - Adoption of new rules drops readmission penalties against safety net hospitals
May 2, 2019 - Kids and teens who consume zero-calorie sweetened beverages do not save calories
May 2, 2019 - Improved procedure for cancer-related erectile dysfunction
May 2, 2019 - Hormone may improve social behavior in autism
May 2, 2019 - Alzheimer’s disease may be caused by infectious proteins called prions
May 2, 2019 - Even Doctors Can’t Navigate Our ‘Broken Health Care System’
May 2, 2019 - Study looks at the impact on criminal persistence of head injuries
May 2, 2019 - Honey ‘as high in sugars as table sugar’
May 2, 2019 - Innovations to U.S. food system could help consumers in choosing healthy foods
May 2, 2019 - FDA Approves Mavyret (glecaprevir and pibrentasvir) as First Treatment for All Genotypes of Hepatitis C in Pediatric Patients
May 2, 2019 - Women underreport prevalence and intensity of their own snoring
May 2, 2019 - Concussion summit focuses on science behind brain injury
May 2, 2019 - Booker’s Argument For Environmental Justice Stays Within The Lines
May 2, 2019 - Cornell research explains increased metastatic cancer risk in diabetics
May 2, 2019 - Mount Sinai study provides fresh insights into cellular pathways that cause cancer
May 2, 2019 - Researchers to study link between prenatal pesticide exposures and childhood ADHD
May 2, 2019 - CoGEN Congress 2019: Speakers’ overviews
May 2, 2019 - A new strategy for managing diabetic macular edema in people with good vision
May 2, 2019 - Sagent Pharmaceuticals Issues Voluntary Nationwide Recall of Ketorolac Tromethamine Injection, USP, 60mg/2mL (30mg per mL) Due to Lack of Sterility Assurance
May 2, 2019 - Screen time associated with behavioral problems in preschoolers
May 2, 2019 - Hormone reduces social impairment in kids with autism | News Center
May 2, 2019 - Researchers synthesize peroxidase-mimicking nanozyme with low cost and superior catalytic activity
May 2, 2019 - Study results of a potential drug to treat Type 2 diabetes in children announced
May 2, 2019 - Multigene test helps doctors to make effective treatment decisions for breast cancer patients
May 2, 2019 - UNC School of Medicine initiative providing unique care to dementia patients
May 2, 2019 - Nestlé Health Science and VHP join forces to launch innovative COPES program for cancer patients
May 2, 2019 - Study examines how our brain generates consciousness and loses it during anesthesia
May 2, 2019 - Transition Support Program May Aid Young Adults With Type 1 Diabetes
May 2, 2019 - Study shows how neutrophils exacerbate atherosclerosis by inducing smooth muscle-cell death
May 2, 2019 - Research reveals complexity of how we make decisions
Uncovering the Structure of HIV Integrase to Inform Drug Discovery

Uncovering the Structure of HIV Integrase to Inform Drug Discovery

In this interview Dr Kushol Gupta, Research Assistant Professor in the Department of Biochemistry and Biophysics at the Perelman School of Medicine, University of Pennsylvania talks to News-Medical and Life Sciences about the importance of light scattering techniques for investigating structure and mechanism of action in his ongoing research into HIV integrase and the development of HIV therapeutics.

Which instrumentation do you consider key to your research?

In the John Foundation Core, we rely upon an array of state-of-the-art instrumentation. All the techniques we use come together and complement each other to provide a deeper understanding of structure and function.

Light scattering measurements are key enablers of our ongoing research. In fact, the most popular and productive workhorses of our toolbox, day to day, are our light scattering instruments, all made by Wyatt Technology.  These include our Dynapro Nanostar dynamic light scattering (DLS) instrument; our SEC-MALS components, the DAWN multi-angle light scattering instrument and the Optilab refractive index detector coupled to size-exclusion chromatography; and our most recent acquisition, our CG-MALS (composition-gradient multi-angle light scattering) instrument, the Calypso.

Over the past decade, the team’s many papers and funded studies have all incorporated some variety of light scattering.

What exactly are these light scattering techniques, and when do you use them?

MALS directly measures the level of light scattering that occurs as a result of the molecular mass of a biopolymer, enabling the independent measurement of its properties in solution. The mass, and consequently the amount of scattered light, increases due to macromolecular interactions between two molecules, such as protein and protein or a protein and a nucleic acid. The phenomenon can be as simple as two monomers that form a dimer or more complex events like polymerization and protein aggregation.

