Since interconversion of thalidomide enantiomers could occur under physiological conditions, special precautions are required to delineate any enantiomer-specific differences in the biological activity of thalidomide.
To this end, we synthesized each thalidomide enantiomer with deuterium substitution of the hydrogen atom bonded to the chiral carbon atom C3, Fig. Deuterated thalidomides have been found to be at least five times more stable than thalidomide with respect to racemization We initially compared the CRBN-binding affinity of the enantiomers in a competitive binding assay with each deuterated thalidomide enantiomer chemically coupled to ferrite beads Fig. CRBN bound to the beads was eluted by the addition of deuterated S - or R - thalidomide in a concentration-dependent manner.
This is consistent with a previous demonstration that non-deuterated S -thalidomide binds CRBN more tightly than the R -enantiomer by competition for thalidomide-immobilized beads The aforementioned conclusion is further supported by results of a cell-based assay, in which deuterated S -thalidomide inhibited the auto-ubiquitylation activity of the CRBN-containing ubiquitin ligase complex more strongly than deuterated R -thalidomide Fig.
Considering their half-lives of racemization, which occurs in a matter of hours with non-deuterated thalidomide under physiological conditions 11 , 12 , 18 , 19 , 21 , 24 , 25 , only a very small fraction of each enantiomer is thought to be racemized during the assay. To confirm this hypothesis, we analyzed the enantiomeric purities of all the thalidomides used before and after the experiments. Supplementary Table 1 and Supplementary Fig. No significant racemization was detected for the non-deuterated thalidomides and no racemization was detected for the deuterated thalidomides.
These results are consistent with a previous report indicating that the racemization of thalidomide is slow under lower or neutral pH conditions, while the racemization is accelerated under higher pH values The half-life of racemization of S -thalidomide was The pH values of the medium solutions used in the current experiments were 5.
Atom numbering is shown in the S -D-Thal chemical structure. The hydrogen atom at the chiral centre C3 atom of the glutarimide moiety is substituted with a deuterium atom. Full-length blots in b and c are presented in Supplementary Fig.
IB, immunoblotting. IP, immunoprecipitation. Full-length blots in a and b are presented in Supplementary Fig. These results collectively suggest that differences in the pharmacological activity of the enantiomers are determined, at least in part, by their differential binding affinity to CRBN.
On the other hand, the effect by deuterated R -thalidomide on the binding was very weak. These results support the notion that the binding affinity of CRBN to the S -enantiomer was stronger than that to the R -enantiomer and, that the difference in binding affinity affects degradation of the down-stream substrates. Both structures gave clear electron densities for the bound thalidomide molecules Fig. S -thalidomide binds this pocket so that the glutarimide ring is docked into the tri-Trp pocket, and the phthalimido group is located outside of the pocket Fig.
Inside the pocket, the glutarimide ring, which is sandwiched between Trp and Trp, makes nonpolar contacts with Trp forming the floor of the pocket and forms two hydrogen bonds with the protein: the glutarimide 6-carbonyl group forms a hydrogen bond to the main-chain amide of Trp and the glutarimide 1-imino group NH forms a hydrogen bond to the main-chain carbonyl group of His Fig.
These characteristics of the binding mode are identical to those observed for R -thalidomide Fig. No significant structural differences were found between the overall structures of TBDs bound to S - and R -thalidomide, as reflected in the extremely small root-mean-square rms deviation 0. Additionally, these structures are similar to the free forms 28 0. In addition to the similarity in the overall structures, the thalidomide-binding sites also exhibit high similarity without any significant structural deviation between the S - and R -thalidomide-bound forms Fig.
Side chains of residues at the tri-Trp pocket are shown as stick models. Residues forming the tri-Trp pocket are shown as stick models cyan for C, blue for N, and red for O.
The S -thalidomide molecule is shown as a stick model yellow for C, blue for N, and red for O. Two hydrogen bonds formed between the protein and the glutarimide moiety of thalidomide are represented as red dotted lines. The structural overlay shows no significant structural differences with a small root-mean-square rms deviation 0.
The rms deviation is 0. The rms deviation is 1. Middle , superposition of S - and R -thalidomide-bound tri-Trp pockets. Side chains of key residues forming the tri-Trp pocket are shown as stick models.
The observed high similarity in the structures of the thalidomide-binding sites, the binding modes, and overall TBD structures of our complexes imply that other factors are responsible for the differences in the binding affinity of S - and R -thalidomides.
The electron density maps of our complex structures are sufficiently clear to define the conformation of the bound thalidomide molecules Fig. We then examined the thalidomide conformation in the CRBN-bound state. Similarly, the 16 R -thalidomide molecules display essentially the same conformation, which is distinct from that of S -thalidomide as described below, and exhibit a small averaged rms deviation of 0. These results suggest that S - and R -thalidomide molecules bound to CRBN have a defined conformation but no variable conformations.
Thus, the phthalimide ring is locked at the pocket entrance and the rotational conformation of the phthalimide ring around the C-N bond between the phthalimide and glutarimide rings is the same in both enantiomers. Overlay of the bound S - and R -thalidomide molecules shows good overlap of the molecules with a relatively small shift in orientation of the phthalimide ring Fig. This shift is caused by the different nature of the glutarimide ring conformation.
The glutarimide ring of S -thalidomide displays a C4- endo puckered conformation, a relaxed and stable five-membered ring conformation, in which the C4-carbon atom is displaced from the plane formed by the rest of the ring atoms. This puckered form closely resembles that observed in the isolated free form of thalidomide 33 , 34 Fig. Compared to the free form, the CRBN-bound form has a small shift 0. Comparison of the bound thalidomide conformations.
The phthalimido 1-carbonyl group labelled with red 1 is docked into the groove formed by the side chains of Trp and Trp The phthalimido 3-carbonyl group labelled with red 3 is docked into the groove formed by Trp and His side chains. The van der Waals surfaces of the two carbonyl groups are shown with dots and labelled with red atom numbers. The grooves for docking with the phthalimido carbonyl groups are indicated with orange arrows.
S -thalidomide superimposed onto R -thalidomide with overlapping imide groups, which are linked to CRBN by direct hydrogen bonds and polar interactions.
The puckered C4 atoms are separated from each other by 1. The phthalimido groups are shifted from each other by a maximum of 1. CRBN-bound S -thalidomide yellow is superimposed onto the free form grey of S -thalidomide in the racemic thalidomide crystal 34 with imide groups overlapped. This conformation is similar to the glutarimide ring of free S -thalidomide, which displays a slightly twisted C4- endo puckered conformation.
The phthalimido group of the CRBN-bound form exhibits a displacement 0. This highly twisted conformation is in sharp contrast with free R -thalidomide grey in the racemic thalidomide crystal In sharp contrast to the relaxed form of the bound S -thalidomide, CRBN-bound R -thalidomide has the glutarimide ring in a twisted conformation, which is distinct from the stereochemically relaxed C4- exo puckered conformation of R -thalidomide Fig. The twisted conformation is forced primarily by the contacts between the phthalimide ring and the entrance of the tri-Trp pocket and accompanies a large displacement 1.
This displacement needs to avoid steric clash of the phthalimidocarbonyl group against the groove formed by Trp and Trp, and enables the protruding carbonyl groups to dock into the grooves Fig. To visualize the steric hindrance, we superimposed the free form of R -thalidomide onto the CRBN-bound form of R -thalidomide found in the crystal Fig.
We found that the C4- exo conformation of the free form causes serious steric clash 2. The twisted conformation of the bound R -thalidomide avoids this clash by shifting the phthalimide ring toward the endo direction 1.
Moreover, close inspection of the glutarimide ring puckering by modelling revealed that R -thalidomide in the C4- exo puckered glutarimide ring conformation causes steric clash of the C4-carbon atom against Trp To avoid this clash, the glutarimide ring is also forced to adopt a twisted conformation. All of these relatively small but significant differences in the binding mode between S - and R -thalidomide molecules suggest that the S -thalidomide bound state of the thalidomide-CRBN complex is more stable than the R -thalidomide bound state.