Dynamic light scattering, or DLS, measures the fluctuations in intensity of scattered light that arise from a molecule’s Brownian motion. The signals are analysed to determine the translational diffusion coefficient and hence the molecule’s size, or hydrodynamic radius.

DLS and MALS have become day-to-day prerequisites for success in the study of biological macromolecules. For those using de-novo techniques like cryo-electron microscopy, X-ray crystallography, or NMR, routine analysis and batch-to-batch validation of recombinant preparations are critical steps towards successful structure determination.

For enzymologists characterizing the activity and inhibition of recombinant preparations, batch to batch validation is key to accurate findings and the assessment of lead targets. For a small angle x-ray and neutron scattering scientist  like myself, it is almost impossible to reliably interpret experimental results without these key experimental measures.

I personally have performed several hundred SEC-MALS experiments here at Penn over the last decade in a wide array of investigations, including protein-nucleic acid interactions, protein-protein interactions in the context of RNA splicing, HIV biology and drug discovery, site-specific recombination and chromatin structure.

How does SEC-MALS work?

SEC-MALS is size-exclusion chromatography in line with multi-angle light scattering. In biochemistry and structural biology studies it is one of the most popular implementations of light scattering.

The samples of interest are passed through a standard chromatography column to separate the macromolecules according to their size. The eluent from this separation then enters into a multi-angle light scattering instrument where there are multiple angles of light scattering recorded every second. The differential refractive index detector can be used to directly determine the concentration of the macromolecule at each time point.

Molecular mass can then be determined from the light scattering and concentration data, without the need for any standard curves or theoretical extinction coefficients. The analysis is independent of retention time and any assumptions about conformation or column interactions.

Can you tell us more about your HIV research?

The HIV AIDS pandemic remains a major global public health problem with 37 million people worldwide currently infected.  It is projected that 30 million people will be taking anti-retroviral regimens by 2020. However, treatment efforts are being hampered by the development of viral resistance to current antiretroviral drugs so there is a need for novel therapeutics.

FDA-approved anti-HIV drugs are strand transfer inhibitors (STIs) that block the integration of HIV DNA into the host genome to form a provirus. The newest generation of these compounds are now being optimized to counteract emerging drug resistance and improve drug half-life.

My primary area of research is the HIV integrase enzyme, which—among other things—catalyses strand transfer. Integrase is one of three enzymes encoded by the HIV genome. It is a 32-kilodalton, three-domain protein.

The most distinguishing feature of the recombinant HIV integrase protein is its propensity to self-associate and form oligomers, which makes its structure difficult to study. Although partial crystal structures are available, the full-length protein has eluded investigators worldwide for many years.

Image Credit:Shutterstick/BioMedical

How does SEC-MALS contribute to such research?

The application of small angle scattering techniques based on x-rays and neutrons, SAXS and SANS, to structural biology and solution biophysics has increased exponentially over the past 15 years. They provide rich information, and are among the key tools we use to learn about molecular structure.

In these approaches, a columnated beam of either x-rays or experimental neutrons is directed at the sample of macromolecules and the intensity of scattering is plotted as a function of the scattering angle.

Using a Fourier transform analysis, we can gain insight into the shape and spatial extent of the molecule. This information can then be directly compared with profiles of known atomic structures to confirm structure and composition in solution.

However, if a sample is even slightly contaminated or partially oligomerized, the interpretation of these results is greatly compromised. This is where characterization and quality control by SEC-MALS have been critical to the growth and application of small-angle scattering techniques, since SAXS and SANS tend to involve expensive beam time and travel.

Among other benefits, SEC-MALS is far more sensitive to the presence of large aggregates than SEC-UV.  In the case of a 440 kDa, Lactocous Lactii Group II intron protein-RNA complex, it detected low levels of large aggregates not detectable by UV. As a result we performed an additional purification step, finally enabling successful SAXS measurements and determination of high resolution cryo-EM structures.

SEC-MALS is key to the success of study of HIV integrase host factor interactions using small angle x-ray scattering. It enabled us to screen out many mutated and truncated forms of recombinant integrase complexes, so we can obtain high quality data of the complex of interest. This was highlighted in our study of the interaction between integrase and host lens epithelium-derived growth factor (LEDGF), which is an important part of the viral life cycle.