As with the previous structures 28 , 29 , our structure of thalidomide-bound CRBN TBD prepared with racemic thalidomide revealed that the bound thalidomide molecules are all S -enantiomers Supplementary Fig.
Comparison of R -thalidomides in the free form and in the CRBN-bound form found in the complex crystal. Yamamoto, T. Synthesis and configurational stability of S - and R -deuteriothalidomides.
Takeuchi, Y. Suzuki, E. Three-step synthesis of R - and S -thalidomides from ornithine. Enantiomer 6 , — Shibata, N. In Topics in Heterocyclic Chemistry , ed. Eguchi, S. Synthesis, configurational stability and stereochemical biological evaluations of S - and R hydroxythalidomides. Suzuki, S. Design and Synthesis of Thalidomide—Deoxyribonucleoside Chimeras. Lee, C. Fluorothalidomide: a characterization of maternal and developmental toxicity in rabbits and mice.
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Chirality 7 , 44—52 Clinical pharmacology of thalidomide. Caner, H. Trends in the development of chiral drugs. Drug Discov. Today 9 , — Nguyen, L. Chiral Drugs: An Overview. Hess, S. Synthesis and immunological activity of water-soluble Thalidomide prodrugs.
Download references. The authors would like to thank Ms. Yuka Suzuki for her contributions during the early stages of this work, and Mr. Toshiaki Honda for his help on the water solubility experiments. You can also search for this author in PubMed Google Scholar. Correspondence to Norio Shibata. Reprints and Permissions. Sci Rep 8, Download citation. Received : 17 August Accepted : 06 November Published : 20 November Anyone you share the following link with will be able to read this content:.
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Clinical pharmacology of thalidomide. Chirality in antirheumatic drugs. Molecule of the Day: Thalidomides. Two important lessons were learned: 1 Chiral drugs need to be evaluated as pure enantiomers because the different enantiomers can have completely different effects.
Birth Defects Research Part C —, Thalidomide was marketed and distributed in 46 countries around the world using different names. Thalidomide became one of the world's largest selling drugs, and was marketed heavily and advertised as completely safe Vargesson, ; Fig.
Indeed, sample packets of the drug were given out to physicians to distribute freely to patients suffering from morning sickness Fig. Precisely how many women were given the drug will never be known. Structure of thalidomide enantiomers and packaging. Both enantiomers, R and S, can rapidly interconvert racemize in body fluids and tissues and form equal concentrations of each form.
Note the safety advice on the packet. Reproduced from Vargesson, BioEssays, ,31,— Soon after thalidomide's release, reports surfaced of patients developing peripheral neuropathy after taking the drug. Reports of occurrences of severe birth defects affecting multiple body systems were also coming to light, that initially were not linked to, or were denied to be due to thalidomide. In addition, there were reports of increased miscarriage rates during this period Lenz, ; Vargesson, , ; McCredie, ; Stephens, Thalidomide was subsequently withdrawn from the UK in Nov and by from most of the world.
As a result, the incidence and occurrence of these severe birth defects was then not seen. Whether this disaster could have been prevented remains unclear. The packaging and information of contraindications of drugs was minimal when compared to the way drugs are sold today Fig. More importantly, the state of drug testing in the s and s was different when compared to today's standards.
Thalidomide was not approved or licensed for use in the USA between and , despite pressure to do so. An FDA physician, Frances Kelsey, responsible for approving drug licenses, had concerns about the safety of the drug, given the reports of peripheral neuropathy in patients taking the drug, but was also concerned about effects during pregnancy.
Kennedy for averting a thalidomide disaster in the USA Vargesson, , The thalidomide disaster completely changed the way drugs are tested. In fact, articles written by Dr Frances Kelsey provided the foundation for modern day drug testing Kelsey, ; Vargesson, Merker et al. Why mice are less sensitive to the drug remains unclear. Since the disaster, drug screening policies have changed to incorporate several species as well as in vitro tests, and there has not been a repeat of the disaster.
The evidence that thalidomide causes birth defects is now undoubted. Oddly, no individual nor Grunenthal itself was successfully prosecuted over the disaster. Grunenthal no longer produces thalidomide, but today is one of the world's largest Pharmaceutical companies producing drugs for pain relief. The survivors of the thalidomide disaster have severe handicaps, and many of the survivors are experiencing early onset age related issues, such as osteoarthritis, joint mobility issues and coronary heart disease, making life more difficult Miller and Stromland, ; Newman, ; Smithells and Newman, ; McCredie, ; Vargesson, , In some countries the distributors of the drug, for example, Distillers in the UK and Australia now Diageo , have been sued for compensation by the survivors.
Compensation has been received from such distributors. In addition Government support has been given to survivors. However the amount of support varies between countries. Today, thalidomide is used to successfully treat a wide range of medical conditions, which include leprosy, multiple myeloma, and cancers, as well as Crohn's disease, HIV, and others. Thalidomide use is carefully monitored using successful schemes like the S. However, tragically a new generation of thalidomide survivors has occurred in Brazil Castilla et al.
Thalidomide is used to treat complications of leprosy in Brazil, which is sadly common and debilitating. Unfortunately the drug is given to patients who share the medicine with others, and who do not understand or are not informed of the dangers, and damaged children are born.
The damage to these children is similar to the damage seen in children between and Between and , thalidomide caused severe birth defects in over 10, children.
Indeed a detailed UK Government sponsored report in UK Government Report, detailed that almost all the tissues and organs of the body could be affected by the drug. Damage was primarily seen to the limbs upper limbs more commonly affected than lower limbs , face, eyes, ears, genitalia, and internal organs, including heart, kidney, and gastrointestinal tract.
The vertebral column was also affected in some survivors and occurrence of facial palsies was also documented. Due to the wide range of damage and conditions that thalidomide exposure can cause, the damage is usually referred to collectively as thalidomide embryopathy or thalidomide syndrome, as some of the damage is not unique to thalidomide embryopathy and can be seen in other human conditions, for example Duane syndrome Smithells and Newman ; Miller and Stromland, Thalidomide embryopathy is a severe condition and affects many tissues, all of which can occur independently in humans but rarely together.
Furthermore, many babies with these malformations are likely to have died in utero and been miscarried or stillborn. The true numbers of babies affected by thalidomide will likely never be known. Damage to the limbs are one of the most common and studied features of thalidomide embryopathy. Phocomelia remains the most striking limb deformity caused by thalidomide, and remains the stereotypical image of thalidomide embryopathy.
Radial dysplasia loss of the radius and thumb is also seen in thalidomide survivors Lenz and Knapp, ; Spiers, ; Taussig, ; Ruffing, ; Smithells, ; Newman, ; Smithells and Newman, The range and type of upper limb deformities induced by thalidomide exposure exhibit a characteristic pattern. The thumb is the first structure to be affected, followed by the radius, humerus, and lastly the ulna Lenz and Knapp, ; Nowack, ; Smithells and Newman, ; McCredie, ; Miller et al.
The majority of thalidomide survivors exhibit some form of limb deformity, which are usually reduction defects and most often symmetrical. Indeed symmetrical limb defects remain one of the hallmarks for the diagnosis of thalidomide embryopathy.
However, several studies indicate that unilateral limb defects can occur in thalidomide survivors and differences in severity can also occur between each side of the body—so does not have to be symmetrical Lenz and Knapp, ; UK Government Report, ; Schmidt and Salzano, ; Schmidt and Salzano, Phocomelia or Amelia are seen, as well as reduction in the long bone lengths. However, abnormalities of the lower limbs are seen less commonly than those of the upper limbs, with lower limb deformities on their own occurring, but being very rare.
The femur is the bone most often affected in the lower limb, and similarly to the ulna, the fibula is usually the final bone to remain normal Nowack, ; Hamanishi, ; Lenz, ; Smithells and Newman, ; Miller and Stromland, ; McCredie, Why the upper limbs are more affected in thalidomide survivors is unclear.