Similarly, in our 2012 bifocal study, SEC-MALS analyses were critical to the characterization of a related retroviral integrase protein-DNA complex. We confirmed using SEC-MALS that the integrase protein binds to viral DNA ends with a stoichiometry of four integrase to two DNA. SEC-MALS enabled us to optimize buffer conditions and limit aggregation.

Image Credit:Shutterstock/Explode

Has SEC-MALS has been critical to the success of your research?

Absolutely. In our most recent structural studies of the interaction of HIV integrase with the complex new class of drugs known as ALLINIS, SEC-MALS along with other techniques provided the breakthrough needed to obtain the full-length protein structure for the first time. This in turn enabled us the investigate the protein-protein interactions that were revealed by our new structure.

The ALLINIs are potent allosteric inhibitors of HIV integrase that are now being evaluated in clinical trials. They were identified though study of the crystal structures of the LEDGF-integrase interaction. It became apparent that small molecules could block this interaction and prevent HIV replication very early in the process.

The LEDGF binding site on the integrase protein was identified from the structure we obtained with the help of SEC-MALS, and molecules that prevent this interaction from occurring were developed using structure-based design. Potential drug candidates were selected through screening for the ability to disrupt the binding of the host factor LEDGF to integrase.

What do your studies with ALLINIS show?

Our studies focus on two ALLINIS (GSK1264 and GSKOO2). Both compounds, share a common linkage connecting a substituted central aromatic ring system. And x-ray crystallography has shown that they both bind to the same site on the catalytic domain of integrase with high affinity.

Both molecules have potent late-replication effects that dramatically disrupt HIV particle organization and alter the distribution of morphology of the viral particles. This is linked to their ability to catalyse the aggregation of HIV integrase independently of temperature and concentration. Such aggregation was monitored by SEC-MALS and shown to require both the catalytic core domain and the C-terminal domain of integrase.

We wanted to know how these drugs worked at the structural level. What are the structural changes that lead to drug-induced integrase aggregation?

To discover this, we needed the structure of the enzyme-drug complex. Since the drug promotes the aggregation of an already marginally soluble protein, this was not conducive to obtaining an ordered crystal lattice for analysis by x-ay crystallography. Instead, we set out to characterize numerous point mutations of the complex using multi-angle light scattering, analytical ultracentrifugation, small-angle x-ray scattering (SAX) to identify which forms of the integrase protein were less responsive to the ALLINIs.

Image Credit:Shutterstock/Sciencepics

Using both SEC-MALS and sedimentation equilibrium analysis, we discovered mutations of integrase that existed solely as dimers. Drug-induced polymerization was greatly attenuated even at elevated drug concentrations. For the first time we were able to crystallize the full-length, intact protein complexed with an ALLINI.

We were able to solve the structure using a molecular replacement research model for the previously determined structure of the catalytic core domain and the C-terminal domain. Extensive protein-protein interactions were observed that have not been seen with any other integrase form. The C terminal domain from one dimer reaches across to the core domain of a neighbouring dimer, much akin to a hand gripping a ball.

Mutations of integrase that conferred resistance to ALLINIS fell into two categories. They either directly disrupted the binding interface or perturbed drug positioning preventing it from binding. By introducing specific integrase mutations, we were able to confirm our new understanding of this drug-stabilized protein-protein interface; again, using SEC-MALS to determine oligomeric forms, in combination with other techniques.

We have conducted a range of SEC-MALS studies to further explore the structure of the integrase-ALLINI complex. The most surprising finding, as underscored by our new crystal structure, is the anticipated polymerization and late replication effects. The ALLINIs were selected and designed to interrupt protein-protein interactions between LEDGF and integrase, yet our results show that the drugs very efficiently stabilize an open polymer of integrase.

These results together provide insights into drug design approaches for this new class of drugs, just entering clinical trials.

Where can our readers go to find out more?

To find out more please visit

About Professor Kushol Gupta

Dr Kushol Gupta is a Research Assistant Professor in the Department of Biochemistry and Biophysics at the Perelman School of Medicine, University of Pennsylvania. He also serves as a director of the Johnson Foundation of structural biology and biophysics core.

Dr Gupta’s research focuses on HIV integrase and the antiviral effects of small molecules that target this protein. The use of multiple biophysical techniques, including x-ray crystallography, small angle x-ray scattering, light scattering, and analytical ultra-centrifugation, provides a deeper understanding of the structure and function of HIV integrase, more effective screening of anti-HIV integrase compounds, and a complete picture of the therapeutic mechanism of potential drug candidates.

Tagged with:

About author

Related Articles