The severity of limb damage seen in thalidomide survivors can vary between each side of individuals. Some cases exhibit bilateral disorders and others have a more severely affected limb on one side of the body. More strikingly, there are several reports of unilateral limb damage in thalidomide survivors, though these cases are rare Lenz and Knapp, ; Somers, ; UK Government Report, ; Schmidt and Salzano, ; Schmidt and Salzano, Characteristic shoulder and hip joint damage occurs in thalidomide embryopathy.
For example, the acromioclavicular joint of the shoulder is more prominent, and sharpened in appearance, when the shoulder is damaged through thalidomide exposure Newman, The hip joint can be hypoplastic or, in some cases, completely absent, as is also true for the pubic bone Newman, Eye and ear damage internal and external is another hallmark used to diagnose thalidomide embryopathy.
Thalidomide can cause small eyes micropthalmia , anophthalmos absence of the eyeball , and poor vision. Eye defects also include aberrant or no lacrimation tear formation , coloboma, and strabismus Smithells, ; Cullen, ; James, ; Newman, ; Schmidt and Salzano, ; Kelsey, ; Miller and Stromland, Ocular defects can occur unilaterally, although there may still be poor vision in the unaffected eye Smithells, Abnormalities in eye movement are also observed and usually occur in conjunction with ear defects and facial muscle weakness Smithells and Newman, Ear defects in thalidomide survivors are usually symmetrical, ranging from absence of the outer anotia to reduction of the outer ear microtia; Ruffing, ; Smithells and Newman, Deafness or reduced hearing, as well as cranial nerve palsies in addition to ear defects, are also observed in some thalidomide survivors Smithells and Leck, ; Livingstone, ; Newman, ; Ruffing, ; Newman, ; Miller and Stromland, The hemangioma is usually observed in the centre of the forehead and can extend over the nose to the upper lip.
Facial palsy and facial asymmetry are also associated with thalidomide embryopathy, likely due to weakened facial muscles and facial nerve damage Newman, , ; Smithells and Newman, ; Miller and Stromland, Various vertebral column issues have been reported in thalidomide survivors, including irregular vertebral spacing; fusion of vertebrae particularly in the lower spinal column, and in some cases progressive kyphosis which often requires surgical intervention throughout life Ruffing, ; Smithells and Newman, Thalidomide survivors can have shorter stature than average, with the shortness due to shorter leg bones, independent of vertebral defects Brook et al.
All of the internal organs can be affected following thalidomide exposure in utero. Damage seen includes malformations of the heart, kidneys, genitals, and gastrointestinal tract Smithells and Newman, ; Ruffing, ; Lenz and Knapp, However, the precise incidence of such deformities is unknown, as such defects are not always outwardly seen or apparent, particularly if they do not present until later in life.
Genital defects, both internal and external, have been reported in thalidomide survivors. These include absence of the testes, testicular abnormalities, and hypospadiasis in males, and in females malformations of the uterus and reproductive tract defects Cuthbert and Speirs, ; Newman, ; Newman, ; Smithells and Newman, Other common problems associated with thalidomide exposure include anorectal stenosis, intestinal atresia, pyloric stenosis, and inguinal hernia Lenz and Knapp, ; Somers, ; Cuthbert and Speirs, ; Nowack, ; Newman, , ; Smithells and Newman, Thalidomide could affect developing neural pathways Miller and Stromland, , possibly by preventing angiogenesis in the brain Hallene et al.
Animal model evidence indicates such damage can occur quite late in fetal development Hallene et al. Exposure before and after this time sensitive window is thought to not cause damage to the embryo Newman, , ; Lenz and Knapp, ; Smithells and Newman, However, early exposure to thalidomide in humans and rats induces miscarriage, and late fetal exposure in rats induces brain damage James, ; Kajii et al.
This indicates there is likely no safe time period to take the drug. Indeed a detailed UK Government report into the thalidomide disaster in the UK indicates that a few children were born unharmed or without outward damage to mothers with confirmed exposure to the drug during pregnancy UK Government Report, Whether these individuals had other problems, not obvious at birth, that became apparent later in life is unknown.
From these interviews, dates of intake and exposure were ascertained and correlated to the damage seen in the infants, to determine the timing of damage. This window correlates with outward damage primarily and severe internal organ damage.
Obviously any other damage that may not present until later in life could not be recorded. Exposure to thalidomide earlier in the time sensitive window results in more severe damage, as evidenced by outward signs, such as effects on the limbs, face, and genitals, and obvious internal damage, such as kidney failure Fig.
Embryogenesis lasts until around week 10—11 when the embryo is fully patterned and formed. The remaining period of pregnancy allows the fetus, as it is now termed, to grow and mature. As thalidomide was used to treat morning sickness, which can occur from week 4 and last until week 12 in some women, and in others throughout pregnancy, this exposure coincides with the major developmental events underpinning the thalidomide disaster.
Given that the majority of studies in humans in the 's assessed damage in severely affected infants, and there are very few studies assessing the damage or late onset damage in adults, it is possible that the drug did cause far more damage than initially described. For example, one littermate could exhibit varying damage to all 4 limbs, another to one limb, and another just a tail anomaly Merker et al.
The mechanism underlying this variance in damage is unknown, but also hints that thalidomide damage in humans could be broader. How and why the drug caused such a range and variability in damage remains unclear, but likely includes individual differences in metabolism and clearance of the drug, as well genetic and environmental factors. Some of the thalidomide induced damage is not unique to thalidomide embryopathy, but can be seen independently in other human conditions, such as facial palsies, Duane syndrome, and autism and limb reduction, for example, radial dysplasia Smithells and Newman, ; Miller and Stromland, ; Lenz and Knapp, Indeed, some of the damage the drug causes can often be confused for other human congenital malformations.
This can make diagnosis challenging on occasion, particularly if the mother cannot recall or admit to using thalidomide during pregnancy. As has another much rarer condition, Roberts Syndrome, also known as pseudothalidomide syndrome, given its striking phocomelia to all four limbs, facial damage, and internal organ damage. A zebrafish model of Roberts syndrome indicates that ESCO2 loss prevents normal development by disrupting the cell cycle Monnich et al.
Patients with Wnt3 mutations also have facial and urogenital damage, also seen in some thalidomide survivors. The similarity of thalidomide embryopathy to other human conditions highlights the difficulties faced in diagnosing conditions.
With the advent of genetic testing, many conditions can now be tested for and ruled out. Another important aspect that helps identify thalidomide embryopathy from genetic conditions is that thalidomide is not a mutagen and defects are not hereditary, that is, passed on to the next generation Ashby et al.
Indeed a recent study in Sweden followed offspring from thalidomide survivors and found no evidence of any injuries or damage in their offspring Stromland et al. However, patients with less severe damage or, for example, not exhibiting bilateral limb damage, were excluded. The precise range and severity of damage this drug caused and causes may never be known. However, with the ongoing thalidomide tragedy in Brazil, the thalidomide survivors, many of whom are children or young adults, offer the possibility of studying the damage caused and its progression, as well as late onset problems.
The new diagnostic algorithm proposed www. Thalidomide was marketed as a nonaddictive, nonbarbiturate sedative that would also be nonlethal if overdoses occurred unlike barbiturates; Fig. The drug is a synthetic derivative of glutamic acid, a naturally occurring amino acid involved in important physiological processes, for example, brain neurotransmission and metabolism.
Thalidomide consists of two linked rings, a glutarimide and pthalimide ring Fig. Thalidomide has a chiral carbon, which is unstable and allows two enantiomers to coexist, which can interswitch between the two states rapidly in bodily fluids and in water Smith et al. However as the drug can convert racemise between enantiomeric states, it is very difficult to make a stable form that is nonteratogenic.
Thalidomide was sold as a racemic mix of both enantiomers Fig.
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